Assessment of multiple displacement amplification in molecular epidemiology.
Well-characterized epidemiological resources are generated with great effort, yet associated patient DNA samples can be limiting. The efficacy of the whole genome amplification (WGA) method, termed multiple displacement amplification (MDA), was assessed for detecting heterozygous sequence variants, mutation scanning, and PCR for challenging segments. Fifteen common polymorphisms from 10 genes located on 8 chromosomes were genotyped by direct sequencing of 300 PCR products from 115 high-quality MDA-amplified DNA samples extracted by different methods. The GC content of these analyzed segments ranges from 30% to 69%. Genotyping results demonstrate 100% accuracy. For heterozygotes, the relative intensity of peaks generated by the two alleles is highly similar for genomic and MDA-amplified genomic DNA, independent of GC content. In contrast, one of four heterozygous loci was mistyped when lower quality MDA-amplified DNA samples were used. The results of single-stranded conformation polymorphism (SSCP)-type of mutation scanningfor seven MDA-amplified DNA samples in four genes were concordant with the genomic DNA samples. PCR on MDA-amplified DNA was routinely successful for challenging 10- and 12-kb segments with GC content ranging from 30% to 80%, demonstrating that rather long segments, which are difficult to amplify with PCR, are amplified well with MDA. These results suggest that MDA is an effective method of WGA with utility in molecular epidemiology. Quality control of the MDA-amplified DNA is critical for high performance.
Yan
Jin
J
City of Hope National Medical Center, Duarte, CA 91010-3000, USA.
Feng
Jinong
J
Hosono
Seiyu
S
Sommer
Steve S
SS
D001483:Base Sequence; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005169:Factor VIII / Q000235:genetics; D005784:Gene Amplification; D017720:Molecular Epidemiology; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D011786:Quality Control
D017931:DNA Primers; D005169:Factor VIII