Unlabeled oligonucleotides as internal temperature controls for genotyping by amplicon melting.

ABSTRACT: Amplicon melting is a closed-tube method for genotyping that does not require probes, real-time analysis, or allele-specific polymerase chain reaction. However, correct differentiation of homozygous mutant and wild-type samples by melting temperature (Tm) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood, amplicon Tm differences of 0.03 to 0.39 degrees C attributable to the extractions were observed. To correct for solution chemistry differences between samples, complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay

SUBMITTER: Seipp MT 

PROVIDER: S-EPMC1899416 | biostudies-literature | 2007 Jul

REPOSITORIES: biostudies-literature