Single-nucleotide-polymorphism genotyping for whole-genome-amplified samples using automated fluorescence correlation spectroscopy

Anal Biochem. 2004 Apr 15;327(2):215-21. doi: 10.1016/j.ab.2004.01.012.

Abstract

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.

MeSH terms

  • Genome, Human
  • Genotype
  • Humans
  • Polymerase Chain Reaction
  • Polymorphism, Single Nucleotide*
  • Receptors, Tumor Necrosis Factor, Type I / genetics
  • Receptors, Tumor Necrosis Factor, Type II / genetics
  • Sequence Analysis, DNA / methods*
  • Spectrometry, Fluorescence / methods*

Substances

  • Receptors, Tumor Necrosis Factor, Type I
  • Receptors, Tumor Necrosis Factor, Type II