Gene Synthesis Error Correction

CCATGGGCAGCAGCcAtcAtcatcATCAT

After PCR (gene synthesis), treat PCR mixture with 5 U mismatch cleaving enzymes: 5 µl aliquot of the PCR reaction E.coli endonuclease V, or 1000 U T4 endonuclease VII (USB) 1 X reaction buffer Water to 20 µl. Incubate at 37 oC in for 30 min to 24 h.

Optional: add E.coli exonuclease I (Fermentas) to digest single stranded DNA.

5X Isothermal (ISO) Buffer:25% PEG 8000, 500 mM Tris HCl pH 8.050 mM MgCl250 mM DTT1 mM each of the 4 dNTPs5 mM NAD. 6 ml of this buffer can be prepared by combining the following: 3 ml of 1 M Tris HCl pH 8.0300 μl of 1 M MgCl2600 μl of 10 mM dNTPs300 μl of 1 M DTT (1.54 g dissolved in dH20 up to 10 ml)1.5 g PEG 8000300 μl of 100 mM NAD (Sigma; 0.66 g dissolved in dH20 up to 10 ml; resuspend by heating at 50°C followed by continuous vortexing)Add water to 6 ml, aliquot 1 ml and store at 80°C. 2X Assembly Master Mix:ISO reaction buffer and the enzymatic activities required for assembly of the component oligonucleotides into the gene products: 5X isothermal (ISO) reaction buffer) T5 exonuclease (Epicentre), PHUSION® DNA polymerase (Finnzymes Oy, Vantaa, Finland) and Taq DNA ligase.800 μl of the 2X assembly master mix, sufficient for 80 reactions, can be prepared by combining the following:320 μl 5X ISO buffer as prepared above6.4 μl of 1 U/ μl T5 exo (diluted 1 : 10 from enzyme stock in lx T5 exo buffer) 20 μl of 2 U/μl PHUSION® polymerase80 μl οf 40 U/μl Taq ligase374 μl dH20 Mix well and store at 20°C, or on ice if to be used immediately.The assembly mixture can be stored at 20°C for at least one year. The enzymes remain active following at least 10 freeze thaw cycles. The mixture is ideal for the assembly of DNA molecules with 20 150 bp overlaps Genes Assembly 10~15 μl oligo mixmix 1 μl of 2 μM each oligo Take 10 μl of this oligo mix Add 10 μl 2X assembly master mix prepared as above. Incubate for 50°C for 1 hour. Then PCR: 2.5 μl assembly reaction10 μl 5× PHUSION® HF Buffer1 μl 10 mM dNTPs35.5 μl water0.5 μl Hot Start PHUSION® Polymerase1 μl 10 μM F1 μl 10 μM R 98° C. for 1 minute, 25 cycles98° C. for 10 seconds 60° C. for 30 seconds72° C. for 1.5 minutes 72° C. for 5 minutes Cel1 nuclease/Exonuclease IIITransfer 8 μl of PCR reaction:98° C. for 2 minutesslow cool to 85° C. at 2° C./secondhold at 85° C. for 2 minutesslow cool to 25° C. at 0.1° C./secondhold at 25° C. for 2 minutesand then hold at 10° C. Then add2 μl SURVEYOR® nuclease incubate at 42° C for 1 hour.Then add1 μl Exonuclease III (diluted 1 to 4000 in 1×HF Buffer) incubated at 37° C. for 1 hour.Use 2 μl of this reaction mix for PCR as described above. SURVEYOR ® nuclease +Exonuclease III > Error rate: 1/5,572 bp, correct clone: 21 out of 28 (75%) Combinations of various endonuclease enzymes alone or together with an exonuclease enzyme or a polymerase enzyme having exonuclease activity to remove inherent mismatches, primarily due to incorrect sequences within oligonucleotides incorporated in the assembled gene products.

SURVEYOR® nuclease (cuts 3′ to mismatch) was used to perform error correction alone or in a two step reaction as described above together with PHUSION® DNA polymerase (Finnzymes, Oy, Finland), which has 3′ to 5′ exonuclease activity. Reaction conditions were 42° C. for 20 minutes with SURVEYOR® nuclease, followed by 37° C. for 20 minutes with PHUSION® DNA polymerase.

T7 endonuclease (cuts 5′ to mismatch) was used to perform error correction alone or in a two step reaction as described above together with T5 exonuclease having 5′ to 3′ exonuclease activity. Reaction conditions were 37° C. for 20 minutes with T7 endonuclease, followed by 37° C. for 20 minutes with T5 exonuclease.

Gene Error correction Method Error Rate Number ofcorrect clones HA None 1/791 bp 2 out of 19 (10.5%)

HA SURVEYOR ® nuclease 1/1,725 bp 8 out of 26 (30.8%)

HA<< SURVEYOR ® nuclease + PHUSION ® DNA polymerase<< 1/2,217 bp<< 13 out of 26 (50%)<<

HA T7 endonuclease 1/973 bp 5 out of 25 (20%)

HA T7 endonuclease + T5 exonuclease 1/1,504 bp 6 out of 21 (28.6%)

Error doped assembly

http://www.biorxiv.org/content/early/2017/01/15/100685<

ErrASE was the most effective at decreasing the error frequency, with two rounds of treatment dropping the error frequency from the doped oligo rate of 46.1 to 8.0 errors/kb. The next most effective enzyme at decreasing error frequency was T7 Endonuclease I (9.1 errors/kb). Based on previous reports in the mutation detection literature, we hypothesized that the addition of a ligase with T7 Endonucluase I would improve correction [39]. We find that the addition of T7 ligase actually decreased assembly quality relative to the no ligase control. In agreement with previous studies, we also find T7 Endonuclase I to be highly sensitive to protocol and concentration as exhibited by the wide range of error frequencies [12, 14]. After T7 Endonuclease I, we found MutS to be the third most effective enzyme at 11.2 errors/kb, with T4 Endonuclease VII, Surveyor, and Endonuclease V following.<

ErrASE https://www.google.com/patents/US20150010953 http://www.novicibiotech.com/ https://www.thermofisher.com/order/catalog/product/A14972<

perfect assemblies sequences<

However, when looking at number of perfect assemblies sequences, MutS was the most effective enzyme treatment. MutS increased the percentage of perfect sequences in the doped oligo from 1.9% to 47.8% (47.6% for 950nM), while ErrASE increased it to 45.6%, and T7 Endonuclease I increased it to 41.7%. In other words, the oligos that are imperfect after the MutS treatment have more errors on average than those after the T7 Endonuclease I and ErrASE treatments.<

in general, all enzymes tested were able to correct insertions and deletions. We find that enzyme performance (as measured by error frequency or number of perfect assembliess) is directly related to the ability to correct mismatches. For example the best performing enzymes, ErrASE, T7 Endonuclease I, and MutS, were able to decrease the median mismatch error rate relative to the error doped input by 6.2 , 5.1 , and 4.2 fold, respectively. In contrast, the worst performing enzyme, Endonuclease V, was unable to decrease the median mismatch error rate relative to the error doped input. We note that these measurements represent a ceiling on mismatch correction, as it is likely that the polymerase added mismatches back into the sample during the sequencing preparation.<

two rounds of ErrASE or MutS treatment resulted in a significantly different change in the median fold reduction of C/G ! G/C mismatches as compared to the bulk median of all other mismatches (15.2 vs 5.4 fold for ErrASE; 5.1 vs 4.1 fold for MutS; Mann Whitney U, p << 0:001). In contrast, two rounds T7 Endonuclease I did not result in significant changes in the median fold reduction of C/G ! G/C mismatches (5.6 vs 5.1 fold; Mann Whitney U, NS). They did however, significantly change the median fold reduction of A/T ! T/A mismatches as compared<

to the bulk median of all other mismatches (12.7 vs 4.2 fold; Mann Whitney U, p << 0:001). Lastly, we found that using a T7 Ligase in conjunction with T7 Endonuclase I actually decreased assembly quality relative to the no ligase control, in contrast to previous reports [39]. These data also suggest that T7 Endonuclase I is sensitive to protocol, as the no ligase control performed worse than the Fuhrmann et al. protocol [12].<

For example, GC or AT rich constructs would be best corrected by ErrASE and T7 Endonuclease I, respectively. Alternatively, MutS can be used for applications such as protein libraries, where the proportion of perfect sequences are paramount<

the relative rate of correction for transitions is likely lower than what is measured here due to polymerase errors. For example, the median fold correction of A/T ! G/C transitions (the most common Taq based error) was significantly different than that of the bulk median for all other mismatches for ErrASE, MutS, and T7 Endonuclase I (2.6 vs 7.1 fold for ErrASE; 2.8 vs 4.4 fold for MutS; 2.5 vs 6.8 fold for T7 Endonuclease I; Mann Whitney U, p << 0:001).<

ErrASE would bet the most effective at correcting GC rich templates, and T7 Endonuclease I is the most effective at correcting AT rich templates. Alternatively, MutS would be appropriate for the most common applications requiring a single sequenced verified perfect assembly<

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CEL I is a mismatch endonuclease isolated from celery. The use of CEL I in a diagnostic method for the detection of mutations in targeted polynucleotide sequences, in particular, those associated with cancer, is disclosed in U.S. Pat. No. 5,869,245. Methods of isolating and preparing CEL I are also disclosed in this patent. However, there is no disclosure in this patent relating to the use of CEL I in DNA sequence reassortment.<

Nucleic acid molecules that encode CEL I are disclosed in PCT Application Publication No. WO 01/62974 A1. As with U.S. Pat. No. 5,869,245, the use of CEL I in a diagnostic method for the detection of mutations in targeted polynucleotide sequences associated with cancer is disclosed. Also similarly, there is no disclosure relating to the use of CEL I in DNA reassortment.<

The use of RES I endonuclease is contemplated in diagnostic methods for the detection of mutations in targeted polynucleotide sequences, in particular, those associated with cancer. Examples of some of these types of diagnostic methods are disclosed in U.S. Pat. No. 5,869,245, Sokurenko, et al., and Del Tito, et al.<

The reactivity of Endonuclease VII of phage T4 with DNA loops of eight, four, or one nucleotide, or any of 8 possible base mismatches in vitro is disclosed in “Endonuclease VII of Phage T4 Triggers Mismatch Correction in Vitro” Solaro, et al., J Mol Biol 230(93)868. The publication reports a mechanism where Endonuclease VII introduces double stranded breaks by creating nicks and counternicks within six nucleotides 3′ of the mispairing. The publication discloses that a time delay between the occurrence of the first nick and the counternick was sufficient to allow the 3′ 5′ exonuclease activity of gp43 to remove the mispairing and its polymerase activity to fill in the gap before the occurrence of the counternick. Nucleotides are erased from the first nick, which is located 3′ of the mismatch on either strand and stops 5′ of the mismatch at the first stable base pair. The polymerase activity proceeds in the 5′ to 3′ direction towards the initial nick, which is sealed by DNA ligase. As a result, very short repair tracks of 3 to 4 nucleotides extend across the site of the former mismatch. The publication concludes with a discussion regarding the various activities Endonuclease VII may have within phage T4. However, the publication does not disclose any practical utility for Endonuclease VII outside of phage T4, and there is no disclosure regarding its applicability in DNA reassortment.<

CEL I enzyme was prepared from celery stalks using the homogenization, ammonium sulfate, and Concanavalin A Sepharose protocol described by Yang et al. (Biochemistry, 39:3533–3541 (2000), incorporated herein by reference. A 1.5 kg sample of chilled celery stalks was homogenized with a juice extractor. One liter of juice was collected, adjusted to 100 mM Tris HCL, pH 7.7 with 100 micromolar phenylmethylsulfonyl fluoride (PMSF), and filtered through two layers of miracloth. Solid (NH4)2SO4 was slowly added to 25% saturation while stirring on ice. After 30 minutes, the suspension was centrifuged at 27,000 g for 1.5 hours at 4° C. The supernatants were collected and adjusted with solid (NH4)2SO4 to 80% saturation while stirring on ice followed by centrifugation at 27,000 g for 2 hours. The pellets were re suspended in buffer B (0.1 M Tris HCL, pH 7.7, 0.5 M KCl, 100 micromolar PMSF) and dialyzed against the same buffer.<

Conconavalin A (ConA) Sepharose affinity chromatography was performed by first incubating the dialyzed sample with 2 ml of ConA resin overnight with gentle agitation. The ConA resin was then packed into a 0.5 cm diameter column and washed with several column volumes of buffer B. Elution was performed using 0.3 M alpha methyl mannoside in buffer B. Fractions were collected in 1 ml aliquots. Fractions were assayed for mismatch cleavage activity on a radiolabeled mismatch substrate by incubating 0.1 microliter of each fraction with the mismatched probe in buffer D (20 mM Tris HCL, pH 7.4, 25 mM KCL, 10 mM MgCl2) for 30 minutes at 45° C. as described by Oleykowski et al. (Nucleic Acids Research 26: 4597–4602 (1998), incorporated herein by reference. Reaction products were visualized by separation on 10% TBE PAGE gels containing 7% urea (Invitrogen), followed by autoradiography. Aliquots of the CEL I fractions having mismatch cleavage activity were stored frozen at −20° C. A series of five fold dilutions of CEL I fraction #5 were then analyzed for mismatch cleavage of radiolabeled mismatch substrate. Reactions were performed either in buffer D, New England BioLabs (NEB) T4 DNA ligase buffer (50 mM Tris HCL, pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mM ATP, 25 microgram/ml BSA), or Gibco/BRL T4 DNA ligase buffer (50 mM Tris HCL, pH 7.6, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 5% (w/v) polyethylene glycol 8000). Reaction products were visualized as above. Cleavage activity in buffer D and in NEB T4 DNA ligase buffer were found to be roughly equivalent, whereas cleavage in the PEG containing Gibco/BRL ligase buffer was enhanced by five to ten fold compared to the other buffers.<

Additional analysis of CEL I activity was carried out using defined heteroduplex DNAs from two different Green Fluorescent Protein (GFP) genes as substrate. This GFP heteroduplex substrate was prepared by annealing single stranded DNAs corresponding to cycle 3 GFP on the sense strand and wild type GFP on the antisense strand. The single stranded DNAs had been synthesized by asymmetric PCR and isolated by agarose gel electrophoresis. After annealing by heating to 90° C. and cooling in the presence of 1×NEB restriction enzyme buffer 2 (10 mM Tris HCL, pH 7.9, 10 MM MgCl2, 50 mM NaCl, 1 mM dithiothreitol), the heteroduplex DNA was isolated by agarose gel electrophoresis followed by excision of the heterduplex band and extraction using Qiaquick DNA spin columns. A total of twenty eight mismatches, one or two nucleotides in length, occur throughout the length of the heteroduplex molecule. The distribution of the mismatches ranges from small clusters of several mismatches separated by one or two nucleotides to mismatches separated by more than thirty base pairs on either side.<

A series of three fold dilutions of CEL I in 1×NEB T4 DNA ligase buffer were prepared and one microliter aliquots of each were incubated in two separate series of 10 microliter reactions, each containing as substrate either 0.5 microgram of a supercoiled plasmid preparation or one hundred nanograms of the cycle3/wild type GFP heteroduplex. All reactions took place in 1×NEB T4 DNA ligase buffer. Reactions were incubated at 45° C. for 30 minutes and run on 1.5% TBE agarose gel in the presence of ethidium bromide.<

Treatment of the supercoiled plasmid preparation with increasing amounts of CEL I resulted in the conversion of supercoiled DNA to nicked circular, then linear molecules, and then to smaller fragments of DNA of random size. Treatment of the mismatched GFP substrate with the CEL I preparation resulted in the digestion of the full length heteroduplex into laddered DNA bands which are likely to represent cleavage on opposite DNA strands in the vicinity of clusters of mismatches. Further digestion resulted in the conversion of the mismatched GFP substrate to smaller DNAs that may represent a limit digest of the heteroduplex DNA by the CEL I preparation.<

This example teaches various mismatch resolution cocktails that conserve the full length GFP Gene.<

