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Denaturing High-Performance Liquid Chromatography
Denaturing high-performance liquid chromatography (DHPLC) is a PCR-based
method that uses liquid chromatography as an alternative to gel electrophoresis.
DHPLC is traditionally used to detect variants in PCR products containing both
allelic forms of a polymorphism (e.g. heterozygotes or a 1:1 mix of both alleles) via
heteroduplex separation and thereby requires separate analyses of multiple indi-
vidual test samples. DHPLC can be used to estimate absolute allele frequencies of
SNPs in pooled DNA samples and also of the difference in allele frequency between
different pooled DNA samples. This technique therefore offers an effi cient and
cheap method for genotyping SNPs in large case-control and family-based associa-
tion samples.
Multiplex Allele-Specifi c Diagnostic Assay
Multiplex Allele-Specifi c Diagnostic Assay (MASDA) employs a two-step approach
to mutation detection. The fi rst step is a DNA probe-based hybridization procedure
in which DNA fragments from a patient sample are screened for the presence of
specifi c mutations. In the second step, the hybridized probe is released from the
patient’s DNA sample, digested into fragments and identifi ed by its unique banding
pattern on gel electrophoresis. Identifi cation of the probe reveals the mutation pres-
ent in the sample DNA. MASDA has several applications in research as well as
clinical laboratories. MASDA is also an important part of genetic profi ling for phar-
maceutical clinical trials.
Representational Oligonucleotide Microarray Analysis
The power of representational oligonucleotide microarray analysis (ROMA) method
is based on two fundamental realities: fi rst, that the whole genome can be simplifi ed
into reproducible fragments sampled throughout the genome; second, that dense
oligonucleotide arrays can quantify the amount of these fragments from different
DNA sources. ROMA is ideally suited for the analysis of gene amplifi cation and
deletions in cancers. It is also an ideal technology to uncover genetic polymor-
phisms in normal populations represented by deletions and duplications.
Restriction Fragment Length Polymorphism
Amplifi cation of a specifi c region of the gene of interest by the PCR followed by
digestion of amplifi ed DNA products with restriction endonucleases is useful in
screening for genetic mutations associated with altered metabolism of drugs or can-
cer susceptibility. Sizes of the digestion products from the study subjects are com-
pared with those generated from a DNA substrate amplifi ed from control subjects.
2 Molecular Diagnostics in Personalized Medicine