Fluorescence-based method for measuring and determining the mechanisms of recombination in quantitative PCR.
We quantitatively measured the amount of recombinant molecules formed during PCR when the break point cluster region (BCR) cDNA was coamplified with a homologous internal standard using Taq polymerase. The products were analysed under denaturing conditions using capillary electrophoresis followed by detection of the fluorescently labelled products and the recombinant molecules were differentiated by their size. Early termination of chain synthesis and reannealing of incomplete fragments, to each other as well as to BCR and internal standard, is one mechanism for generating recombinants during PCR since prolonging extension time reduced, but did not totally suppress recombinant molecule formation. Template switching by the extending chain is another mechanism since recombinant molecules could be detected even after only one round of primer extension. The latter mechanism is probably facilitated by increasing number of templates. Thus, the large increase of recombinant molecules formed in plateau phase is mediated by direct amplification of the recombinants and de novo synthesis by template switching. The effect of additives on recombination could be quantitatively measured and both betaine and DMSO were effective in suppressing recombination. Thus, prolonging extension time, reducing the number of amplification cycles and incorporating additives in the PCR reaction, reduced recombinant molecule formation.
Shammas
F V
FV
Department of Clinical Chemistry, Rogaland Central Hospital, 4011 Stavanger, Norway. shammas@online.no
Heikkilä
R
R
Osland
A
A
D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D019075:Electrophoresis, Capillary; D005453:Fluorescence; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D012015:Reference Standards
D017931:DNA Primers; D018076:DNA, Complementary