Mismatched GFP substrate was treated with various concentrations of CEL I in the presence of cocktails of enzymes that together constitute a synthetic mismatch resolution system. The enzymes used were CEL I, T4 DNA polymerase, Taq DNA polymerase and T4 DNA ligase. CEL I activity should nick the heteroduplex 3′ of mismatched bases. T4 DNA polymerase contains 3′ 5′ exonuclease for excision of the mismatched base from the nicked heteroduplex. T4 DNA polymerase and Taq DNA polymerase contain DNA polymerase capable of filling the gap. T4 DNA ligase seals the nick in the repaired molecule. Taq DNA polymerase also has 5′ flap ase activity.<

Matrix experiments were performed to identify the reaction conditions that would serve to resolve mismatches in the GFP heteroduplex substrate. In one experiment, cycle 3/wild type GFP heteroduplex was incubated in a matrix format with serial dilutions of CEL I fraction number five (described above) at eight different concentrations. Each reaction contained 100 nanograms of heteroduplex substrate and 0.2 microliters of T4 DNA ligase (Gibco BRL) in 1×NEBT4 DNA ligase buffer and dNTPs at 250 micromolar each, in a reaction volume of 10 microliters. In all, the matrix contained 96 individual reactions. One full set of reactions was incubated at room temperature for 30 minutes while another full set was incubated at 37° C. for 30 minutes.<

After incubation, PCR was used to amplify the GFP gene from each reaction. Aliquots from each PCR were then digested with HindIII and HpaI and electrophoresed on 3% agarose gels with ethidium bromide. Only cycle 3 GFP has a HindIII site and only wild type encodes a HpaI site.<

If DNA mismatch resolution occurred at either the HindIII or HpaI mismatched sites, then a proportion of the PCR product would be expected to contain both sites, yielding a novel band. The band was observed in all samples, including the negative control samples that had neither CEL I, nor T4 DNA polymerase, nor Taq DNA polymerase. The results suggested that a basal level of background recombination may have occurred at some point in the experiment other than in the GRAMMR reaction; possibly in the PCR step. PCR mediated recombination is known to occur at some frequency between related sequences during amplification [reference Paabo, et al., DNA damage promotes jumping between templates during enzymatic amplification. J Biol Chem 265(90)4718–4721].<

In another experiment, 200 nanograms of cycle 3/wild type GFP heteroduplex was treated with CEL I and T4 DNA polymerase in various concentrations along with 2.5 units of Taq DNA polymerase in the presence or absence of T4 DNA ligase (0.2 units; Gibco BRL). Each reaction contained 1×NEB T4 DNA ligase buffer with 0.05 mM each dNTP in a final volume of 20 microliters. Reactions were incubated for 30 minutes at 37° C. and 10 microliters were run on a 2% TBE agarose gel in the presence of ethidium bromide. Results showed that in the presence of DNA ligase, but in the absence of T4 DNA polymerase, increasing amounts of CEL I caused greater degradation of the heteroduplexed DNA, but that this effect could be counteracted by increasing the amount of T4 DNA polymerase in the reaction. These results indicated that the various components of the complete reaction could act together to conserve the integrity of the full length gene through DNA mismatch resolution.<

Another matrix experiment was conducted to expand on these results and to identify additional conditions for DNA mismatch resolution for this synthetic system. 60 nanograms of cycle3/wild type GFP heteroduplex were treated with CEL I and T4 DNA polymerase at various concentrations in the presence of 2.5 units of Taq DNA polymerase and 0.2 units of T4 DNA ligase in 1×NEB T4 DNA ligase buffer containing 0.5 mM of each dNTP in a reaction volume of 10 microliters. Each set of reactions was incubated for 1 hour at either 20° C., 30° C., 37° C., or at 45° C. All reactions were then run on a 1.5% TBE agarose gels in the presence of ethidium bromide. The results showed that the GFP heteroduplex was cleaved into discrete fragments by the CEL I preparation alone. The success of DNA mismatch resolution was initially gauged by the degree to which the apparent full length integrity of the GFP sequence was maintained by the other components of the mismatch resolution system in the presence of CEL I. Conditions of enzyme concentration and temperature were identified that conserved a high proportion of the DNA as full length molecules in this assay. Namely, one microliter of the CEL I fraction five preparation (described in Example 1) with one microliter (1 unit) of the T4 DNA polymerase in the presence of the other reaction components which were held constant in the experiment. It was found that as the reaction temperature increased, the degradative activity of CEL I increased accordingly. Furthermore, it was shown that the other components of the repair reaction acted to conserve the integrity of the full length DNA at 20° C., 30° C., and 37° C., but was remarkably less efficient at conserving the full length DNA at 45° C. From these results, we concluded that under these experimental conditions, incubation at 45° C. was not optimal for the process of GRAMMR, and that incubation at 20° C., 30° C., and 37° C. were permissible.<

Another experiment was performed in which alternative enzymes were used for the DNA mismatch resolution reaction. Instead of T4 DNA ligase, Taq DNA ligase was used. Pfu DNA polymerase (Stratagene) was employed in a parallel comparison to a set of reactions that contained T4 DNA polymerase as the 3′ exonuclease/polymerase. Reactions were carried out in Taq DNA ligase buffer containing 8 units of Taq DNA ligase (NEB), 2.5 units Taq DNA polymerase, 0.5 mM of each dNTP, various dilutions of CEL I, and either T4 DNA polymerase or Pfu DNA polymerase). Reactions were run on a 1.5% TBE agarose gels in the presence of ethidium bromide. It was found that in the presence of the Pfu DNA polymerase, Taq DNA polymerase, and Taq DNA ligase, the full length integrity of the CEL I treated substrate DNA was enhanced compared to DNA incubated with CEL I alone. This result shows that enzymes with functionally equivalent activities can be successfully substituted into the GRAMMR reaction. EXAMPLE 3 Restoration of Restriction Sites to GFP Heteroduplex DNA after DNA Mismatch Resolution (GRAMMR)<

This experiment teaches the operability of genetic reassortment by DNA mismatch resolution (GRAMMR) by demonstrating the restoration of restriction sites.<

The full length products of a twenty fold scale up of the GRAMMR reaction, performed at 37° C. for one hour, using the optimal conditions found above (the 1× reaction contained sixty nanograms of heteroduplex DNA, one microliter of CEL I fraction five (described in Example 1), one unit T4 DNA polymerase in the presence of 2.5 units of Taq DNA polymerase and 0.2 units of T4 DNA ligase in 1×NEB T4 DNA ligase buffer containing 0.5 mM of each dNTP in a reaction volume of 10 microliters) were gel isolated and subjected to restriction analysis by endonucleases whose recognition sites overlap with mismatches in the GFP heteroduplex, thereby rendering those sites in the DNA resistant to restriction enzyme cleavage. The enzymes used were BamHI, HindIII, HpaI, and XhoI. Negative controls consisted of untreated GFP heteroduplex. Positive controls consisted of Cycle 3 or wild type GFP sequences, individually. All controls were digested with the same enzymes as the product of the DNA mismatch resolution reaction. All samples were run on a 2% TBE agarose gel in the presence of ethidium bromide.<

After treatment with the mismatch resolution cocktail, a proportion of the DNA gained sensitivity to BamHI and XhoI restriction endonucleases, indicating that DNA mismatch resolution had occurred. The HpaI cut samples could not be interpreted since a low level of cleavage occurred in the negative control. The HindIII, BamHI and XhoI sites displayed different degrees of cleavage in the GRAMMR treated samples. Restoration of the XhoI site was more extensive than that of the BamHI site, which was in turn, more extensive than restoration at HindIII site.<

The extent to which cleavage occurs is indicative of the extent to which mismatches in the DNA have been resolved at that site. Differences in mismatch resolution efficiency may relate to the nature or density of mismatches present at those sites. For example, the XhoI site spans a three mismatch cluster, whereas the BamHI site spans two mismatches and the HindIII site spans a single mismatch. EXAMPLE 4 GRAMMR Reassorted GFP Genes<

This example demonstrates that GRAMMR can reassort sequence variation between two gene sequences in a heteroduplex and that there are no significant differences in GRAMMR products that were directly cloned, or PCR amplified prior to cloning.<

The GRAMMR treated DNA molecules of Example 3 were subsequently either directly cloned by ligation into pCR Blunt II TOPO (Invitrogen), or amplified by PCR and ligated into pCR Blunt II TOPO according to the manufacturer's instructions, followed by transformation into E. coli. After picking individual colonies and growing in liquid culture, DNA was prepared and the sequences of the GFP inserts were determined. As negative controls, the untreated GFP heteroduplex substrate was either directly cloned or PCR amplified prior to cloning into the plasmid.<

In GRAMMR, reassortment of sequence information results from a process of information transfer from one strand to the other. These sites of information transfer are analogous to crossover events that occur in recombination based DNA shuffling methods. For the purposes of relating the results of these reassortment experiments, however, the GRAMMR output sequences are described in terms of crossovers. Sequences of twenty full length GFP clones that were derived from the GRAMMR treated GFP genes were analyzed. Four of these clones were derived from DNA that had been directly cloned into pZeroBlunt [ref] following GRAMMR treatment (no PCR amplification). The other sixteen sequences were cloned after PCR amplification. Analysis of these full length GFP sequences revealed that all twenty sequences had undergone sequence reassortment having between one and ten crossovers per gene. A total of 99 crossovers were found in this set of genes, giving an average of about 5 crossovers per gene. With the distance between the first and last mismatches of about 590 nucleotides, an overall frequency of roughly one crossover per 120 base pairs was calculated. Within this set of twenty clones, a total of seven point mutations had occurred within the sequences situated between the PCR primer sequences, yielding a mutation frequency of roughly 0.05%.<

Thirty five clones that had not been subjected to GRAMMR treatment were sequenced. Of these controls, fourteen were derived from direct cloning and twenty one were obtained after PCR amplification using the GFP heteroduplex as template. Of these thirty five non GRAMMR treated control clones, eight were recombinants, ranging from one to three crossovers, with most being single crossover events. A total of twenty five point mutations had occurred within the sequences situated between the PCR primers, yielding a mutation frequency of roughly 0.1%.<

No significant differences were observed between the GRAMMR treated products that were either directly cloned or PCR amplified. Notably, though, in the non GRAMMR treated controls, the frequency of recombinants was higher in the PCR amplified DNAs than in the directly cloned DNAs. This higher frequency is consistent with results obtained by others in which a certain level of recombination was found to be caused by “jumping PCR.” [Paabo, et al., DNA damage promotes jumping between templates during enzymatic amplification. J Biol Chem 265(90)4718–4721]. EXAMPLE 5 Heteroduplex Substrate Preparation for Plasmid on Plasmid Genetic Reassortment By DNA Mismatch Resolution (POP GRAMMR) of GFP Plasmids<

This example teaches that heteroduplex substrate for Genetic Reassortment by DNA Mismatch Resolution can be in the form of intact circular plasmids. Cycle 3 GFP and wild type GFP heteroduplex molecules were prepared plasmid on plasmid (POP) format. In this format, the GFP sequences were reasserted within the context of a circular double stranded plasmid vector backbone. This made possible the recovery of the reasserted product by direct transformation of E. coli using an aliquot of the GRAMMR reaction. Consequently, neither PCR amplification nor other additional manipulation of the GRAMMR treated DNA was necessary to obtain reasserted clones.<

Mismatched DNA substrate for POP GRAMMR reactions was generated containing wild type GFP (SEQ ID NO:01) and Cycle 3 GFP (SEQ ID NO:02), resulting in the two pBluescript based plasmids, pBSWTGFP (SEQ ID NO:03) and pBSC3GFP (SEQ ID NO:04), respectively. The GFPs were inserted between the KpnI and EcoRI sites of the pBluescript polylinker so that the only sequence differences between the two plasmids occurred at sites where the wild type and Cycle 3 GFPs differ from one another. Both plasmids were linearized by digestion of the plasmid backbone with SapI, cleaned up using a DNA spin column, mixed, amended to 1×PCR buffer (Barnes, 1994; PNAS, 91, 2216–2220), heated in a boiling water bath for three minutes, and slow cooled to room temperature to anneal the denatured DNA strands. Denaturing and annealing these DNAs led to a mixture of duplexes, the re formation of parental duplexes, and the formation of heteroduplexes from the annealing of strands from each of the two input plasmids. Parental duplexes were deemed undesirable for GRAMMR and were removed by digestion with restriction enzymes that cut in one or the other parental duplex but not in the heteroduplexed molecules. PmlI and XhoI were chosen for this operation since PmlI cuts only in the wild type GFP sequence and XhoI cuts only Cycle 3 GFP. After treatment with these enzymes, the products were resolved on an agarose gel. The full length, uncut heteroduplex molecules were resolved from the PmlI and XhoI cut parental homoduplexes in an agarose gel and purified by excision of the band and purification with a DNA spin column.<

The resulting population of heteroduplexed molecules was treated with DNA ligase to convert the linear DNA into circular, double stranded DNA heteroduplexes. After confirmation by agarose gel shift analysis, the circular double stranded GFP heteroduplexed plasmid was used as substrate for GRAMMR reactions. Examples of the resulting clones are included as SEQ ID NO:05, SEQ ID NO:06, SEQ ID NO:07, and SEQ ID NO:08. EXAMPLE 6 Exemplary Reaction Parameters for Genetic Reassortment by DNA Mismatch Resoluton<

CEL I and T4 DNA Polymerase Concentrations Compared<

The GRAMMR reaction involves the interaction of numerous enzymatic activities. Several parameters associated with the GRAMMR reaction were examined, such as CEL I concentration, T4 DNA polymerase concentration, reaction temperature, substitution of T4 DNA polymerase with T7 DNA polymerase, the presence of Taq DNA polymerase, and the source of the CEL I enzyme. A matrix of three different CEL I concentrations versus two concentrations of T4 DNA polymerase was set up to examine the limits of the in vitro DNA mismatch resolution reaction.<

Twenty one nanograms (21 ng) of the circular double stranded heteroduplexed plasmid, prepared as described above, was used as substrate in a series of ten microliter reactions containing 1×NEB ligase buffer, 0.5 mM each dNTP, 1.0 unit Taq DNA polymerase, 0.2 units T4 DNA ligase (Gibco/BRL), either 1.0 or 0.2 units T4 DNA polymerase, and either 0.3, 0.1, or 0.03 microliters of a CEL I preparation (fraction 5, described in Example 1). Six reactions representing all six combinations of the two T4 DNA polymerase concentrations with the three CEL I concentrations were prepared, split into equivalent sets of five microliters, and incubated at either 20 degrees C. or 37 degrees C. A control reaction containing no CEL I and 0.2 unit of T4 DNA polymerase with the other reaction components was prepared and incubated at 37 degrees C. After 30 minutes, one microliter aliquots of each reaction were transformed into competent DH5 alpha E. coli which were then plated on LB amp plates. Colonies were picked and cultured. Plasmid DNA was extracted and examined by restriction fragment length polymorphism analysis (RFLP) followed by sequence analysis of the GFP gene sequences. RFLP analysis was based on differences in several restriction enzyme recognition sites between the wild type and Cycle 3 GFP genes. The RFLP results showed that throughout the CEL I/T4 DNA polymerase/temperature matrix, reassortment of restriction sites, that is GRAMMR, had occurred, and that no such reassortment had occurred in the zero CEL I control clones. DNA sequence analysis confirmed that reassortment had occurred in all of the CEL I containing samples. Sequencing also confirmed that the zero CEL I controls were not reasserted, with the exception of a single clone of the 16 control clones, which had a single base change from one gene sequence to the other, presumably resulting either from repair in E. coli or from random mutation. The sequences of several exemplary GRAMMR reassorted GFP clones are shown; all of which came from the reaction containing 0.3 microliters of the CEL I preparation and 1.0 unit of T4 DNA polymerase incubated at 37 degrees C. The parental wild type and Cycle 3 GFP genes are shown first for reference. EXAMPLE 7 Taq DNA Polymerase is Not Required for Genetic Reassortment by DNA Mismatch Resolution<

