Editing Recombinase Polymerase Amplification (section)

== Enzymes ==

Coding sequences for uvsx, uvsy, gp32, Bsu, and Nfo were amplified from genomic DNA (DSMZ, Braunschweig Germany), fused to hexahistidine-tags ('''N-terminal for uvsY, Bsu, and Nfo, C-terminal for uvsX and gp32'''), and cloned into suitable expression vectors. Overexpression and purification were done by standard protocols using nickel-NTA resin (Qiagen, Valencia, California, United States). Human genomic DNA was from Promega (Madison, Wisconsin, United States), '''B. subtilis DNA''' was from ATCC,

uvsX: T4 DNA-binding protein<br/>uvsY: T4 uvsY protein, a recombination accessory protein<br/>Bsu polymerase: B. subtilis DNA

Bacillus subtilis (Ehrenberg) Cohn (ATCC® 23857D-5™)

ATCC® Number: 23857D-5 ™<br/>Strain Designations: Genomic DNA from Bacillus subtilis Strain 168 [ATCC® 23857™]

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Bacillus subtilis (Ehrenberg) Cohn (ATCC® 82D-5™)

ATCC® Number: 82D-5 ™<br/>Strain Designations: Genomic DNA from strain AMC [ATCC 82]

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Bacillus subtilis subsp. spizizenii Nakamura et al. (ATCC® 6633D-5™)

ATCC® Number: 6633D-5 ™<br/>Deposited As Bacillus subtilis (Ehrenberg) Cohn<br/>Strain Designations: Genomic DNA from strain NRS 231 [ATCC 6633]

<br/>gp32: T4 single-stranded DNA binding protein<br/>double-strand–specific Escherichia coli endonuclease IV<br/>(Nfo)<br/>N-terminal for uvsY, Bsu, and Nfo, C-terminal for uvsX and gp32

RPA Reaction

Reactions were performed at 37 oC for 60 min or as indicated. Standard conditions were 50 mM Tris (pH 7.9), 100 mM potassium acetate, 14 mM magnesium acetate, 2 mM DTT, 5% Carbowax20M, 200 lM dNTPs, 3 mM ATP, 50 mM phosphocreatine, 100 ng/ul creatine kinase, and 30 ng/ul Bsu; in the multiplex experiment, Carbowax20M was at 5.5%. Recombinase protein concentrations were 900 ng/ul gp32, 120 ng/ul uxsX, and 30 ng/ul uvsY. Primers used for amplification of human targets were apoB4, apoB300, sry3, sry4, pbdg5, pbdg6 (300 nM each), for B. subtilis, bs1 and bs2 were used (300 nM each) in the experiment in Figure 2B. For&nbsp;MRSA amplification, primers used were 500 nM sccIII, 100 nM orfX (Figure 4A) or 265 nM sccI/II, 265 nM sccIII, 70 nM orfX (Figure 5) or 240 nM Bio-sccI/II, 240 nM Bio-sccIII, 120 nM orfX (Figure 6). Reaction volumes were 20 ll, except for the experiment shown in Figure 2A (40 ll).



Reactions were performed at 37 oC for 120 min, and conditions used&nbsp;were 50 mM Tris (pH 8.4), 80 mM potassium acetate, 10 mM&nbsp;magnesium acetate, 2 mM DTT, 3 mM ATP, 200 lM dNTPs, 20 mM&nbsp;phosphocreatine, 100 ng/ul creatine kinase, 5% Carbowax20M, 600 ng/ul gp32, 125 ng/ul uvsX, 25 ng/ul uvsY, and 30 ng/ul Bsu. And 450 copies&nbsp;of human DNA were used as template in 30-ul reactions (per sample&nbsp;the equivalent of 10-ul reaction volume was loaded on the gel).

Reactions were performed at 37 8C for 120 min. Conditions&nbsp;used were 50 mM Tris/Cl (pH 8.4), 80 mM potassium acetate, 10 mM&nbsp;magnesium acetate, 2 mM DTT, 3 mM ATP, 200 &nbsp;lM dNTPs, 20 mM&nbsp;phosphocreatine, 100 ng/ul creatine kinase, 5% Carbowax20M, 600&nbsp;ng/ul gp32, 200 ng/ul uvsX, 60 ng/ul uvsY, and 30 ng/ul Bsu&nbsp;polymerase.
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Real-time monitoring. Real-time RPA was performed in a plate&nbsp;reader (Flx-800; BioTek, Winooski, Vermont, United States) in the&nbsp;presence of SybrGreenI (1:50,000; Molecular Probes, Eugene, Oregon,&nbsp;United States) or fluorophore/quencher probes (Eurogentec, San&nbsp;Diego, California, United States). Three probes were used:

<br/>SATamra1 59-tgttaattgaacaagtgtacagagcatt(T)a(H)ga(q1)tatgcgtggag-biotin-39<br/>SATamra2 59-tgttaattgagcaagtgtatagagcatt(T)a(H)ga(q2)tatgcgtggag-biotin-39

BSFlc 59-catgattggatgaataagctgcagc(F)g(H)t(q3)aaaggaaactta-biotin-3&nbsp;



Here, (T) is dT-TAMRA, (F) is dT-fluorescein, (H) is THF, (q1) is&nbsp;dT-BHQ1, (q2) is dT-BHQ2, and (q3) is dT-DDQ1.

Probes were used&nbsp;at 60 nM SATamra1 (MRSAIII experiment) or at 45 nM SATamra1, 45&nbsp;nM SATamra2, 60 nM BSFlc (multiplex experiment).



Nfo was used at&nbsp;200 ng/ul. Excitation/detection was at 485/525 nm (SybrGreenI, BSFlc)&nbsp;or 530/575 nm (SATamra1/2). Measurements were taken every 30 or&nbsp;45 s (multiplex experiment). Fluorescence probe data were normalised (subtraction of water control average) and preamplification&nbsp;baseline adjusted. The logarithm of the read-out was plotted against<br/>reaction time.

<br/>Lateral-flow strip detection. For lateral-flow strip experiments, two&nbsp;probes were used at 75 nM each:<br/>Lfs1 59-FAM-ccacatcaaatgatgcgggttgtgttaat(H)gaacaagtgtacagag-ddC-39<br/>Lfs2 59-FAM-ccacatcaaatgatgcgggttgtgttaat(H)gagcaagtgtatagag-ddC-39<br/>The 59-biotinylated forms of sccI/II and sccIII were utilised as&nbsp;primers. For each reaction (20 ll), 1 ll was diluted with 5 ll of&nbsp;running buffer (PBS/3% Tween) and applied directly to HybriDetect&nbsp;strips (Milenia Biotec, Bad Nauheim, Germany) according to the&nbsp;manufacturer’s instructions.