Editing Preservation of Saliva

The composition of the nucleic acid-preserving solution used in Examples 4-6 is 33 mM TRIS-HCl, 0.67 M urea, 0.67 M LiCl, 0.6% sodium dodecyl sulfate, 3.3 mM CDTA, 30% ethanol, and 0.25 M sodium ascorbate, all adjusted to a final pH of 8.0. In the examples, the composition is mixed with an equal volume of saliva. Subsequent to these experiments, it has been found that a composition which is 0.3 M TRIS-HCl, 0.67 M urea, 0.67 M NaOAc, 0.6% sodium dodecyl sulfate, 3.3 mM CDTA, 30% ethanol, and 0.1 M sodium ascorbate, all adjusted to a final pH of 8.0, stabilizes DNA for longer periods of time.



Chinese

Tris-HCl with pH being 6-8.5 (5-20mmol/L), EDTA (Ethylene Diamine Tetraacetic Acid) (0.5-2mmol/L), NaOAc (2.5-3.5mol/L), cane sugar (0.1-0.4mol/L), N-acetyl-5-methoxytryptamine (1-3mmol/L), propylparaben (1-4mg/mL), diazonium imidazolidinyl urea (1-3mg/mL), protease K(10mu g/mL)



Participants were asked to take series of mouth swabs before a meal, preferably in a mouth without food remains, by rubbing a maximum of five consecutive cotton buds (Antonides C.V.), with a time interval of at least four hours, along the inside of the mouth. After rubbing, the mouth swab was placed in a Falcon tube, containing 0.5 ml of STE buffer (100 mM NaCl, 10 mM Tris and 10 mM EDTA) with proteinase K (0.1 mg/ml) and sodium dodecyl sulphate (0.5%) per mouth swab.



The total DNA yield (human and nonhuman) was measured with the Picogreen® dsDNA Quantitation Kit (Molecular Probes, Inc). Bacterial DNA yield present in the mouth swab sample was estimated by a semi-quantitative method using real-time polymerase chain reaction (PCR). A region of the 16S rRNA gene, which is known to be conserved across a wide variety of micro organisms and is not found in humans15 was amplified using a Roche LightCycler™ (Roche) and SYBR Green I dye (Molecular Probes, Inc.). The nucleotide sequences of the primers are as follows: primer 1, 5’-CTACGGGAGGCAGCAG-3’; primer 2, 5’- ATTACCGCGGCTGCTGG-3’15. The estimated bacterial DNA content of the mouth swab samples divided by the measured total DNA yield represents the estimated proportion of bacterial DNA in the mouth swab sample.

The mouth swabs were collected from participants by post. Mouth swabs were taken and stored by the participants in STE buffer (100 mM NaCl, 10 mM EDTA, 10 mM Tris) including 0±1 mg}ml proteinase K and 0±5% SDS. When the swabs arrived by post in the TNO laboratories, the proteinase K concentration was increased to 0±2 mg}ml and the swabs were incubated at 65 °C for 2 h. After centrifugation of the samples as described (Meulenbelt et al., 1995), 0±2 volume of 8 M KAc was added to the lysate and mixed well but gently. The mixture was kept on ice for 15 min. One volume of chloroform:isoamylalcohol (24:1) was added and placed in a top-over for half an hour. Subsequently, samples were centrifuged and isopropanol was added to the supernatant for precipitation of genomic DNA. DNA of 1911 individuals was obtained using this mouth swab procedure and the average yield per cotton bud was 2±0 µg (SD 1±4) genomic DNA. Depending on the subject taking the mouth swab, the DNA yield is quite variable

We have isolated human genomic DNA of family members (ages between 4 and 72 years), young twins (ages between 2 mo and 5 years), and random controls by using mouth swabs. Mouth swabs of the family members and random controls were taken by the participants themselves, and parents took mouth swabs of their twins, following a written protocol. Although twin pairs were sometimes only a few months old, problems were not encountered by parents obtaining samples from their children. At least 10 consecutive samples can be taken from one subject without a significant loss in yield per cotton swab. A second round of 10 cotton swabs can be taken after -4 h, giving a maximal result of 20 samples in 1 d. The mouth swab sample should be taken in a clean mouth without food remains. After rubbing, the cotton swab, sample-end first, should be placed in a Falcon tube, containing 0.5 ml of STE buffer (100 mM NaCl, 10 mM TrisHCL [pH 8.0] and 10 mM EDTA) with proteinase K (0.2 mg/ml) and SDS (0.5%) per cotton swab. In this way, samples can be kept at least 3 wk without decrease in yield or quality of DNA. Highmolecular-weight human genomic DNA (>50 kb) was isolated with an average yield of 2.1 ± 0.2 gg per cotton swab from 81 individuals by using mouth swab samples that were placed in lysis buffer immediately after wiping. DNA isolations of another 181 individuals were performed with mouth swab samples that were kept dry after wiping, that is, without lysis buffer. High-molecular-weight human genomic DNA was also isolated from these individuals. The average DNA yield of these sam ples, however, was 1.3 ± 0.05 ,ug per cotton swab. Mouth swab samples which were kept dry for >7 d before isolation gave an extra decrease in DNA yield. The storage of cotton swabs in lysis buffer after wiping is therefore the better procedure. Variation of DNA yield within each method is primarily caused by the difference in pressure exerted during the mouth swab sampling.
[[Category:Protocols]]