This experiment teaches that Taq DNA Polymerase does not dramatically, if at all, contribute or interfere with the functioning of Genetic Reassortment by DNA Mismatch Resolution (GRAMMR). Taq DNA polymerase is reported to have a 5′ flap ase activity, and had been included in the teachings of the previous examples as a safeguard against the possible formation and persistence of undesirable 5′ flaps in the heteroduplexed DNA undergoing GRAMMR.<

GRAMMR reactions were set up, as in Example 6, with twenty one nanograms of the circular double stranded heteroduplexed GFP plasmid substrate in ten microliter reactions containing 1×NEB ligase buffer, 0.5 mM each dNTP, 0.2 units T4 DNA ligase, 1.0 unit T4 DNA polymerase, 1.0 microliter of a CEL I preparation (fraction 5, described in Example 1), and either 2.5 units, 0.5 units of Taq DNA polymerase, or no Taq DNA polymerase. After 30 minutes, one microliter aliquots of each reaction were transformed into competent DH5 alpha E. coli which were then plated on LB amp plates. Colonies were picked and cultured. Plasmid DNA was extracted and examined by RFLP analysis followed by sequence analysis of the GFP gene sequences. The RFLP results showed that reassortment of restriction sites, that is, GRAMMR, had occurred both in the presence and the absence of Taq DNA polymerase in the GRAMMR reaction. DNA sequence analysis confirmed these results. Therefore, the data shows that Taq DNA polymerase was unnecessary for GRAMMR. EXAMPLE 8 Alternate Proofreading DNA Polymerases for Genetic Reassortment by DNA Mismatch Resolution<

This experiment teaches that Genetic Reassortment by DNA Mismatch Resolution is not limited to the use of T4 DNA polymerase, and that alternate DNA polymerases can be substituted for it.<

Reactions were set up, as in Example 6, with twenty one nanograms of the circular double stranded heteroduplexed GFP plasmid substrate in ten microliter reactions containing 1×NEB ligase buffer, 0.5 mM each dNTP, 0.2 units T4 DNA ligase (Gibco/BRL), 10 units or 2 units of T7 DNA polymerase, 1.0 microliter of a CEL I preparation (fraction 5, described in Example 1), and 2.5 units of Taq DNA polymerase. After 30 minutes, one microliter aliquots of each reaction were transformed into competent DH5 alpha E. coli which were then plated on LB amp plates. Colonies were picked and cultured. Plasmid DNA was extracted and examined by RFLP analysis followed by sequence analysis of the GFP gene sequences. The RFLP results showed that reassortment of restriction sites, that is GRAMMR, had occurred in both T7 DNA polymerase containing reactions. DNA sequence analysis confirmed these results. Therefore, the data shows that T7 DNA polymerase can substitute for T4 DNA polymerase for GRAMMR. In addition, it shows that individual components and functionalities can be broadly substituted in GRAMMR, while still obtaining similar results. EXAMPLE 9 Use of Cloned CEL I in the GRAMMR Reaction<

This example teaches that CEL I from a cloned source can be used in place of native CEL I enzyme purified from celery in Genetic Reassortment By DNA Mismatch Resolution without any noticeable change in results.<

The cDNA of CEL I was cloned from celery RNA. The gene was inserted into a TMV viral vector and expressed. Transcripts of the construct were used to infect Nicotiana benthamiana plants. Infected tissue was harvested, and the CEL I enzyme was purified. The GRAMMR results obtained using the purified enzyme were compared to those using CEL I purified from celery, and were found to be similar.<

Reactions were set up using twenty one nanograms of the circular double stranded heteroduplexed GFP plasmid substrate in ten microliters containing 1×NEB ligase buffer, 0.5 mM each dNTP, 0.2 units T4 DNA ligase (Gibco/BRL), 1 unit of T4 DNA polymerase, and either 1.0 microliter of CEL I purified from celery (fraction 5, described in Example 1), or 0.3 microliters of CEL I purified from a cloned source. After 30 minutes, one microliter aliquots of each reaction were transformed into competent DH5 alpha E. coli which were then plated on LB amp plates. Colonies were picked and cultured. Plasmid DNA was extracted and examined by RFLP analysis followed by sequence analysis of the GFP gene sequences. The RFLP results showed that reassortment of restriction sites, that is, GRAMMR had occurred in both celery derived CEL I, as well as cloned CEL I containing reactions. DNA sequence analysis confirmed these results. Therefore, the data shows CEL I from a cloned source can be used in lieu of CEL I from celery for GRAMMR. In addition, the data demonstrates that it is CEL I activity that is part of the GRAMMR method, rather than a coincidental effect resulting from the purifying steps used in extracting CEL I from celery. EXAMPLE 10 Molecular Breeding of Tobamovirus 30K Genes in a Viral Vector.<

In the preceding examples, Genetic Reassortment by DNA Mismatch Resolution has been taught to be useful for reasserting sequences that are highly homologous, for example, wtGFP and Cycle 3 GFP are 96% identical. The present example teaches that GRAMMR can be used to reassort more divergent nucleic acid sequences, such as genes encoding tobamovirus movement protein genes.<

Heteroduplexes of two tobamovirus movement protein (MP) genes that are approximately 75% identical were generated. The heteroduplex substrate was prepared by annealing partially complementary single stranded DNAs of opposite strandedness synthesized by asymmetric PCR; one strand encoding the movement protein gene from the tobacco mosaic virus U1 type strain (TMV U1) (SEQ ID NO:09), and the other strand encoding the movement protein gene from tomato mosaic virus (ToMV) (SEQ ID NO:10). The sequences of the two partially complementary movement protein genes were flanked by 33 nucleotides of absolute complementarity to promote annealing of the DNAs at their termini and to facilitate PCR amplification and cloning. The annealing reaction took place by mixing 2.5 micrograms of each single stranded DNA in a 150 microliter reaction containing 333 mM NaCl, 33 mM MgCl2, 3.3 mM dithiothreitol, 166 mM Tris HCl, pH 7, and incubating at 95° C. for one minute followed by slow cooling to room temperature. GRAMMR was performed by incubating 5 microliters of the heteroduplex substrate in a 20 microliter reaction containing 1×NEB ligase buffer, 0.5 mM each dNTP, 0.4 units T4 DNA ligase (Gibco/BRL), 2.0 units of T4 DNA polymerase, and CEL I. The CEL I was from a cloned preparation and the amount that was used varied from 2 microliters of the prep, followed by five serial 3 fold dilutions. A seventh preparation with no CEL I was prepared, which served as a control.<

After one hour at room temperature, DNA was purified from the reactions using Strataprep spin DNA purification columns (Stratagene, LaJolla, Calif.) and used as templates for PCR reactions using primers designed to anneal to the flanking primer binding sites of the two sequences. PCR products from each reaction were purified using Strataprep columns, digested with AvrII and PacI, and ligated into the movement protein slot of similarly cut pGENEWARE MP Avr Pac. This plasmid contained a full length infectious tobamovirus GFP clone modified with AvrII and PacI sites flanking the movement protein gene to permit its replacement by other movement protein genes. After transformation of DH5 alpha E. coli and plating, colonies were picked, cultures grown, and DNA was extracted. The movement protein inserts were subjected to DNA sequence analysis from both directions and the sequence data confirmed that in the majority of inserts derived from the GRAMMR treated material were reasserted sequences made up of both TMV U1 and ToMV movement protein gene sequences. The DNA sequences of several exemplary GRAMMR MP clones are shown as SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. EXAMPLE 11 GRAMMR Reassortment to Generate Improved Arsenate Detoxifying Bacteria<

Arsenic detoxification is important for mining of arsenopyrite containing gold ores and other uses, such as environmental remediation. Plasmid pGJ103, containing an arsenate detoxification operon (Ji and Silver, 1992) (Ji, G. and Silver, S., Regulation and expression of the arsenic resistance operon from Staphylococcus aureus plasmid pI258, J. Bacteriol. 174, 3684–3694 (1992), incorporated herein by reference), is obtained from Prof. Simon Silver (U. of Illinois, Chicago, Ill.). E. coli TG1 containing pGJ103, containing the pI258 ars operon cloned into pUC19, has a MIC (minimum inhibitory concentration) of 4 μg/ml on LB ampicillin agar plates. The ars operon is amplified by mutagenic PCR [REF], cloned into pUC19, and transformed into E. coli TG1. Transformed cells are plated on a range of sodium arsenate concentrations (2, 4, 8, 16 mM). Colonies from the plates with the highest arsenate levels are picked. The colonies are grown in a mixed culture with appropriate arsenate selection. Plasmid DNA is isolated from the culture. The plasmid DNA is linearized by digestion with a restriction endonuclease that cuts once into the pUC19 plasmid backbone. The linearized plasmids are denatured by heating 10 min. at 94° C. The reaction is allowed to cool to promote annealing of the single strands. Partially complementary strands that hybridize have non basepaired nucleotides at the sites of the mismatches. Treatment with CEL I (purified by the method of Example 9) causes nicking of one or the other polynucleotide strand 3′ of each mismatch. The presence of a polymerase containing a 3′ to 5′ exonuclease (“proofreading”) activity, such as T4 DNA polymerase allows excision of the mismatch, and subsequent 5′ to 3′ polymerase activity fills in the gap using the other strand as a template. T4 DNA ligase then seals the nick by restoring the phosphate backbone of the repaired strand. The result is a randomization of mutations among input strands to give output strands with potentially improved properties. These output polynucleotides are transformed directly into E. coli TG1 and the cells are plated at higher arsenate levels; 8, 16, 32, 64 mM. Colonies are picked from the plates with the highest arsenate levels and another round of reassortment is performed as above except that resulting transformed cells are plated at 32, 64, 128, 256 mM arsenate. The process can then be repeated one or more times with the selected clones in an attempt to obtain additional improvements. EXAMPLE 12 Cloning, Expression and Purification of CEL I Endonuclease<

This example teaches the preparation of nucleic acid molecules that were used for expressing CEL I endonuclease from plants, identified herein as, p1177 MP4CEL I Avr, and p1177 MP4 CEL I 6HIS. In particular, this example refers to disclosures taught in U.S. Pat. Nos. 5,316,931, 5,589,367, 5,866,785, and 5,889,190, incorporated herein by reference.<

The aforementioned clones were deposited with the American Type Culture Collection, Manassas, Va. 20110 2209 USA. The deposits were received and accepted on Dec. 13, 2001, and assigned the following Patent Deposit Designation numbers, PTA 3926 (p1177 MP4 celI Avr, and PTA 3927 (p1177 MP4 celI 6HIS).<

1. Celery RNA Extraction:<

Celery was purchased from a local market. Small amounts of celery tissue (0.5 to 0.75 grams) were chopped, frozen in liquid nitrogen, and ground in a mortar and pestle in the presence of crushed glass. After addition of 400 microliters of Trizol and further grinding, 700 microliters of the extract were removed and kept on ice for five minutes. Two hundred microliters of chloroform were then added and the samples were centrifuged, left at room temperature for three minutes, and re centrifuged at 15,000 g for 10 minutes. The aqueous layer was removed to a new tube and an equal volume of isopropanol was added. Tubes were inverted to mix and left at room temperature for 10 minutes followed by centrifugation at 15,000 g for ten minutes at 4° C. The pellet was washed twice in 400 microliters of 70% ethanol, once in 100% ethanol, air dried, and resuspended in 40 microliters of distilled water. One microliter of RNasin was added and 3.5 microliters was run on a 1% agarose gel to check the quality of the RNA prep (Gel picture). The remainder was stored at −70° C. until further use.<

2. CEL I Gene Cloning and Expression by a Viral Vector:<

The total RNA from celery was subjected to reverse transcription followed by PCR to amplify the cDNA encoding the CEL I gene sequence. In separate reactions, eleven microliters of the total celery RNA prep was mixed with one microliter (50 picomoles) of either CelI Avr R, CelI 6H R, or with two microliters of oligo dT primer. CelI Avr R was used to prime cDNA and amplify the native CEL I sequence at the 3′ end of the gene, while CelI 6H R was used to add a sequence encoding linker peptide and a 6 His tag to the 3′ terminus of the CEL I gene. The samples were heated to 70° C. for one minute and quick chilled on ice prior to the addition of 4 microliters of 5× Superscript II buffer, two microliters of 0.1M DTT, 1 microliter of 10 mM each dNTP, and 1 microliter of Superscript II (Gibco/BRL) to each reaction. The reactions were incubated at 42° C. for one hour.<

PCR amplification of the CEL I cDNA sequence was performed using the method of W. M. Barnes (Proc Nati Acad Sci. USA, 1994 Mar 15;91(6):2216–20) with a Taq Pfu mixture or with Pfu alone. The RT reaction primed with CelI Avr R was used as template for a PCR using primers CelI Pac F (as the forward primer) paired with CelI Avr R (as the reverse primer). In other PCRs, the RT reaction that was primed with oligo dT was used as template for both of the above primer pairs. All PCR reactions were performed in 100 microliters with 30 cycles of annealing at 50° C. and two minutes of extension at 72° C. Aliquots of the resulting reactions were analyzed by agarose gel electrophoresis. Reactions in which Pfu was used as the sole polymerase showed no product. All reactions performed with the Taq/Pfu mixtures yielded product of the expected size. However, those amplified from cDNA primed with Cel I specific primer pairs gave more product than reactions amplified from cDNA primed with oligo dT. DNAs from the PCR reactions that gave the most product were purified using a Zymoclean DNA spin column kit and digested with PacI and AvrII, gel isolated, and ligated into PacI and AvrII digested plasmid pRT130, a tobamovirus based GENEWARE® vector. 2 microliters of each ligation were transformed into DH5a competent E. coli and cultured overnight on LB amp agar plates. Colonies were picked and grown overnight in liquid culture, and plasmid DNA was isolated using a Qiagen plasmid prep kit. 12 clones from each construct were screened by digestion with PacI and AvrII and 11 of 12 of each set were positive for insert of the correct size. Ten of the clones for each construct were transcribed in vitro and RNA was inoculated to N. benthamiana plants. In addition, the CEL I gene inserts in both sets often clones were subjected to sequence analysis. Several clones containing inserts encoding the native form of CEL I had sequence identical to the published CEL I sequence in WO 01/62974 A1. One clone containing an insert encoding CEL I fused to a 6 Histidine sequence was identical to the published CEL I sequence. One clone of each (pRT130 celI Avr B3 and pRT130 cell 6His A9, respectively) was selected for further work. The CEL I encoding sequences in these clones were subsequently transferred to another GENEWARE vector. It should be noted that applicant's designations for each of the clones were shortened in the deposit to the aforementioned deposit with the American Type Culture Collection, that is, p1177 MP4 celI Avr B3 is referred to as p1177 MP4 celI Avr; and p1177 MP4 cell 6HisA9 is referred to asp1177 MP4 celI 6His. The clone p1177 MP4 celI Avr contained the CEL I open reading frame extending from nucleotide 5765 to 6655; and the clone p1177 MP4 celI 6His A9 contained the CEL I open reading frame extending from nucleotide 5765–6679.<

3. Assay of Cloned CEL I Activities.<

To determine whether the GENEWARE constructs containing Cel I sequences could produce active CEL I enzyme, samples of pRT130 celI Avr and pRT130 celI 6His and GFP GENEWARE control infected plants were harvested and homogenized in a small mortar and pestle in Tris HCl at pH 8.0. Extracts were clarified and assayed for supercoiled DNA nicking activity. Each supercoiled DNA nicking assay was performed in a reaction containing 0.5 micrograms of a supercoiled plasmid prep of a pUC19 derivative in 1×NEB ligase buffer in a total volume of 10 microliters. The amounts of plant extract added to the reactions were 0.1 microliter, 0.01 microliter, or 0.001 microliter, incubated at 42° C. for 30 minutes, and run on a 1% TBE agarose gel in the presence of ethidium bromide. Little or no nicking activity was detected in the GFP GENEWARE control infected plant extract whereas extracts from plants infected with the CEL I GENEWARE constructs showed appreciable amounts of activity against the plasmid DNA substrate.<

