Whole genome amplification on DNA from filter paper blood spot samples: an evaluation of selected systems.
As the number of single-nucleotide polymorphism (SNP) screening and other mutation scanning studies have increased explosively, following the development of high-throughput instrumentation, it becomes even more important to have sufficient template DNA. The source of DNA is often limited, especially in epidemiological studies, which require many samples as well as enough DNA to perform numerous SNP screenings or mutation scannings. Therefore, the aim is to solve the problem of stock DNA limitation. This need has been an important reason for the development of whole genome amplification (WGA) methods. Several systems are based on Phi29 polymerase multiple displacement amplification (MDA) or on DNA fragmentation (OmniPlex). Using TaqMan SNP genotyping assays, we have tested four WGA systems -- AmpliQ Genomic Amplifier Kit, GenomiPhi, Repli-g, and GenomePlex -- on DNA extracted from Guthrie cards to evaluate the amplification bias, concordance- and call rates, cost efficiency, and flexibility. All systems successfully amplified picograms of DNA from Guthrie cards to micrograms of product without loss of heterozygosity and with minimal allelic bias. A modified AmpliQ set up was chosen for further evaluation. In all, 2,000 SNP genotyping results from amplified and nonamplified samples were compared and the concordance rates between the samples were 99.7%. The call rate using the TaqMan system was 99.8%. DNA extracted from Guthrie cards and amplified with one of the four evaluated WGA systems is applicable in epidemiological genetic screenings. System choice should be based on requirements for system flexibility, product yield, and use in subsequent analysis.
Sørensen
Karina Meden
KM
Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen S, Denmark. kms@ssi.dk
Jespersgaard
Cathrine
C
Vuust
Jens
J
Hougaard
David
D
Nørgaard-Pedersen
Bent
B
Andersen
Paal Skytt
PS
D000483:Alleles; D001769:Blood; D004247:DNA / Q000235:genetics*; D016678:Genome; D006801:Humans; D010209:Paper; D016133:Polymerase Chain Reaction / Q000379:methods*
D004247:DNA