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

356 Mazars and Theillet
Fig. 1. Schematic representation of thermal asymmetric PCR.
These PCR fragments comprised the following: (1) exons 7 and 8 of the p53 gene
and (2) exon 1 of HRAS. This latter sequence is particularly GC-rich, and as part
of another study, primer A was synthesized with a 40-bp GC stretch (to make a GC
clamp). In conclusion, thermal asymmetric PCR allows direct sequencing of both
strands with high reproducibility and reduced risk of contamination.
2. Materials
All solutions should be made according to the standard required for molecular
biology, such as molecular biology-grade reagents and sterile distilled water. All
reagents for sequencing are available commercially.
2.1. PCR Amplification
1. Primers were synthesized on Applied Amplifications, and PCR was performed on a
Perkin–Elmer Cetus thermal cycler.
2. 1× Taq polymerase buffer: 10 mM Tris-HCl, pH 8.3, 2 mM MgCl 2 , 50 mM KCl, 0.01%
gelatin; 100 µM of dNTPs.
3. Taq polymerase was purchased from Perkin–Elmer and used at 1 U/reaction.