Additional activity assays were performed on extracts of plants inoculated with pRT130 celI Avr B3 and pRT130 celI 6His A9. In these assays, intracellular fluid was washed from infected leaves and assayed separately from material obtained from the remaining washed leaf tissues. Assays were performed as described above with the exception that the incubation was at 37° C. for one hour. Samples were run on a 1% TBE agarose gel in the presence of ethidium bromide and photographed.<

4. Purification of 6His Tagged CEL I from Infected N. Benthamiana Plants.<

N. benthamiana plants were inoculated with RNA transcripts from pRT130 celI 6His A9 at 20–21 days post sowing. Tissues were harvested from 96 infected plants at 10 days post inoculation and subjected to intracellular fluid washes. Briefly, infected leaf and stem material was vacuum infiltrated for 30 seconds twice with chilled infiltration buffer (50 mM phosphate pH 4 in the presence of 7 mM β ME). Infiltrated tissues were blotted to adsorb excess buffer and secreted proteins were recovered by centrifugation at 2500×g for 20 min using basket rotor (Beckman). PMSF was added to the extracted intracellular fluid (IF) containing recombinant CEL_I to a final concentration of 1 mM, and incubated at 25° C. for 15 min with stirring. After addition of Imidazole (pH 6.0) and NaCl to the extract to the final concentration of 5 mM and 0.5 M respectively, IF was adjusted to pH 5.2 and filtered through 1.2μ Sartorius GF membrane (Whatman) to remove most of the Rubisco and green pigments. Immediately after clarification, pH was adjusted to 7.0 using concentrated NaOH solution and incubated on ice for 20 min to allow non proteinaceous material to precipitate. IF was further clarified using 0.8μ or 0.65/0.45μ Sartorius GF (Whatman). Recombinant CEL I was purified from the clarified IF by metal chelating affinity chromatography using Ni2+ Fast Flow Sepharose (Amersham Pharmacia Biotech, NJ) equilibrated with binding buffer (50 mM phosphate, 0.5 M NaCl; pH 7.0) containing 5 mM imidazole, with a linear velocity of 300 cm/hr. Unbound protein was washed with 20 mM imidazole/binding buffer, and CEL I was eluted from Ni2+ Sepharose with a linear gradient of 20 to 400 M imidazole in the binding buffer. Fractions still containing imidazole were assayed for supercoiled DNA nicking activity as described above but were found to have negligible activity. The same fractions were then dialyzed against 0.1 M Tris HCl, pH 8.0 in the presence of ZnCl2 using 10 kD MWCOF dialysis tubing (Pierce) and assayed again. The supercoiled DNA nicking activity was restored after this dialysis.<

IF and purified CEL I protein were analyzed using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) precast Tris glycine gels (Invitrogen, Carlbad, Calif.) in the buffer system of Laemmli with a Xcell II Mini Cell apparatus (Invitrogen, Carlsbad, Calif.). The protein bands were visualized by Coomassie brilliant blue and by silver staining. SDS PAGE Gels were scanned and analyzed using Bio Rad gel imager.<

Mass Spectrometry of Purified CEL I<

The average molecular mass of the purified CEL I was determined by matrix assisted laser/desorption ionization time of flight mass spectrometry (MALDI TOF). An aliquot of CEL I was diluted 1:10 with 50% acetonitrile/water and mixed with sinapinic acid matrix (1:1 v/v) using a PE Biosystem DE Pro mass spectrometer. The mass spectrometry was performed using an accelerating voltage of 25 kV and in the positive linear ion mode.<

Mass Spectrometry of Peptides Isolated from Purified CEL I.<

CEL I was separated on SDS PAGE on a 14% gel and stained with Coomassie brilliant blue. A single homogenous band was visible. This band was excised and de stained completely. Protein was reduced in the presence of 10 mM DDT in 50% acetonitrile for 30 min at 37° C. and reduced sulfhydro groups were blocked in the presence of 28 mM iodoacetamide in 50% acetonitrile for 30 min at 24° C. in absence of light. Gel pieces were washed with 50% acetonitrile and after partial dehydration, the excised CEL I band was macerated in a solution of high purity trypsin (Promega). The proteolytic digestion was allowed to continue at 37° C. for 16 h. The resulting peptides were eluted from gel pieces with a 50% acetonitrile and 0.1% tri fluoro acetic acid (TEA) concentrated in a SpeedVac. The peptides were analyzed by MALDI TOF. Mixed tryptic digests were crystallized in a matrix of α cyano 4 hydroxycinnamic acid and analyzed by using a PerSeptive Biosystem DE STR MALDI TOF mass spectrometer equipped with delayed extraction operated in the reflector positive ion mode and accelerating voltage of 20 kV. Expected theoretical masses were calculated by MS digest (Protein Prospector) or GPMAW program (Lighthouse Data, Odense, Denmark). For tandem mass spectrometry (nano electrospray ionization (ESI), peptide samples were diluted with 5% acetonitrile/0.1% formic acid and subjected to LC MS/MS, analyzed on a quadropole orthogonal time of flight mass spectrometry instrument (micromass, inc., Manchester, UK). The data were processed by Mslynx and database was searched by Sonar.<

Virally expressed, recombinant CEL I was secreted to the IF. Clarified IF extracted material was used to purify the His tag CEL I activity. CEL I was purified using one step Ni2+ affinity chromatography separation. A highly purified homogeneous single protein band was purified as determined by Coomassie stained SDS PAGE and mass spectrometry. The size of mature proteins and percent glycosylation concur with what has been reported for the CEL I protein isolated from celery (Yang et al., 2000). The purified CEL I has an average molecular mass of 40 kD as determined by MALDI TOF mass spectrometry, indicates 23.5% glycosylation by mass. CEL I has four potential glycosylation cites at amino acid positions 58, 116, 134, and 208. A mono isotopic mass of 2152.6086 (2152.0068 Theoretical) Da corresponding to the mass of the peptide 107–125 (K)DMCVAGAIQNFTSQLGHFR(H) that was recovered by MALDI TOF, indicates that asparagine 116 is not glycosylated. Together, these gel analyses and mass spectrometry data indicate that a significant fraction of the CEL I protein was recoverable from the intracellular space, and that the protein was correctly processed in the N. benthamiana plant.<

For subsequent experiments, the 6 His tagged CEL I gene was produced using p1177 MP4 celI 6His A9. This clone was transcribed and inoculated onto N. benthamiana plants, which were harvested 8 days post infection. The plant material was combined with 2 volumes of extraction buffer (500 mM NaCl, 100 mM NaPi, 25 mM Tris pH 8.0, 7 mM Beta mercaptoethanol, 2 mM PMSF) and vacuum infiltrated. Following buffer infiltration the tissue was macerated in a juice extractor, the resulting green juice adjusted to 4% w/v polyethyleneglycol, and let stand at 4° C. for one hour. The green juice was clarified by either centrifugation at low speed (3500×g) for 20 minutes or combined with perlite (2% w/v) and filtered through a 1.2 μm filter. The tagged CEL I can be selectively purified from the clarified green juice by metal affinity chromatography. The green juice was either combined with nickel NTA resin, and batch binding of the CEL I performed, or purification was performed in column format, where the green juice was permitted to flow through a bed of nickel NTA resin. For binding, the clarified green juice was adjusted to 10% w/v glycerol and 10 mM imidazole. Following binding the resin was washed extensively with wash buffer (330 mM NaCl, 100 mM NaPi, pH 8.0, 10 mM imidazole) and the bound CEL I enzyme eluted from the nickel NTA resin in 2 resin bed volumes of 1× phosphate buffered saline (PBS) containing 400 mM imidazole. The CEL I preparation was subsequently dialyzed against 1×PBS to remove the imidazole, assayed for activity, and stored at 4° C. or at −20° C. with or without glycerol until use. EXAMPLE 13 Cloning, Expression and Use of RES I Endonuclease<

This example teaches the construction of a cDNA library from Selaginella lepidophylla, the identification of a nucleic acid sequence from the library that encodes an endonuclease, and the expression of the new endonuclease, herein designated as “RES I.”<

RNA was extracted from tissues of the resurrection plant, Selaginella lepidophylla, using the Trizol method, and oligo dT primed cDNA that was prepared using standand methodology. Resulting cDNAs were ligated into a GENEWARE based cloning vector and the ligation products were transformed into competent E. coli cells. Bacterial colonies containing GENEWARE cDNA clones were picked at random and grown as liquid cultures prior to DNA prepping and determination of the cloned cDNA sequences. The sequence files for the cloned Selaginella cDNAs were loaded into a database which was then searched by BLAST analysis for sequences that had similarity to the DNA sequence of the CEL I gene. BLAST analysis was also performed on other DNA sequence databases containing sequences of cDNAs obtained from other species.<

BLAST hits that showed some level of homology to the celery CEL I sequence were identified in libraries from several species and the corresponding GENEWARE cDNA clones were re arrayed into a single set of GENEWARE cDNA clones. This set of cDNA clones was then transcribed in vitro to generate infectious GENEWARE transcripts which were then inoculated onto leaves on Nicotiana benthamiana plants for expression analysis of the cDNA sequences encoded within the GENEWARE viral genome. At seven days post inoculation, leaf samples were taken from the infected plants and homogenized in two volumes of water. The extracts were then assayed for supercoiled DNA nicking and cleavage activity.<

Each supercoiled DNA nicking assay was performed in a reaction containing 0.5 micrograms of a supercoiled plasmid prep of a pUC19 derivative in 1×NEB T4 DNA ligase buffer in a total volume of 10 microliters. The amounts of plant extract added to the reactions were 1 microliter, 0.33 microliter, or 0.011 microliter, incubated at 37° C. for 30 minutes, and run on a 1% TAE agarose gel in the presence of Gelstar fluorescent DNA staining reagent. Little or no nicking activity was detected in uninfected plant extracts whereas only extracts from plants infected with GENEWARE constructs containing cDNAs for a single gene from Selaginella lepidophylla showed appreciable amounts of activity against the plasmid DNA substrate.<

A sample of the aforementioned Selaginella lepidophylla gene, as shown in FIG. 3 (SEQ ID NO:16), was mailed to the American Type Culture Collection, Manassas, Va. 20110 2209 USA on Jul. 29, 2002. The deposit was received and accepted on Jul. 30, 2002 and assigned the following Patent Deposit Designation number, PTA 4562.<

The complete gene sequences of these clones were determined and PCR primers were designed to amplify the open reading frame minus any non coding 5′ and 3′ sequences and to add a six histidine tail to the C terminus of the encoded protein. The primers were then used to amplify the ORF from one of the active full length Selaginella clones. The resulting PCR product was then cloned into the GENEWARE vector pDN4 between the PacI and AvrII sites for expression in planta. The resulting clone, pLSB2225, was sequenced to confirm that the gene had been inserted correctly, and then transcribed in vitro followed by inoculation of the infectious transcripts onto N. benthamiana plants. Seven days post inoculation, infected plant extracts were made as above and assayed for supercoiled DNA nicking and digestion activity to confirm the activity of the cloned enzyme.<

Each supercoiled DNA nicking assay was performed in a reaction containing 0.5 micrograms of a supercoiled plasmid prep of a pUC19 derivative in 1×NEB E. coli DNA ligase buffer in the presence of 50 mM KCl in a total volume of 10 microliters. The amounts of plant extract added to the reactions were 0.2 microliter, 0.04 microliter, 0.008 microliter, or 0.0016 microliter, incubated at 37° C. for 30 minutes, and run on a 0.8% TAE agarose gel in the presence of Gelstar fluorescent DNA staining reagent. Little or no nicking activity was detected in uninfected plant extracts whereas extracts from plants infected with the GENEWARE Selaginella construct pLSB2225 showed appreciable amounts of activity against the plasmid DNA substrate.<

After positive results were obtained in that assay, extracts of pLSB2225 infected plants were used in a GRAMMR shuffling experiment to test the ability of this enzyme to operate as a component of the mismatch resolution reaction in place of the GENEWARE produced CEL I enzyme of celery origin. EXAMPLE 14 Use of RES I in the GRAMMR Reaction<

This example teaches that RES I can be used in place of native CEL I enzyme purified from celery in Genetic Reassortment By DNA Mismatch Resolution without any noticeable change in results.<

GRAMMR shuffling was performed between the wildtype Aequorea victoria GFP gene (Prasher, et al., Gene111(92)229) in a pBS derivative (Stratagene, La Jolla, Calif.) encoded by pBSWTGFP (SEQ ID NO:03) and a variant with mutations to increase fluorescence intensity in E. coli, and to alter the emission wavelength to blue light emission (Crameri, et al., Nat Biotechnol 14(96)315; Heim et al., PNAS91 (94)12501; Yang, et al., J Biol Chem 273(98)8212). This variant gene, encoded by the plasmid pBSC3BFP, as shown in FIG. 5 (SEQ ID NO: ), encodes a fluorescent protein that emits bright blue light when excited by longwave UV light.<

The GRAMMR reactions were performed on GFP/c3BFP heteroduplexes in a circular, double stranded plasmid DNA context. The circular, whole plasmid heteroduplex DNA substates were prepared by first linearizing pBSWTGFP (SEQ ID NO:03) and pBSC3BFP (FIG. 5, SEQ ID NO:4) by digestion with Kpn I and NgoM IV, respectively, then purifying the digested DNA using DNA spin columns. Next, 200 nanograms of each of the two linearized plasmids were mixed and brought to 1×SSPE (180 nM NaCl, 10 mM NaH.sub2PO4, 1 mM EDTA at pH 7.4) in a volume of 20 microliters. The mixture was then incubated at 95 degrees Celsius for 4 minutes, plunged into icewater where it remained for 10 minutes prior to incubation at 37 degrees Celsius. After 30 minutes, the annealed DNA sample was then transferred back to ice where it was held until use in GRAMMR reactions.<

Two independent series of shuffling reactions were performed to compare CEL I with RES I in their abilities to facilitate sequence shuffling by GRAMMR. Each GRAMMR reaction contained 1 unit of T4 DNA polymerase, 2 units of E. coli DNA ligase, and 5 nanomoles of each dNTP in 1×NEB E. coli ligase buffer supplemented with KCl to 50 mM. Two separate enzyme dilution series were then performed. To each of two series of tubes containing aliquots of the above cocktail, one microliter aliquots of GENEWARE expressed CEL I or RES I extracts at dilutions of 1/3, 1/9, 1/27, 1/81, or 1/243 were added. An endonuclease free control reaction was also prepared. To each of the reactions, one microliter aliquots containing 20 nanograms of the annealed DNA heteroduplex substrate were added and the reactions incubated at room temperature for one hour and on ice for 30 minutes prior to transformation into competent E. coli.<

Green fluorescent protein (GFP) and blue fluorescent protein (BFP) could be visualized in the resulting colonies by long wave UV illumination. The parental wild type GFP has dim green fluorescence, and the parental c3BFP gave bright blue fluorescence. In the genes encoding these fluorescent proteins, the sequences that determine the emission color and those that govern fluorescence intensity are at different positions from one another. It is expected that DNA shuffling would result in the “de linking” of the sequences that determine the emission color from those that govern fluorescence intensity. As a consequence, the resultant progeny would be expected to exhibit reassortment of the functional properties of emission color and intensity. Therefore a measure of the extent of the DNA shuffling that had taken place in each reaction could be scored by examining the color and intensity of fluorescence from the bacterial colonies on the corresponding plates. In the zero nuclease control, only dim green and bright blue colonies were observed. However, on plates with cells transformed with DNAs from the reactions containing either CEL I or RES I, some bright green as well as some dim blue colonies were observed, indicating that shuffling of DNA sequences had taken place. DNA sequence analysis confirmed that this was indeed the case and that on average, the recovery of shuffled clones was greater than 85% for both CEL I and RES I and that the number and distribution of information transfer events was similar for both enzymes. However, it appeared that the activity of RES I in this experiment was several fold higher than that of CEL I, as indicated by the low transformation efficiency of reactions treated with the higher concentrations of the RES I preparation. EXAMPLE 15 Molecular Breeding of Highly Divergent Tobamovirus 30K Genes in Viral Vectors Using Plasmid on Plasmid Genetic Reassortment By DNA Mismatch Resolution (POP GRAMMR)<

