Biomedical Engineering Reference
In-Depth Information
7
Analysis of Polymerase Chain Reaction Products
by Denaturing High-Performance Liquid Chromatography
Ching-Wan Lam
Summary
Denaturing high-performance liquid chromatography (DHPLC) analysis is an ion-
pair reversed-phase high performance liquid chromatography for performing analytical
separations of DNA based on temperature: analysis is performed at a temperature suffi-
cient to partially denature DNA heteroduplexes. The technology detects single-base
changes as efficiently as short deletions and insertions. The chance that a mutation can-
not be detected is 0.5%.
Key Words:
Denaturing HPLC; mutation analysis; heteroduplex; automation.
1. Introduction
Denaturing high-performance liquid chromatography (DHPLC) analysis is
an ion-pair reversed-phase high performance liquid chromatography for per-
forming analytical separations of DNA based on temperature: analysis is
performed at a temperature sufficient to partially denature (melt) DNA hetero-
duplexes
(
1
)
. The melted heteroduplexes are resolved from the corresponding
homoduplexes by ion-pair reversed-phase high performance liquid chroma-
tography. The procedure is referred as temperature-modulated heteroduplex
chromatography (TMHC). THMC relies on the physical changes in DNA mol-
ecules induced by mismatched heteroduplex formation. Heteroduplex DNA is
generated by denaturing and re-annealing a mixed population of reference or
“wild-type” sample and “mutant” DNA (
Fig. 1
). The heteroduplex DNA frag-
ments form as a result of the base-pairing of a single-stranded, mutated DNA
with a single-stranded, “wild-type” DNA. The two strands will not form
hydrogen bonds at the mutation site because the basepairs are mismatched,
thus giving the heteroduplex different melting properties than the homoduplex.
At a critical temperature, heterduplexes will begin to “melt” or denature, mak-
