Directed termination of the polymerase chain reaction: kinetics and applications in mutation detection.
We describe a PCR-based method (DT-PCR) that integrates both DNA amplification and directed chain termination into a single-step process. This method exploits unbalanced nucleotide concentrations to induce the polymerase chain reaction to terminate at specific nucleotide sites, leading to the generation of two sets of nested termination fragments from genomic DNA. The kinetic mechanism underlying the termination process is outlined and the application of this method to the detection and characterization of mutations in fragments as long as 1 kb is described. The method is effective for the analysis of both haploid and diploid genomes, and not only allows the recognition of both indels (insertions and deletions) and nucleotide substitutions, but also enables the determination of their position in a single-step fashion.
Chen
J Z
JZ
Department of Zoology, University of Guelph, ON, Canada. jjchen@uoguelph.ca
Hebert
P D
PD
D000818:Animals; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D014644:Genetic Variation; D005838:Genotype; D007059:Ictaluridae / Q000235:genetics; D007700:Kinetics; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D013997:Time Factors
D017931:DNA Primers