Example 10 taught the reassortment of movement protein (MP) genes from several divergent strains of tobamovirus (approximately 75% identical; cloned into the pGENEWARE MP Avr Pac vector) using Genetic Reassortment by DNA Mismatch Resolution (GRAMMR). This example teaches the use of Plasmid on plasmid GRAMMR (POP GRAMMR) for reasserting even more highly divergent species.<

Starting parental MP genes from the tobamoviruses TMV Cg (FIG. 6, SEQ ID NO:19), TMV Ob (FIG. 7, SEQ ID NO:20), TMV U2 (FIG. 8, SEQ ID NO:21), TMV U1 (SEQ ID NO:09), and tomato mosaic virus (ToMV) (SEQ ID NO:10) were used. The plasmid of pGENEWARE ToMV MP was linearized by digestion with Sma I. The plasmids of pGENEWARE containing the MP genes from either TMV Cg, TMV Ob, TMV U2, or TMV U11 were digested with Stu I. The digested pGENEWARE MP constructs were purified using DNA spin columns. The following heterduplex pairs were generated: pGENEWARE Cg MP and pGENEWARE ToMV MP, pGENEWARE TMV Ob MP and pGENEWARE ToMV MP, pGENEWARE TMV U2 MP and pGENEWARE ToMV MP, pGENEWARE TMV U1 MP and pGENEWARE ToMV MP. The heteroduplexes of these MP gene sequences are approximately 47%, 58%, 62%, and 75% identical, respectively. Heteroduplex DNA was generated by mixing 200 nanograms of each of the two linearized plasmids in 1×SSPE (180 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, at pH 7.4) in a volume of 20 microliters. The mixture was incubated at 95 degrees Celsius for 4 minutes, plunged into ice water where it remained for 10 minutes prior to incubation at 37 degrees Celsius. After 30 minutes, the annealed DNA sample was then transferred back to ice where it was held until use in GRAMMR reactions.<

Each 10 microliter GRAMMR reaction contained 1 unit of T4 DNA polymerase, 2 units of E. coli DNA ligase, and 0.5 mm of each dNTP in 1×NEB E. coli DNA ligase buffer supplemented with KCl to 50 mM. A one microliter aliquot of CEL I (diluted 1/3, 1/9, 1/27, 1/81, 1/243, or 1/729) was next added. An endonuclease free control reaction was also prepared. To each of the reactions, a one microliter aliquot containing 20 nanograms of the annealed DNA heteroduplex substrate was added and the reactions were incubated at room temperature for one hour and on ice for 30 minutes prior to transformation into competent E. coli.<

Cleavage of Mismatched DNA Substrate by CEL I<

This example teaches the preparation of CEL I enzyme and its use in the cleavage of mismatched DNA substrate.<

CEL I enzyme was prepared from celery stalks using the homogenization, ammonium sulfate, and Concanavalin A Sepharose protocol described by Yang et al. (Biochemistry, 39:3533 3541 (2000), incorporated herein by reference. A 1.5 kg sample of chilled celery stalks was homogenized with a juice extractor. One liter of juice was collected, adjusted to 100 mM Tris HCL, pH 7.7 with 100 micromolar phenylmethylsulfonyl fluoride (PMSF), and filtered through two layers of miracloth. Solid (NH4)2SO4 was slowly added to 25% saturation while stirring on ice. After 30 minutes, the suspension was centrifuged at 27,000 g for 1.5 hours at 4° C. The supernatants were collected and adjusted with solid (NH4)2SO4 to 80% saturation while stirring on ice followed by centrifugation at 27,000 g for 2 hours. The pellets were re suspended in buffer B (0.1 M Tris HCL, pH 7.7, 0.5 M KCl, 100 micromolar PMSF) and dialyzed against the same buffer.<

Conconavalin A (ConA) Sepharose affinity chromatography was performed by first incubating the dialyzed sample with 2 ml of ConA resin overnight with gentle agitation. The ConA resin was then packed into a 0.5 cm diameter column and washed with several column volumes of buffer B. Elution was performed:using 0.3 M alpha methyl mannoside in buffer B. Fractions were collected in 1 ml aliquots. Fractions were assayed for mismatch cleavage activity on a radiolabeled mismatch substrate by incubating 0.1 microliter of each fraction with the mismatched probe in buffer D (20 mM Tris HCL, pH 7.4, 25 mM KCL, 10 mM MgCl2) for 30 minutes at 45° C. as described by Oleykowski et al. (Nucleic Acids Research 26: 4597 4602 (1998), incorporated herein by reference. Reaction products were visualized by separation on 10% TBE PAGE gels containing 7% urea (Invitrogen), followed by autoradiography. Aliquots of the CEL I fractions having mismatch cleavage activity were stored frozen at −20° C. A series of five fold dilutions of CEL I fraction #5 were then analyzed for mismatch cleavage of radiolabeled mismatch substrate. Reactions were performed either in buffer D, New England BioLabs (NEB) T4 DNA ligase buffer (50 mM Tris HCL, pH 7.5, 10 mM MgCl2, 10 mM dithiothreitol (DTT), 1 mM ATP, 25 microgram/ml BSA), or Gibco/BRL T4 DNA ligase buffer (50 mM Tris HCL, pH 7.6, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 5% (w/v) polyethylene glycol 8000). Reaction products were visualized as above. Cleavage activity in buffer D and in NEB T4 DNA ligase buffer were found to be roughly equivalent, whereas cleavage in the PEG containing Gibco/BRL ligase buffer was enhanced by five to ten fold compared to the other buffers.<

Additional analysis of CEL I activity was carried out using defined heteroduplex DNAs from two different Green Fluorescent Protein (GFP) genes as substrate. This GFP heteroduplex substrate was prepared by annealing single stranded DNAs corresponding to cycle 3 GFP on the sense strand and wild type GFP on the antisense strand. The single stranded DNAs had been synthesized by asymmetric PCR and isolated by agarose gel electrophoresis. After annealing by heating to 90° C. and cooling in the presence of 1×NEB restriction enzyme buffer 2 (10 mM Tris HCL, pH 7.9, 10 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol), the heteroduplex DNA was isolated by agarose gel electrophoresis followed by excision of the heteroduplex band and extraction using Qiaquick DNA spin columns. A total of twenty eight mismatches, one or two nucleotides in length, occur throughout the length of the heteroduplex molecule. The distribution of the mismatches ranges from small clusters of several mismatches separated by one or two nucleotides to mismatches separated by more than thirty base pairs on either side.<

A series of three fold dilutions of CEL I in 1×NEB T4 DNA ligase buffer were prepared and one microliter aliquots of each were incubated in two separate series of 10 microliter reactions, each containing as substrate either 0.5 microgram of a supercoiled plasmid preparation or one hundred nanograms of the cycle3/wild type GFP heteroduplex. All reactions took place in 1×NEB T4 DNA ligase buffer. Reactions were incubated at 45° C. for 30 minutes and run on 1.5% TBE agarose gel in the presence of ethidium bromide.<

Treatment of the supercoiled plasmid preparation with increasing amounts of CEL I resulted in the conversion of supercoiled DNA to nicked circular, then linear molecules, and then to smaller fragments of DNA of random size. Treatment of the mismatched GFP substrate with the CEL I preparation resulted in the digestion of the full length heteroduplex into laddered DNA bands which are likely to represent cleavage on opposite DNA strands in the vicinity of clusters of mismatches. Further digestion resulted in the conversion of the mismatched GFP substrate to smaller DNAs that may represent a limit digest of the heteroduplex DNA by the CEL I preparation. EXAMPLE 2 Conservation of Full Length GFP Gene with Mismatch Resolution Cocktails<

This example teaches various mismatch resolution cocktails that conserve the full length GFP Gene.<

Mismatched GFP substrate was treated with various concentrations of CEL I in the presence of cocktails of enzymes that together constitute a synthetic mismatch resolution system. The enzymes used were CEL I, T4 DNA polymerase, Taq DNA polymerase and T4 DNA ligase. CEL I activity should nick the heteroduplex 3′ of mismatched bases. T4 DNA polymerase contains 3′ 5′ exonuclease for excision of the mismatched base from the nicked heteroduplex. T4 DNA polymerase and Taq DNA polymerase contain DNA polymerase capable of filling the gap. T4 DNA ligase seals the nick in the repaired molecule. Taq DNA polymerase also has 5′ flap ase activity.<

Matrix experiments were performed to identify the reaction conditions that would serve to resolve mismatches in the GFP heteroduplex substrate. In one experiment, cycle 3/wild type GFP heteroduplex was incubated in a matrix format with serial,dilutions of CEL I fraction number five (described above) at eight different concentrations. Each reaction contained 100 nanograms of heteroduplex substrate and 0.2 microliters of T4 DNA ligase (Gibco BRL) in 1×NEBT4 DNA ligase buffer and dNTPs at 250 micromolar each, in a reaction volume of 10 microliters. In all, the matrix contained 96 individual reactions. One full set of reactions was incubated at room temperature for 30 minutes while another full set was incubated at 37° C. for 30 minutes.<

After incubation, PCR was used to amplify the GFP gene from each reaction. Aliquots from each PCR were then digested with HindIII and HpaI and electrophoresed on 3% agarose gels with ethidium bromide. Only cycle 3 GFP has a HindIII site and only wild type encodes a HpaI site.<

If DNA mismatch resolution occurred at either the HindIII or HpaI mismatched sites, then a proportion of the PCR product would be expected to contain both sites, yielding a novel band. The band was observed in all samples, including the negative control samples that had neither CEL I, nor T4 DNA polymerase, nor Taq DNA polymerase. The results suggested that a basal level of background recombination may have occurred at some point in the experiment other than in the GRAMMR reaction; possibly in the PCR step. PCR mediated recombination is known to occur at some frequency between related sequences during amplification [reference Paabo, et al., DNA damage promotes jumping between templates during enzymatic amplification. J Biol Chem 265(90)4718 4721].<

In another experiment, 200 nanograms of cycle 3/wild type GFP heteroduplex was treated with CEL I and T4 DNA polymerase in various concentrations along with 2.5 units of Taq DNA polymerase in the presence or absence of T4 DNA ligase (0.2 units; Gibco BRL). Each reaction contained 1×NEB T4 DNA ligase buffer with 0.05 mM each dNTP in a final volume of 20 microliters. Reactions were incubated for 30 minutes at 37° C. and 10 microliters were run on a 2% TBE agarose gel in the presence of ethidium bromide. Results showed that in the presence of DNA ligase, but in the absence of T4 DNA polymerase, increasing amounts of CEL I caused greater degradation of the heteroduplexed DNA, but that this effect could be counteracted by increasing the amount of T4 DNA polymerase in the reaction. These results indicated that the various components of the complete reaction could act together to conserve the integrity of the full length gene through DNA mismatch resolution.<

Another matrix experiment was conducted to expand on these results and to identify additional conditions for DNA mismatch resolution for this synthetic system. 60 nanograms of cycle3/wild type GFP heteroduplex were treated with CEL I and T4 DNA polymerase at various concentrations in the presence of 2.5 units of Taq DNA polymerase and 0.2 units of T4 DNA ligase in 1×NEB T4 DNA ligase buffer containing 0.5 mM of each dNTP in a reaction volume of 10 microliters. Each set of reactions was incubated for 1 hour at either 20° C., 30° C., 37° C., or at 45° C. All reactions were then run on a 1.5% TBE agarose gels in the presence of ethidium bromide. The results showed that the GFP heteroduplex was cleaved into discrete fragments by the CEL I preparation alone. The success of DNA mismatch resolution was initially gauged by the degree to which the apparent full length integrity of the GFP sequence was maintained by the other components of the mismatch resolution system in the presence of CEL I. Conditions of enzyme concentration and temperature were identified that conserved a high proportion of the DNA as full length molecules in this assay. Namely, one microliter of the CEL I fraction five preparation (described in Example 1) with one microliter (1 unit) of the T4 DNA polymerase in the presence of the other reaction components which were held constant in the experiment. It was found that as the reaction temperature increased, the degradative activity of CEL I increased accordingly. Furthermore, it was shown that the other components of the repair reaction acted to conserve the integrity of the full length DNA at 20° C., 30° C., and 37° C., but was remarkably less efficient at conserving the full length DNA at 45° C. From these results, we concluded that under these experimental conditions, incubation, at 45° C. was not optimal for the process of GRAMMR, and that incubation at 20° C., 30° C., and 37° C. were permissible.<

Another experiment was performed in which alternative enzymes were used for the DNA mismatch resolution reaction. Instead of T4 DNA ligase, Taq DNA ligase was used. Pfu DNA polymerase (Stratagene) was employed in a parallel comparison to a set of reactions that contained T4 DNA polymerase as the 3′ exonuclease/polymerase. Reactions were carried out in Taq DNA ligase buffer containing 8 units of Taq DNA ligase (NEB), 2.5 units Taq DNA polymerase, 0.5 mM of each dNTP, various dilutions of CEL I, and either T4 DNA polymerase or Pfu DNA polymerase). Reactions were run on a 1.5% TBE agarose gels in the presence of ethidium bromide. It was found that in the presence of the Pfu DNA polymerase, Taq DNA polymerase, and Taq DNA ligase, the full length integrity of the CEL I treated substrate DNA was enhanced compared to DNA incubated with CEL I alone. This result shows that enzymes with functionally equivalent activities can be successfully substituted into the GRAMMR reaction. EXAMPLE 3 Restoration of Restriction Sites to GFP Heteroduplex DNA after DNA Mismatch Resolution (GRAMMR)<

This experiment teaches the operability of genetic reassortment by DNA mismatch resolution (GRAMMR) by demonstrating the restoration of restriction sites.<

The full length products of a twenty fold scale up of the GRAMMR reaction, performed at 37° C. for one hour, using the optimal conditions found above (the 1× reaction contained sixty nanograms of heteroduplex DNA, one microliter of CEL I fraction five (described in Example 1), one unit T4 DNA polymerase in the presence of 2.5 units of Taq DNA polymerase and 0.2 units of T4 DNA ligase in 1×NEB T4 DNA ligase buffer containing 0.5 mM of each dNTP in a reaction volume of 10 microliters) were gel isolated and subjected to restriction analysis by endonucleases whose recognition sites overlap with mismatches in the GFP heteroduplex, thereby rendering those sites in the DNA resistant to restriction enzyme cleavage. The enzymes used were BamHI, HindIII, HpaI, and XhoI. Negative controls consisted of untreated GFP heteroduplex. Positive controls consisted of Cycle 3 or wild type GFP sequences, individually. All controls were digested with the same enzymes as the product of the DNA mismatch resolution reaction. All samples were run on a 2% TBE agarose gel in the presence of ethidium bromide.<

After treatment with the mismatch resolution cocktail, a proportion of the DNA gained sensitivity to BamHI and XhoI restriction endonucleases, indicating that DNA mismatch resolution had occurred. The HpaI cut samples could not be interpreted since a low level of cleavage occurred in the negative control. The HindIII, BamHI and XhoI sites displayed different degrees of cleavage in the GRAMMR treated samples. Restoration of the XhoI site was more extensive than that of the BamHI site, which was in turn, more extensive than restoration at HindIII site.<

The extent to which cleavage occurs is indicative of the extent to which mismatches in the DNA have been resolved at that site. Differences in mismatch resolution efficiency may relate to the nature or density of mismatches present at those sites. For example, the XhoI site spans a three mismatch cluster, whereas the BamHI site spans two mismatches and the HindIII site spans a single mismatch. EXAMPLE 4 GRAMMR Reassorted GFP Genes<

This example demonstrates that GRAMMR can reassort sequence variation between two gene sequences in a heteroduplex and that there are no significant differences in GRAMMR products that were directly cloned, or PCR amplified prior to cloning.<

