Single-nucleotide-polymorphism genotyping for whole-genome-amplified samples using automated fluorescence correlation spectroscopy.
Whole-genome amplification (WGA) methods were adopted for single-nucleotide-polymorphism (SNP) typing to minimize the amount of genomic DNA that has to be used in typing for thousands of different SNPs in large-scale studies; 5-10 ng of genomic DNA was amplified by a WGA method (improved primer-extension-preamplification-polymerase chain reaction (I-PEP-PCR), degenerated oligonucleotide primer-PCR (DOP-PCR), or multiple displacement amplification (MDA)). Using 1/100 to 1/500 amounts of the whole-genome-amplified products as templates, subsequent analyses were successfully performed. SNPs were genotyped by the sequence-specific primer (SSP)-PCR method followed by fluorescence correlation spectroscopy (FCS). The typing results were evaluated for four different SNPs on tumor necrosis factor receptor 1 and 2 genes (TNFR1 and TNFR2). The genotypes determined by the SSP-FCS method using the WGA products were 100% in concordance with those determined by nucleotide sequencing using genomic DNAs. We have already carried out typing of more than 300 different SNPs and are currently performing 7,500-10,000 typings per day using WGA samples from patients with several common diseases. WGA coupled with FCS allows specific and high-throughput genotyping of thousands of samples for thousands of different SNPs.
Bannai
Makoto
M
Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. bannai@m.u-tokyo.ac.jp
Higuchi
Kaori
K
Akesaka
Tamao
T
Furukawa
Masako
M
Yamaoka
Megumi
M
Sato
Kazuhisa
K
Tokunaga
Katsushi
K
D015894:Genome, Human; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D047888:Receptors, Tumor Necrosis Factor, Type I; D047889:Receptors, Tumor Necrosis Factor, Type II