The effects of sequence length and oligonucleotide mismatches on 5' exonuclease assay efficiency.

ABSTRACT: Although increasingly used for DNA quantification, little is known of the dynamics of the 5' exonuclease assay, particularly in relation to amplicon length and mismatches at oligonucleotide binding sites. In this study we used seven assays targeting the c-myc proto-oncogene to examine the effects of sequence length, and report a marked reduction in efficiency with increasing fragment length. Three of the assays were further tested on 15 mammalian species to gauge the effect of sequence differences on performance. We show that the effects of probe and primer binding site mismatches are complex, with single point mutations often having little effect on assay performance, while multiple mismatches to the probe caused the greatest reduction in efficiency. The usefulness of the assays in predic

SUBMITTER: Smith S 

PROVIDER: S-EPMC137155 | biostudies-literature | 2002 Oct

REPOSITORIES: biostudies-literature