The GRAMMR treated DNA molecules of Example 3 were subsequently either directly cloned by ligation into pCR Blunt II TOPO (Invitrogen), or amplified by PCR and ligated into pCR Blunt II TOPO according to the manufacturer's instructions, followed by transformation into E. coli. After picking individual colonies and growing in liquid culture, DNA was prepared and the sequences of the GFP inserts were determined. As negative controls, the untreated GFP heteroduplex substrate was either directly cloned or PCR amplified prior to cloning into the plasmid.<

In GRAMMR, reassortment of sequence information results from a process of information transfer from one strand to the other. These sites of information transfer are analogous to crossover events that occur in recombination based DNA shuffling methods. For the purposes of relating the results of these reassortment experiments, however, the GRAMMR output sequences are described in terms of crossovers. Sequences of twenty full length GFP clones that were derived from the GRAMMR treated GFP genes were analyzed. Four of these clones were derived from DNA that had been directly cloned into pZeroBlunt [ref] following. GRAMMR treatment (no PCR amplification). The other sixteen sequences were cloned after PCR amplification. Analysis of these full length GFP sequences revealed that all twenty sequences had undergone sequence reassortment having between one and ten crossovers per gene. A total of 99 crossovers were found in this set of genes, giving an average of about 5 crossovers per gene. With the distance between the first and last mismatches of about 590 nucleotides, an overall frequency of roughly one crossover per 120 base pairs was calculated. Within this set of twenty clones, a total of seven point mutations had occurred within the sequences situated between the PCR primer sequences, yielding a mutation frequency of roughly 0.05%.<

Thirty five clones that had not been subjected to GRAMMR treatment were sequenced. Of these controls, fourteen were derived from direct cloning and twenty one were obtained after PCR amplification using the GFP heteroduplex as template. Of these thirty five non GRAMMR treated control clones, eight were recombinants, ranging from one to three crossovers, with most being single crossover events. A total of twenty five point mutations had occurred within the sequences situated between the PCR primers, yielding a mutation frequency of roughly 0.1%.<

No significant differences were observed between the GRAMMR treated products that were either directly cloned or PCR amplified. Notably, though, in the non GRAMMR treated controls, the frequency of recombinants was higher in the PCR amplified DNAs than in the directly cloned DNAs. This higher frequency is consistent with results obtained by others in which a certain level of recombination was found to be caused by “jumping PCR.” [Paabo, et al., DNA damage promotes jumping between templates during enzymatic amplification. J. Biol Chem 265(90)4718 4721]. EXAMPLE 5 Heteroduplex Substrate Preparation for Plasmid on Plasmid Genetic Reassortment By DNA Mismatch Resolution (POP GRAMMR) of GFP Plasmids<

This example teaches that heteroduplex substrate for Genetic Reassortment by DNA Mismatch Resolution can be in the form of intact circular plasmids. Cycle 3 GFP and wild type GFP heteroduplex molecules were prepared plasmid on plasmid (POP) format. In this format, the GFP sequences were reasserted within the context of a circular double stranded plasmid vector backbone. This made possible the recovery of the reasserted product by direct transformation of E. coli using an aliquot of the GRAMMR reaction. Consequently, neither PCR amplification nor other additional manipulation of the GRAMMR treated DNA was necessary to obtain reasserted clones.<

Mismatched DNA substrate for POP GRAMMR reactions was generated containing wild type GFP (SEQ ID NO:01) and Cycle 3 GFP (SEQ ID NO:02), resulting in the two pBluescript based plasmids, pBSWTGFP (SEQ ID NO:03) and pBSC3GFP (SEQ ID NO:04), respectively. The GFPs were inserted between the KpnI and EcoRI sites of the pBluescript polylinker so that the only sequence differences between the two plasmids occurred at sites where the wild type and Cycle 3 GFPs differ from one another. Both plasmids were linearized by digestion of the plasmid backbone with SapI, cleaned up using a DNA spin column, mixed, amended to 1×PCR buffer (Barnes, 1994; PNAS, 91, 2216 2220), heated in a boiling water bath for three minutes, and slow cooled to room temperature to anneal the denatured DNA strands. Denaturing and annealing these DNAs led to a mixture of duplexes, the re formation of parental duplexes, and the formation of heteroduplexes from the annealing of strands from each of the two input plasmids. Parental duplexes were deemed undesirable for GRAMMR and were removed by digestion with restriction enzymes that cut in one or the other parental duplex but not in the heteroduplexed molecules. PmlI and XhoI were chosen for this operation since PmlI cuts only in the wild type GFP sequence and XhoI cuts only Cycle 3 GFP. After treatment with these enzymes, the products were resolved on an agarose gel. The full length, uncut heteroduplex molecules were resolved from the PmlI and XhoI cut parental homoduplexes in an agarose gel and purified by excision of the band and purification with a DNA spin column.<

The resulting population of heteroduplexed molecules was treated with DNA ligase to convert the linear DNA into circular, double stranded DNA heteroduplexes. After confirmation by agarose gel shift analysis, the circular double stranded GFP heteroduplexed plasmid was used as substrate for GRAMMR reactions. Examples of the resulting clones are included as SEQ ID NO:05, SEQ ID NO:06, SEQ ID NO:07, and SEQ ID NO:08. EXAMPLE 6 Exemplary Reaction Parameters for Genetic Reassortment by DNA Mismatch Resoluton CEL I and T4 DNA Polymerase Concentrations Compared<

The GRAMMR reaction involves the interaction of numerous enzymatic activities. Several parameters associated with the GRAMMR reaction were examined, such as CEL I concentration, T4 DNA polymerase concentration, reaction temperature, substitution of T4 DNA polymerase with T7 DNA polymerase, the presence of Taq DNA polymerase, and the source of the CEL I enzyme. A matrix of three different CEL I concentrations versus two concentrations of T4 DNA polymerase was set up to examine the limits of the in vitro DNA mismatch resolution reaction.<

Twenty one nanograms (21 ng) of the circular double stranded heteroduplexed plasmid, prepared as described above, was used as substrate in a series of ten microliter reactions containing 1×NEB ligase buffer, 0.5 mM each dNTP, 1.0 unit Taq DNA polymerase, 0.2 units T4 DNA ligase (Gibco/BRL), either 1.0 or 0.2 units T4 DNA polymerase, and either 0.3, 0.1, or 0.03 microliters of a CEL I preparation (fraction 5, described in Example 1). Six reactions representing all six combinations of the two T4 DNA polymerase concentrations with the three CEL I concentrations were prepared, split into equivalent sets of five microliters, and incubated at either 20 degrees C. or 37 degrees C. A control reaction containing no CEL I and 0.2 unit of T4 DNA polymerase with the other reaction components was prepared and incubated at 37 degrees C. After 30 minutes, one microliter aliquots of each reaction were transformed into competent DH5 alpha E. Coli which were then plated on LB amp plates. Colonies were picked and cultured. Plasmid DNA was extracted and examined by restriction fragment length polymorphism analysis (RFLP), followed by sequence analysis of the GFP gene sequences. RFLP analysis was based on differences in several restriction enzyme recognition sites between the wild type and Cycle 3 GFP genes. The RFLP results showed that throughout the CEL I/T4 DNA polymerase/temperature matrix, reassortment of restriction sites, that is GRAMMR, had occurred, and that no such reassortment had occurred in the zero CEL I control clones. DNA sequence analysis confirmed that reassortment had occurred in all of the CEL I containing samples. Sequencing also confirmed that the zero CEL I controls were not reassorted, with the exception of a single clone of the 16 control clones, which had a single base change from one gene sequence to the other, presumably resulting either from repair in E. coli or from random mutation. The sequences of several exemplary GRAMMR reassorted GFP clones are shown; all of which came from the reaction containing 0.3 microliters of the CEL I preparation and 1.0 unit of T4 DNA polymerase incubated at 37 degrees C. The parental wild type and Cycle 3 GFP genes are shown first for reference. EXAMPLE 7 Taq DNA Polymerase is Not Required for Genetic Reassortment by DNA Mismatch Resolution<

This experiment teaches that Taq DNA Polymerase does not dramatically, if at all, contribute or interfere with the functioning of Genetic Reassortment by DNA Mismatch Resolution (GRAMMR). Taq DNA polymerase is reported to have a 5′ flap ase activity, and had been included in the teachings of the previous examples as a safeguard against the possible formation and persistence of undesirable 5′ flaps in the heteroduplexed DNA undergoing GRAMMR.<

GRAMMR reactions were set up, as in Example 6, with twenty one nanograms of the circular double stranded heteroduplexed GFP plasmid substrate in ten microliter reactions containing 1×NEB ligase buffer, 0.5 mM each dNTP, 0.2 units T4 DNA ligase, 1.0 unit T4 DNA polymerase, 1.0 microliter of a CEL I preparation (fraction 5, described in Example 1), and either 2.5 units, 0.5 units of Taq DNA polymerase, or no Taq DNA polymerase. After 30 minutes, one microliter aliquots of each reaction were transformed into competent DH5 alpha E. coli which were then plated on LB amp plates. Colonies were picked and cultured. Plasmid DNA was extracted and examined by RFLP analysis followed by sequence analysis of the GFP gene sequences. The RFLP results showed that reassortment of restriction sites, that is, GRAMMR, had occurred both in the presence and the absence of Taq DNA polymerase in the GRAMMR reaction. DNA sequence analysis confirmed these results. Therefore, the data shows that Taq DNA polymerase was unnecessary for GRAMMR. EXAMPLE 8 Alternate Proofreading DNA Polymerases for Genetic Reassortment by DNA Mismatch Resolution<

This experiment teaches that Genetic Reassortment by DNA Mismatch Resolution is not limited to the use of T4 DNA polymerase, and that alternate DNA polymerases can be substituted for it.<

Reactions were set up, as in Example 6, with twenty one nanograms of the circular double stranded heteroduplexed GFP plasmid substrate in ten microliter reactions containing 1×NEB ligase buffer, 0.5 mM each dNTP, 0.2 units T4 DNA ligase (Gibco/BRL), 10 units or 2 units of T7 DNA polymerase, 1.0 microliter of a CEL I preparation (fraction 5, described in Example 1), and 2.5 units of Taq DNA polymerase. After 30 minutes, one microliter aliquots of each reaction were transformed into competent DH5 alpha E. coli which were then plated on LB amp plates. Colonies were picked and cultured. Plasmid DNA was extracted and examined by RFLP analysis followed by sequence analysis of the GFP gene sequences. The RFLP results showed that reassortment of restriction sites, that is GRAMMR, had occurred in both T7 DNA polymerase containing reactions. DNA sequence analysis confirmed these results. Therefore, the data shows that T7 DNA polymerase can substitute for T4 DNA polymerase for GRAMMR. In addition, it shows that individual components and functionalities can be broadly substituted in GRAMMR, while still obtaining similar results. EXAMPLE 9 Use of Cloned CEL I in the GRAMMR Reaction<

This example teaches that CEL I from a cloned source can be used in place of native CEL I enzyme purified from celery in Genetic Reassortment By DNA Mismatch Resolution without any noticeable change in results.<

The cDNA of CEL I was cloned from celery RNA. The gene was inserted into a TMV viral vector and expressed. Transcripts of the construct were used to infect Nicotiana benthamiana plants. Infected tissue was harvested, and the CEL I enzyme was purified. The GRAMMR results obtained using the purified enzyme were compared to those using CEL I purified from celery, and were found to be similar.<

Reactions were set up using twenty one nanograms of the circular double stranded heteroduplexed GFP plasmid substrate in ten microliters containing 1×NEB ligase buffer, 0.5 mM each dNTP, 0.2 units T4 DNA ligase (Gibco/BRL), 1 unit of T4 DNA polymerase, and either 1.0 microliter of CEL I purified from celery (fraction 5, described in Example 1), or 0.3 microliters of CEL I purified from a cloned source. After 30 minutes, one microliter aliquots of each reaction were transformed into competent DH5 alpha E. coli which were then plated on LB amp plates. Colonies were picked and cultured. Plasmid DNA was extracted and examined by RFLP analysis followed by sequence analysis of the GFP gene sequences. The RFLP results showed that reassortment of restriction sites, that is, GRAMMR had occurred in both celery derived CEL I, as well as cloned CEL I containing reactions. DNA sequence analysis confirmed these results. Therefore, the data shows CEL I from a cloned source can be used in lieu of CEL I from celery for GRAMMR. In addition, the data demonstrates that it is CEL I activity that is part of the GRAMMR method, rather than a coincidental effect resulting from the purifying steps used in extracting CEL I from celery. EXAMPLE 10 Molecular Breeding of Tobamovirus 30K Genes in a Viral Vector<

In the preceding examples, Genetic Reassortment by DNA Mismatch Resolution has been taught to be useful for reassorting sequences that are highly homologous, for example, wtGFP and Cycle 3 GFP are 96% identical. The present example teaches that GRAMMR can be used to reassort more divergent nucleic acid sequences, such as genes encoding tobamovirus movement protein genes.<

Heteroduplexes of two tobamovirus movement protein (MP) genes that are approximately 75% identical were generated. The heteroduplex substrate was prepared by annealing partially complementary single stranded DNAs of opposite strandedness synthesized by asymmetric PCR; one strand encoding the movement protein gene from the tobacco mosaic virus U1 type strain (TMV U1) (SEQ ID NO:10), and the other strand encoding the movement protein gene from tomato mosaic virus (ToMV) (SEQ ID NO:09). The sequences of the two partially complementary movement protein genes were flanked by 33 nucleotides of absolute complementarity to promote annealing of the DNAs at their termini and to facilitate PCR amplification and cloning. The annealing reaction took place by mixing 2.5 micrograms of each single stranded DNA in a 150 microliter reaction containing 333 mM NaCl, 33 mM MgCl2, 3.3 mM dithiothreitol, 166 mM Tris HCl, pH 7, and incubating at 95° C. for one minute followed by slow cooling to room temperature. GRAMMR was performed by incubating 5 microliters of the heteroduplex substrate in a 20 microliter reaction containing 1×NEB ligase buffer, 0.5 mM each dNTP, 0.4 units T4 DNA ligase (Gibco/BRL), 2.0 units of T4 DNA polymerase, and CEL I. The CEL I was from a cloned preparation and the amount that was used varied from 2 microliters of the prep, followed by five serial 3 fold dilutions. A seventh preparation with no CEL I was prepared, which served as a control.<

After one hour at room temperature, DNA was purified from the reactions using Strataprep spin DNA purification columns (Stratagene, LaJolla, Calif.) and used as templates for PCR reactions using primers designed to anneal to the flanking primer binding sites of the two sequences. PCR products from each reaction were purified using Strataprep columns, digested with AvrII and PacI, and ligated into the movement protein slot of similarly cut pGENEWARE MP Avr Pac. This plasmid contained a full length infectious tobamovirus GFP clone modified with AvrII and PacI sites flanking the movement protein gene to permit its replacement by other movement protein genes. After transformation of DH5 alpha E. coli and plating, colonies were picked, cultures grown, and DNA was extracted. The movement protein inserts were subjected to DNA sequence analysis from both directions and the sequence data confirmed that in the majority of inserts derived from the GRAMMR treated material were reasserted sequences made up of both TMV U1 and ToMV movement protein gene sequences. The DNA sequences of several exemplary GRAMMR MP clones are shown as SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15. EXAMPLE 11 GRAMMR Reassortment to Generate Improved Arsenate Detoxifying Bacteria<

