Optimization of annealing temperature to reduce bias caused by a primer mismatch in multitemplate PCR.
To reduce PCR bias derived from a primer mismatch, the effect of the annealing temperature on the product ratio was investigated by denaturing gradient gel electrophoresis analysis of PCR products from a mixture of perfect-match and one-mismatch templates. These templates were generated by PCR from Pediococcus acidilactici for one mismatch and Micrococcus luteus for the perfect match. PCRs showed that the bias was reduced at lower temperatures. An environmental sample was also examined.
Ishii
K
K
Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Hachiohji, Tokyo 192-0397, Japan. ishii-kousuke@c.metro-u.ac.jp
Fukui
M
M
D020137:Base Pair Mismatch; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis*; D004587:Electrophoresis, Agar Gel / Q000379:methods; D016982:Micrococcus luteus / Q000235:genetics; D010376:Pediococcus / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D013696:Temperature; D013698:Templates, Genetic
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S