Abstract
General DNA analysis includes detection of targets, as well as identification and quantification of specific variants. Genomic DNA exists in the form of homoduplexes, with all corresponding base pairs being complementary, A:T and C:G. We call a double-stranded DNA (dsDNA) molecule a heteroduplex when it contains any noncomplementary base pairs. Conformational and thermodynamic changes produced by mismatched bases facilitate the detection of heteroduplexes. Denaturation of samples followed by hybridization to promote heteroduplex formation has been used to screen diploid DNA for heterozygous variations. This form of heteroduplex analysis has focused on detection, and although it may involve a quantitative threshold, the result is essentially binary. Recent work in this area (1) and elsewhere(2) has explored new implementations and applications for a more truly quantitative form of heteroduplex analysis. One setting in which heteroduplexes commonly arise is during PCR. Although the extension process of PCR replicates genomic homoduplexes with high accuracy, if the original sample is heterozygous, heteroduplexes arise during the plateau stage when dsDNA is more likely to be formed by hybridization than extension. Denaturation and hybridization associates complementary and near-complementary strands almost randomly. Any of various methods (listed below) that can detect the presence of heteroduplexes can then be used to detect heterozygous sequence variations. If heteroduplexes are not detected, variations, if any, are known to be homozygous. Homozygous variants may sometimes be distinguished by differences in melting temperature, but these differences may be near the limit of detection even with high-resolution techniques. Indeed, 4% of human single nucleotide polymorphisms (SNPs) exhibit a symmetry that makes homozygous variants thermodynamically identical up to the nearest-neighbor approximation (3). In this case, a 2-stage mixing strategy allows heteroduplex analysis to be used instead of melting temperature analysis for the detection …
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.