Arsenic detoxification is important for mining of arsenopyrite containing gold ores and other uses, such as environmental remediation. Plasmid pGJ103, containing an arsenate detoxification operon (Ji and Silver, 1992) (Ji, G. and Silver, S., Regulation and expression of the arsenic resistance operon from Staphylococcus aureus plasmid pI258, J. Bacteriol. 174, 3684 3694 (1992), incorporated herein by reference), is obtained from Prof. Simon Silver (U. of Illinois, Chicago, Ill.). E. coli TG1 containing pGJ103, containing the pI258 ars operon cloned into pUC19, has a MIC (minimum inhibitory concentration) of 4 μg/ml on LB ampicillin agar plates. The ars operon is amplified by mutagenic PCR [REF], cloned into pUC19, and transformed into E. coli TG1. Transformed cells are plated on a range of sodium arsenate concentrations (2, 4, 8, 16 mM). Colonies from the plates with the highest arsenate levels are picked. The colonies are grown in a mixed culture with appropriate arsenate selection. Plasmid DNA is isolated from the culture. The plasmid DNA is linearized by digestion with a restriction endonuclease that cuts once into the pUC19 plasmid backbone. The linearized plasmids are denatured by heating 10 min. at 94° C. The reaction is allowed to cool to promote annealing of the single strands. Partially complementary strands that hybridize have non basepaired nucleotides at the sites of the mismatches. Treatment with CEL I (purified by the method of Example 9) causes nicking of one or the other polynucleotide strand 3′ of each mismatch. The presence of a polymerase containing a 3′ to 5′ exonuclease (“proofreading”) activity, such as T4 DNA polymerase allows excision of the mismatch, and subsequent 5′ to 3′ polymerase activity fills in the gap using the other strand as a template. T4 DNA ligase then seals the nick by restoring the phosphate backbone of the repaired strand. The result is a randomization of mutations among input strands to give output strands with potentially improved properties. These output polynucleotides are transformed directly into E. coli TG1 and the cells are plated at higher arsenate levels; 8, 16, 32, 64 mM. Colonies are picked from the plates with the highest arsenate levels and another round of reassortment is performed as above except that resulting transformed cells are plated at 32, 64, 128, 256 mM arsenate. The process can then be repeated one or more times with the selected clones in an attempt to obtain additional improvements. EXAMPLE 12 Cloning, Expression and Purification of CEL I Endonuclease<

This example teaches the preparation of nucleic acid molecules that were used for expressing CEL I endonuclease from plants, identified herein as, p1177MP4 CELI Avr, and p1177MP4 CELI 6HIS. In particular, this example refers to disclosures taught in U.S. Pat. Nos. 5,316,931, 5,589,367, 5,866,785, and 5,889,190, incorporated herein by reference.<

The aforementioned clones were deposited with the American Type Culture Collection, Manassas, Va. 20110 2209 USA. The deposits were received and accepted on Dec. 13, 2001, and assigned the following Patent Deposit Designation numbers, PTA 3926 (p1177MP4 celI Avr, and PTA 3927 (p1177MP4 celI 6HIS).<

1. Celery RNA Extraction:<

Celery was purchased from a local market. Small amounts of celery tissue (0.5 to 0.75 grams) were chopped, frozen in liquid nitrogen, and ground in a mortar and pestle in the presence of crushed glass. After addition of 400 microliters of Trizol and further grinding, 700 microliters of the extract were removed and kept on ice for five minutes. Two hundred microliters of chloroform were then added and the samples were centrifuged, left at room temperature for three minutes, and re centrifuged at 15,000 g for 10 minutes. The aqueous layer was removed to a new tube and an equal volume of isopropanol was added. Tubes were inverted to mix and left at room temperature for 10 minutes followed by centrifugation at 15,000 g for ten minutes at 4° C. The pellet was washed twice in 400 microliters of 70% ethanol, once in 100% ethanol, air dried, and resuspended in 40 microliters of distilled water. One microliter of RNasin was added and 3.5 microliters was run on a 1% agarose gel to check the quality of the RNA prep (Gel picture). The remainder was stored at −70° C. until further use.<

2. CEL I Gene Cloning and Expression by a Viral Vector:<

The total RNA from celery was subjected to reverse transcription followed by PCR to amplify the cDNA encoding the CEL I gene sequence. In separate reactions, eleven microliters of the total celery RNA prep was mixed with one microliter (50 picomoles) of either CelI Avr R, CelI 6H R, or with two microliters of oligo dT primer. CelI Avr R was used to prime cDNA and amplify the native CEL I sequence at the 3′ end of the gene, while CelI 6H R was used to add a sequence encoding linker peptide and a 6 His tag to the 3′ terminus of the CEL I gene. The samples were heated to 70° C. for one minute and quick chilled on ice prior to the addition of 4 microliters of 5× Superscript II buffer, two microliters of 0.1M DTT, 1 microliter of 10 mM each dNTP, and 1 microliter of Superscript II (Gibco/BRL) to each reaction. The reactions were incubated at 42° C. for one hour.<

PCR amplification of the CEL I cDNA sequence was performed using the method of W. M. Barnes (Proc Natl Acad Sci. USA, 1994 March 15:91(6):2216 20) with a Taq Pfu mixture or with Pfu alone. The RT reaction primed with CelI Avr R was used as template for a PCR using primers CelI Pac F (as the forward primer) paired with CelI Avr R (as the reverse primer). In other PCRs, the RT reaction that was primed with oligo dT was used as template for both of the above primer pairs. All PCR reactions were performed in 100 microliters with 30 cycles of annealing at 50° C. and two minutes of extension at 72° C. Aliquots of the resulting reactions were analyzed by agarose gel electrophoresis. Reactions in which Pfu was used as the sole polymerase showed no product. All reactions performed with the Taq/Pfu mixtures yielded product of the expected size. However, those amplified from cDNA primed with Cel I specific primer pairs gave more product than reactions amplified from cDNA primed with oligo dT. DNAs from the PCR reactions that gave the most product were purified using a Zymoclean DNA spin column kit and digested with PacI and AvrII, gel isolated, and ligated into PacI and AvrII digested plasmid pRT 130, a tobamovirus based GENEWARE® vector. 2 microliters of each ligation were transformed into DH5α competent E. coli and cultured overnight on LB amp agar plates. Colonies were picked and grown overnight in liquid culture, and plasmid DNA was isolated using a Qiagen plasmid prep kit. 12 clones from each construct were screened by digestion with PacI and AvrII and 11 of 12 of each set were positive for insert of the correct size. Ten of the clones for each construct were transcribed in vitro and RNA was inoculated to N. benthamiana plants. In addition, the CEL I gene inserts in both sets often clones were subjected to sequence analysis. Several clones containing inserts encoding the native form of CEL I had sequence identical to the published CEL I sequence in WO 01/62974 A1. One clone containing an insert encoding CEL I fused to a 6 Histidine sequence was identical to the published CEL I sequence. One clone of each (pRT130 celI Avr B3 and pRT130 celI 6His A9, respectively) was selected for further work. The CEL I encoding sequences in these clones were subsequently transferred to another GENEWARE vector. It should be noted that applicant's designations for each of the clones were shortened in the deposit to the aforementioned deposit with the American Type Culture Collection, that is, p1177MP4 celI Avr B3 is referred to as p1177MP4 celI Avr; and p1177MP4 celI 6His A9 is referred to as p1177MP4 celI 6His. The clone p1177MP4 celI Avr contained the CEL I open reading frame extending from nucleotide 5765 to 6655; and the clone p1177MP4 celI 6His A9 contained the CEL I open reading frame extending from nucleotide 5 765 6679.<

3. Assay of Cloned CEL I Activities.<

To determine whether the GENEWARE constructs containing Cel I sequences could produce active CEL I enzyme, samples of pRT130 celI Avr and pRT 130 celI 6His, and GFP GENEWARE control infected plants were harvested and homogenized in a small mortar and pestle in Tris HCl at pH 8.0. Extracts were clarified and assayed for supercoiled DNA nicking activity. Each supercoiled DNA nicking assay was performed in a reaction containing 0.5 micrograms of a supercoiled plasmid prep of a pUC19 derivative in 1×NEB ligase buffer in a total volume of 10 microliters. The amounts of plant extract added to the reactions were 0.1 microliter, 0.01 microliter, or 0.001 microliter, incubated at 42° C. for 30 minutes, and run on a 1% TBE agarose gel in the presence of ethidium bromide. Little or no nicking activity was detected in the GFP GENEWARE control infected plant extract whereas extracts from plants infected with the CEL I GENEWARE constructs showed appreciable amounts of activity against the plasmid DNA substrate.<

Additional activity assays were performed on extracts of plants inoculated with pRT130 celI Avr B3 and pRT130 celI 6His A9. In these assays, intracellular fluid was washed from infected leaves and assayed separately from material obtained from the remaining washed leaf tissues. Assays were performed as described above with the exception that the incubation was at 37° C. for one hour. Samples were run on a 1% TBE agarose gel in the presence of ethidium bromide and photographed.<

4. Purification of 6His Tagged CEL I from Infected N. benthamiana plants.<

N. benthamiana plants were inoculated with RNA transcripts from pRT130 celI 6His A9 at 20 21 days post sowing. Tissues were harvested from 96 infected plants at 10 days post inoculation and subjected to intracellular fluid washes. Briefly, infected leaf and stem material was vacuum infiltrated for 30 seconds twice with chilled infiltration buffer (50 mM phosphate pH 4 in the presence of 7 mM β ME). Infiltrated tissues were blotted to adsorb excess buffer and secreted proteins were recovered by centrifugation at 2500×g for 20 min using basket rotor (Beckman). PMSF was added to the extracted intracellular fluid (IF) containing recombinant CEL_I to a final concentration of 1 mm, and incubated at 25° C. for 15 min with stirring. After addition of Imidazole (pH 6.0) and NaCl to the extract to the final concentration of 5 mM and 0.5 M respectively, IF was adjusted to pH 5.2 and filtered through 1.2 μ Sartorius GF membrane (Whatman) to remove most of the Rubisco and green pigments. Immediately after clarification, pH was adjusted to 7.0 using concentrated NaOH solution and incubated on ice for 20 min to allow non proteinaceous material to precipitate. IF was further clarified using 0.8 μ or 0.65/0.45 μ Sartorius GF (Whatman). Recombinant CEL I was purified from the clarified IF by metal chelating affinity chromatography using Ni2+ Fast Flow Sepharose (Amersham Pharmacia Biotech, N.J.) equilibrated with binding buffer (50 mM phosphate, 0.5 M NaCl; pH 7.0) containing 5 mM imidazole, with a linear velocity of 300 cm/hr. Unbound protein was washed with 20 mM imidazole/binding buffer, and CEL I was eluted from Ni2+ Sepharose with a linear gradient of 20 to 400 M imidazole in the binding buffer. Fractions still containing imidazole were assayed for supercoiled DNA nicking activity as described above but were found to have negligible activity. The same fractions were then dialyzed against 0.1 M Tris HCl, pH 8.0 in the presence of ZnCl2 using 10 kD MWCOF dialysis tubing (Pierce) and assayed again. The supercoiled DNA nicking activity was restored after this dialysis.<

IF and purified CEL I protein were analyzed using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS PAGE) precast Tris glycine gels (Invitrogen, Carlsbad, Calif.) in the buffer system of Laemmli with a Xcell II Mini Cell apparatus (Invitrogen, Carlsbad, Calif.). The protein bands were visualized by Coomassie brilliant blue and by silver staining. SDS PAGE Gels were scanned and analyzed using Bio Rad gel imager.<

Mass Spectrometry of Purified CEL I<

The average molecular mass of the purified CEL I was determined by matrix assisted laser/desorption ionization time of flight mass spectrometry (MALDI TOF). An aliquot of CEL I was diluted 1:10 with 50% acetonitrile/water and mixed with sinapinic acid matrix (1:1 v/v) using a PE Biosystem DE Pro mass spectrometer. The mass spectrometry was performed using an accelerating voltage of 25 kV and in the positive linear ion mode.<

Mass Spectrometry of Peptides Isolated from Purified CEL I.<

CEL I was separated on SDS PAGE on a 14% gel and stained with Coomassie brilliant blue. A single homogenous band was visible. This band was excised and de stained completely. Protein was reduced in the presence of 10 mM DDT in 50% acetonitrile for 30 mm at 37° C. and reduced sulfhydro groups were blocked in the presence of 28 mM iodoacetamide in 50% acetonitrile for 30 mm at 24° C. in absence of light. Gel pieces were washed with 50% acetonitrile and after partial dehydration, the excised CEL I band was macerated in a solution of high purity trypsin (Promega). The proteolytic digestion was allowed to continue at 37° C. for 16 h. The resulting peptides were eluted from gel pieces with a 50% acetonitrile and 0.1% tri fluoro acetic acid (TFA) concentrated in a Speed Vac. The peptides were analyzed by MALDI TOF. Mixed tryptic digests were crystallized in a matrix of α cyano 4 hydroxycinnamic acid and analyzed by using a PerSeptive Biosystem DE STR MALDI TOF mass spectrometer equipped with delayed extraction operated in the reflector positive ion mode and accelerating voltage of 20 kV. Expected theoretical masses were calculated by MS digest (Protein Prospector) or GPMAW program (Lighthouse Data, Odense, Denmark). For tandem mass spectrometry (nano electrospray ionization (ESI), peptide samples were diluted with 5% acetonitrile/0.1% formic acid and subjected to LC MS/MS, analyzed on a quadrupole orthogonal time of flight mass spectrometry instrument (Micromass UK Ltd, Manchester, UK). The data were processed by Mslynx and the database was searched by Sonar.<

Virally expressed, recombinant CEL I was secreted to the IF. Clarified IF extracted material was used to purify the His tag CEL I activity. CEL I was purified using one step Ni2+ affinity chromatography separation. A highly purified homogeneous single protein band was purified as determined by Coomassie stained SDS PAGE and mass spectrometry. The size of mature proteins and percent glycosylation concur with what has been reported for the CEL I protein isolated from celery (Yang et al., 2000). The purified CEL I has an average molecular mass of 40 kD as determined by MALDI TOF mass spectrometry, indicates 23.5% glycosylation by mass. CEL I has four potential glycosylation cites at amino acid positions 58, 116, 134, and 208. A mono isotopic mass of 2152.6086 (2152.0068 Theoretical) Da corresponding to the mass of the peptide 107 125 (K)DMCVAGAIQNFTSQLGHFR (H) that was recovered by MALDI TOF, indicates that asparagine 116 is not glycosylated. Together, these gel analyses and mass spectrometry data indicate that a significant fraction of the CEL I protein was recoverable from the intracellular space, and that the protein was correctly processed in the N. benthamiana plant.<

For subsequent experiments, the 6 His tagged CEL I gene was produced using p1177MP4 celI 6His A9. This clone was transcribed and inoculated onto N. benthamiana plants, which were harvested 8 days post infection. The plant material was combined with 2 volumes of extraction buffer (500 mM NaCl, 100 mM NaPi, 25 mM Tris pH 8.0, 7 mM Beta mercaptoethanol, 2 mM PMSF) and vacuum infiltrated. Following buffer infiltration the tissue was macerated in a juice extractor, the resulting green juice adjusted to 4% w/v polyethyleneglycol, and let stand at 4° C. for one hour. The green juice was clarified by either centrifugation at low speed (3500×g) for 20 minutes or combined with perlite (2% w/v) and filtered through a 1.2 μm filter. The tagged CEL I can be selectively purified from the clarified green juice by metal affinity chromatography. The green juice was either combined with nickel NTA resin, and batch binding of the CEL I performed, or purification was performed in column format, where the green juice was permitted to flow through a bed of nickel NTA resin. For binding; the clarified green juice was adjusted to 10% w/v glycerol and 10 mM imidazole. Following binding the resin was washed extensively with wash buffer (330 mM NaCl, 100 mM NaPi, pH 8.0, 10 mM imidazole) and the bound CEL I enzyme eluted from the nickel NTA resin in 2 resin bed volumes of 1× phosphate buffered saline (PBS) containing 400 mM imidazole. The CEL I preparation was subsequently dialyzed against 1×PBS to remove the imidazole, assayed for activity, and stored at 4° C. or at −20° C. with or without glycerol until use. EXAMPLE 13 Cloning, Expression and Use of RES I Endonuclease<

This example teaches the construction of a cDNA library from Selaginella lepidophylla, the identification of a nucleic acid sequence from the library that encodes an endonuclease, and the expression of the new endonuclease, herein designated as “RES I.”<

