Abstract
Denaturing high performance liquid chromatography (dHPLC) is a fast and reliable technique for the DNA variation screening (1,2).It can detect in minutes with close to 100% sensitivity and specificity single-base substitutions as well as small deletions and insertions in DNA fragments ranging from 80-1500 base pairs in size (3,4).In partially denaturing HPLC, typically 2–10 chromosomes are compared as a mixture of PCR products. Upon mixing, denaturing and reannealing of amplicons containing one or more mismatches, not only the original homoduplices are formed again but, simultaneously, the sense and anti-sense strands of either homoduplex form two heteroduplices. Heteroduplices denature more extensively at elevated column temperatures in the range of 48-67°C; they are retained less on the chromatographic separation matrix, allowing the separation of homo- and heteroduplex species by ion-pair reversed-phase HPLC (IP-RP-HPLC) (5). Characteristic peak patterns both for homozygous and heterozygous samples are obtained.
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Premstaller, A., Oefner, P.J. (2003). Denaturing High-Performance Liquid Chromatography. In: Kwok, PY. (eds) Single Nucleotide Polymorphisms. Methods in Molecular Biology™, vol 212. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-327-5:015
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DOI: https://doi.org/10.1385/1-59259-327-5:015
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-968-1
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