Flexible use of high-density oligonucleotide arrays for single-nucleotide polymorphism discovery and validation

Genome Res. 2001 Aug;11(8):1418-24. doi: 10.1101/gr.171101.

Abstract

A method for identifying and validating single nucleotide polymorphisms (SNPs) with high-density oligonucleotide arrays without the need for locus-specific polymerase chain reactions (PCR) is described in this report. Genomic DNAs were divided into subsets with complexity of ~10 Mb by restriction enzyme digestion and gel-based fragment size resolution, ligated to a common adaptor, and amplified with one primer in a single PCR reaction. As a demonstration of this approach, a total of 124 SNPs were located in 190 kb of genomic sequences distributed across the entire human genome by hybridizing to high-density variant detection arrays (VDA). A set of independent validation experiments was conducted for these SNPs employing bead-based affinity selection followed by hybridization of the affinity-selected SNP-containing fragments to the same VDA that was used to identify the SNPs. A total of 98.7% (74/75) of these SNPs were confirmed using both DNA dideoxynucleotide sequencing and the VDA methodologies. With flexible sample preparation, high-density oligonucleotide arrays can be tailored for even larger scale genome-wide SNP discovery as well as validation.

Publication types

  • Comparative Study

MeSH terms

  • Cell Line
  • DNA / genetics
  • DNA / isolation & purification
  • Female
  • Gene Expression Profiling / methods
  • Genome, Human*
  • Humans
  • Male
  • Oligonucleotide Array Sequence Analysis / methods*
  • Oligonucleotides / genetics
  • Oligonucleotides / isolation & purification
  • Polymorphism, Single Nucleotide / genetics*
  • Reproducibility of Results
  • Sequence Analysis, DNA / methods

Substances

  • Oligonucleotides
  • DNA