RNA was extracted from tissues of the resurrection plant, Selaginella lepidophylla, using the Trizol method, and oligo dT primed cDNA that was prepared using standard methodology. Resulting cDNAs were ligated into a GENEWARE based cloning vector and the ligation products were transformed into competent E coli cells. Bacterial colonies containing GENEWARE cDNA clones were picked at random and grown as liquid cultures prior to DNA prepping and determination of the cloned cDNA sequences. The sequence files for the cloned Selaginella cDNAs were loaded into a database which was then searched by BLAST analysis for sequences that had similarity to the DNA sequence of the CEL I gene. BLAST analysis was also performed on other DNA sequence databases containing sequences of cDNAs obtained from other species.<

BLAST hits that showed some level of homology to the celery CEL I sequence were identified in libraries from several species and the corresponding GENEWARE cDNA clones were re arrayed into a single set of GENEWARE cDNA clones. This set of cDNA clones was then transcribed in vitro to generate infectious GENEWARE transcripts which were then inoculated onto leaves on Nicotiana benthamiana plants for expression analysis of the cDNA sequences encoded within the GENEWARE viral genome. At seven days post inoculation, leaf samples were taken from the infected plants and homogenized in two volumes of water. The extracts were then assayed for supercoiled DNA nicking and cleavage activity.<

Each supercoiled DNA nicking assay was performed in a reaction containing 0.5 micrograms of a supercoiled plasmid prep of a pUC19 derivative in 1×NEB T4 DNA ligase buffer in a total volume of 10 microliters. The amounts of plant extract added to the reactions were 1 microliter, 0.33 microliter, or 0.011 microliter, incubated at 37° C. for 30 minutes, and run on a 1% TAE agarose gel in the presence of Gelstar fluorescent DNA staining reagent. Little or no nicking activity was detected in uninfected plant extracts whereas only extracts from plants infected with GENEWARE constructs containing cDNAs for a single gene from Selaginella lepidophylla showed appreciable amounts of activity against the plasmid DNA substrate.<

A sample of the aforementioned Selaginella lepidophylla gene, as shown in FIG. 3 (SEQ ID NO:16), was mailed to the American Type Culture Collection, Manassas, Va. 20110 2209 USA on Jul. 29, 2002. The deposit was received and accepted on Jul. 30, 2002., and assigned the following Patent Deposit Designation number, PTA 4562.<

The complete gene sequences of these clones were determined and PCR primers were designed to amplify the open reading frame minus any non coding 5′ and 3′ sequences and to add a six histidine tail to the C terminus of the encoded protein. The primers were then used to amplify the ORF from one of the active full length Selaginella clones. The resulting PCR product was then cloned into the GENEWARE vector pDN4 between the PacI and AvrII sites for expression in planta. The resulting clone, pLSB2225, was sequenced to confirm that the gene had been inserted correctly, and then transcribed in vitro followed by inoculation of the infectious transcripts onto N. benthamiana plants. Seven days post inoculation, infected plant extracts were made as above and assayed for supercoiled DNA nicking and digestion activity to confirm the activity of the cloned enzyme.<

Each supercoiled DNA nicking assay was performed in a reaction containing 0.5 micrograms of a supercoiled plasmid prep of a pUC19 derivative in 1×NEB E. coli DNA ligase buffer in the presence of 50 mM KCl in a total volume of 10 microliters. The amounts of plant extract added to the reactions were 0.2 microliter, 0.04 microliter, 0.008 microliter, or 0.0016 microliter, incubated at 37° C. for 30 minutes, and run on a 0.8% TAE agarose gel in the presence of Gelstar fluorescent DNA staining reagent. Little or no nicking activity was detected in uninfected plant extracts whereas extracts from plants infected with the GENEWARE Selaginella construct pLSB2225 showed appreciable amounts of activity against the plasmid DNA substrate.<

After positive results were obtained in that assay, extracts of pLSB2225 infected plants were used in a GRAMMR shuffling experiment to test the ability of this enzyme to operate as a component of the mismatch resolution reaction in place of the GENEWARE produced CEL I enzyme of celery origin. EXAMPLE 14 Use of RES I in the GRAMMR Reaction<

This example teaches that RES I can be used in place of native CEL I enzyme purified from celery in Genetic Reassortment By DNA Mismatch Resolution without any noticeable change in results.<

GRAMMR shuffling was performed between the wild type Aequorea Victoria GFP gene (Prasher, et al., Gene 111(92)229) in a pBS derivative (Stratagene, La Jolla, Calif.) encoded by pBSWTGFP (SEQ ID NO:03) and a variant with mutations to increase fluorescence intensity in E. coli, and to alter the emission wavelength to blue light emission (Crameri, et al., Nat Biotechnol 14(96)315; Heim et al., PNAS91(94)12501; Yang, et al., J Biol Chem 273(98)8212). This variant gene, encoded by the plasmid pBSC3BFP, as shown in FIG. 5 (SEQ ID NO:18), encodes a fluorescent protein that emits bright blue light when excited by longwave UV light.<

The GRAMMR reactions were performed on GFP/c3BFP heteroduplexes in a circular, double stranded plasmid DNA context. The circular, whole plasmid heteroduplex DNA substrates were prepared by first linearizing pBSWTGFP (SEQ ID NO:03) and pBSC3BFP (FIG. 5, SEQ ID NO:4) by digestion with Kpn I and NgoM IV, respectively, then purifying the digested DNA using DNA spin columns. Next, 200 nanograms of each of the two linearized plasmids were mixed and brought to 1×SSPE (180 nM NaCl, 10 mM NaH2PO4, 1 mM EDTA at pH 7.4) in a volume of 20 microliters. The mixture was then incubated at 95 degrees Celsius for 4 minutes, plunged into icewater where it remained for 10 minutes prior to incubation at 37 degrees Celsius. After 30 minutes, the annealed DNA sample was then transferred back to ice where it was held until use in GRAMMR reactions.<

Two independent series of shuffling reactions were performed to compare CEL I with RES I in their abilities to facilitate sequence shuffling by GRAMMR. Each GRAMMR reaction contained 1 unit of T4 DNA polymerase, 2 units of E. coli DNA ligase, and 5 nanomoles of each dNTP in 1×NEB E. coli ligase buffer supplemented with KCl to 50 mM. Two separate enzyme dilution series were then performed. To each of two series of tubes containing aliquots of the above cocktail, one microliter aliquots of GENEWARE expressed CEL I or RES I extracts at dilutions of 1/3, 1/9, 1/27, 1/81, or 1/243 were added. An endonuclease free control reaction was also prepared. To each of the reactions, one microliter aliquots containing 20 nanograms of the annealed DNA heteroduplex substrate were added and the reactions incubated at room temperature for one hour and on ice for 30 minutes prior to transformation into competent E. coli.<

Green fluorescent protein (GFP) and blue fluorescent protein (BFP) could be visualized in the resulting colonies by long wave UV illumination. The parental wild type GFP has dim green fluorescence, and the parental c3BFP gave bright blue fluorescence. In the genes encoding these fluorescent proteins, the sequences that determine the emission color and those that govern fluorescence intensity are at different positions from one another. It is expected that DNA shuffling would result in the “de linking” of the sequences that determine the emission color from those that govern fluorescence intensity. As a consequence, the resultant progeny would be expected to exhibit reassortment of the functional properties of emission color and intensity. Therefore a measure of the extent of the DNA shuffling that had taken place in each reaction could be scored by examining the color and intensity of fluorescence from the bacterial colonies on the corresponding plates. In the zero nuclease control, only dim green and bright blue colonies were observed. However, on plates with cells transformed with DNAs from the reactions containing either CEL I or RES I, some bright green as well as some dim blue colonies were observed, indicating that shuffling of DNA sequences had taken place. DNA sequence analysis confirmed that this was indeed the case and that on average, the recovery of shuffled clones was greater than 85% for both CEL I and RES I and that the number and distribution of information transfer events was similar for both enzymes. However, it appeared that the activity of RES I in this experiment was several fold higher than that of CEL I, as indicated by the low transformation efficiency of reactions treated with the higher concentrations of the RES I preparation. EXAMPLE 15 Molecular Breeding of Highly Divergent Tobamovirus 30K Genes in Viral Vectors Using Plasmid on Plasmid Genetic Reassortment By DNA Mismatch Resolution (POP GRAMMR)<

Example 10 taught the reassortment of movement protein (MP) genes from several divergent strains of tobamovirus (approximately 75% identical; cloned into the pGENEWARE MP Avr Pac vector) using Genetic Reassortment by DNA Mismatch Resolution (GRAMMR). This example teaches the use of Plasmid on plasmid GRAMMR (POP GRAMMR) for reassorting even more highly divergent species.<

Starting parental MP genes from the tobamoviruses TMV Cg (FIG. 6, SEQ ID NO:18), TMV Ob (FIG. 7, SEQ ID NO:19), TMV U2 (FIG. 8, SEQ ID NO:20), TMV U1 (SEQ ID NO:10), and tomato mosaic virus (ToMV) (SEQ ID NO:09) were used. The plasmid of pGENEWARE ToMV MP was linearized by digestion with Sma I. The plasmids of pGENEWARE containing the MP genes from either TMV Cg, TMV Gb, TMV U2, or TMV U1 were digested with Stu I. The digested pGENEWARE MP constructs were purified using DNA spin columns. The following heterduplex pairs were generated: pGENEWARE Cg MP and pGENEWARE ToMV MP, pGENEWARE TMV Ob MP and pGENEWARE ToMV MP, pGENE WARE TM V U2 MP and pGENE WARE ToMV MP, pGENEWARE TMV U1 MP and pGENEWARE ToMV MP. The heteroduplexes of these MP gene sequences are approximately 47%, 58%, 62%, and 75% identical, respectively. Heteroduplex DNA was generated by mixing 200 nanograms of each of the two linearized plasmids in 1×SSPE (180 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, at pH 7.4) in a volume of 20 microliters. The mixture was incubated at 95 degrees Celsius for 4 minutes, plunged into ice water where it remained for 10 minutes prior to incubation at 37 degrees Celsius. After 30 minutes, the annealed DNA sample was then transferred back to ice where it was held until use in GRAMMR reactions.<

Each 10 microliter GRAMMR reaction contained 1 unit of T4 DNA polymerase, 2 units of E. coli DNA ligase, and 0.5 mM of each dNTP in 1×NEB E. coli DNA ligase buffer supplemented with KCl to 50 mM. A one microliter aliquot of CEL I (diluted 1/3, 1/9, 1/27, 1/81, 1/243, or 1/729) was next added. An endonuclease free control reaction was also prepared. To each of the reactions, a one microliter aliquot containing 20 nanograms of the annealed DNA heteroduplex substrate was added and the reactions were incubated at room temperature for one hour and on ice for 30 minutes prior to transformation into competent E. coli.<

from 7078211, 7273739

Other protocols

After PCR (gene synthesis), treat PCR mixture with 5 U mismatch cleaving enzymes: 5 µl aliquot of the PCR reaction E.coli endonuclease V, or 1000 U T4 endonuclease VII (USB) 1 X reaction buffer Water to 20 µl. Incubate at 37 oC in for 30 min to 24 h.

Optional: add E.coli exonuclease I (Fermentas) to digest single stranded DNA.

TthMuts Buffer

50 mM Tris HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 5 mM beta mercaptoethanol, 10% (v/v) glycerol and 20 mM MgCl2] and T.thermophilus MutS protein

Incubate at 35 or 60 oC for 30 min.

Oligo design: http://helixweb.nih.gov/dnaworks/

Step 1: Gene assembly Mix equal volumes of each oligo at either 20 or 40 uM, dilute this this mixture 10 fold in a 50 ul PCR reaction containing 2 mM dNTP and 1.25 unit polymerase.

PCR: 4 min denaturation step of 95 °C. 5~20 cycles: 95 °C for 30 s 50 °C for 30~45 s 72 °C for 30 s. Then 72 °C for 5 min

Step 2: Gene amplification 1, dilute gene assembly reaction mixture (template) at least 10 fold in 50 ul final vol. 2, Add two outermost oligos at 1 uM final concentration. 3, Add other components. PCR condition is as above except 1 Kb/per min for extention. 15~20 cycles.

For synthesis of genes with high G+C content: initial denaturation step is 98 °C for 30~60 s 98 °C for 20 s for each subsequent denaturation steps 5~30 cycles for gene assembly 15~20 cycles for gene amplification

DNA polymerases: PrimeSTAR HS DNA polymerase > HiFi DNA polymerase > Accuprime pfx DNA polymerase > Herculase HS DNA polymerase > PfuTurbo HS DNA polymerase > Fail Safe DNA polymerase.

1, Purify PCR product using Qiagen kit and elute DNA with 50 ul of T7 endonuclease I buffer (50 mM potassium acetate, 20 mM Tris acetate, pH 7.9, 10 mM magnesium acetate, 1 mM dithiothreitol).

2, Denature DNA at 94 °C for 3 min, anneal at 75°C for 5 min.

3, Add 30 U T7 endonuclease I (New England Biolabs).

4, Incubate at 37°C for 1 h then 55 °C for another hour.

5, Purify desired DNA fragment by agarose gel electrophoresis and Qiagen kit.

6, Optional: Clone the fragment into a vector.

Design oligos using http://helixweb.nih.gov/dnaworks/

First Step: 1. Mix oligos, 15 nM each in 20 ul total volume. 2. Add 1 mM dNTP 3. Add 1 unit of polymerase 4. Run PCR for 10 cycles

Second Step: 1. Add 2 ul first step PCR product 2. Add 1 uM F and R primer 3. Add 1 mM dNTP 4. Add 1 unit polymerase 5. Add water to 50 ul 6. Run PCR for 25 cycles

Preferred polymerases Phusion > PfuTurbo > Pfx 50 > Pfu Ultra II

1, Design oligos using DNAWorks (http://helixweb.nih.gov/dnaworks/), select the least missmatched oligos ( length is 50 bp ~ 60 bp).

2, Order oligos from Integrated DNA Technologies.

3, Make 10 uM oligo stocks.

4, Pool 1 ul of 10 uM oligos to cover ~ 400 to 750 bp (to assemble a building block).

5, PCR using 1 to 5 ul of oligo mix (step 4) as template, 1 ul Phusion hot start DNA polymerase in 48 ul reaction for 5 cycles: 98 oC for 2min. 5 cycles of 98 oC for 30, 58 oC for 30, 72 oC for 30 to 1 min per kb.

6, Add 1 ul of 10 uM end (outer) primers (F & R), continue PCR for 20 more cycles:

20 cycles of 98 oC for 30, 55 oC for 30, 72 oC for 30 to 1 min per kb. 1 cycle: 98 oC for 30, 55 oC for 30', 72 oC for 10 min. 7, Run 5 to 10 ul PCR product on agarose gel. Optional: clone PCR product into a cloning vector.

8, To amplify a gene with length > 750 bp, mix PCR products (2.5 ul) from step 6, or verified clone from step 7, and PCR using 1 ul of 10 uM end (outer) primers (F & R).

9, Clone into a cloning vector.

10, Sequencing. error rate: up to 10 mutations can be found from a clone. But sometimes you can get one correct clone from the first sequencing reaction on any lucky day.

1, Recombinant 6XHis MBP MutS protein from Excellgen is supplied with storage buffer: 50 mM Tris“HCl, pH 7.5, 100 mM NaCl, 0.1 mM EDTA and 0.1 mM DTT, 50% glycerol

The protein should be stored at 20 oC.

2, After first round PCR, purify PCR fragments using Qiagen QIAquick PCR purification kit with elution in dH2O.

3, Dilute PCR product to 250 ng/ul in 10 mM Tris HCl, pH 7.8, 50 mM NaCl and heat to 95 oC for 5 min followed by cooling at 0.1oC/s to 25 oC.

4, Add binding buffer (20 mM Tris HCl, pH 7.8, 10 mM NaCl, 5 mM MgCl2, 1 mM DTT and 5% glycerol) to annealed PCR product, adjust DNA concentration to 11.5 ng/ul, add 6XHis MBP MutS dimers to 950 nM.

5, Incubate the mixture at room temperature for 10 min, then add an equal volume of amylose resin (NEB) preequilibrated with 1X binding buffer, and incubate for 30 min at room temperature.

6, Gently spin down beads and save supernatant for subsequent processing (second round PCR, cloning etc).