John M. S. Bartlett.pdf - Bio-Nica.info

Thermal Asymmetric PCR 359
4. Notes
1. Instability of diluted solution of primers conserved at –20°C can sometimes be problematic.
We recommend storing oligonucleotides as a dried powder and resuspending them in
water prior to use.
2. Estimation of the yield of single-strand DNA produced can be achieved by Southern
blotting: run a 2% agarose gel, blot following standard conditions, and probe with one of
the PCR primers. Two bands should appear if you use higher T m primer or only one band
if you use lower T m primer (corresponding to double-stranded DNA). An alternative
strategy is to add α-dCTP[ 32 P] for the second step of amplification at high temperature:
labeled single-stranded DNA should be exclusively produced.
3. We also sequenced PCR fragments after SSCP analysis. In this case, shifted SSCP bands
were excised from the gel with a sterile razor blade and eluted in 50 µL of distilled water
for 1 h at 65°C. A 1.5-µL aliquot of the eluate was subjected to thermal asymmetric PCR.
4. A good sequence can be obtained even if no primer is added for the sequencing reaction.
The reason is that the low T m primer from PCR is not completely removed by Centricon
30 purification.
5. The present protocol has been optimized for classical radioactivity labeled DNA sequencing,
but should easily be adapted to automated fluorescent sequencing using fluorescent
dye terminators.
References
1. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. F., Higuchi, R., Horn, R. T., et al. (1988)
Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.
Science 239, 487– 491.
2. Gyllensten, U. B. and Erlich, H. A. (1988) Generation of single-stranded DNA by the
polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus.
Proc. Natl. Acad. Sci. USA 85, 7652–7656.
3. Wilson, R. K., Chen, C., and Hood, L. (1990) Optimization of asymmetric polymerase
chain reaction for rapid fluorescent DNA sequencing. BioTechniques 8, 184–189.