Genome complexity reduction for SNP genotyping analysis

Efficient single nucleotide polymorphism (SNP) genotyping methods are necessary to accomplish many current gene discovery goals. A crucial element in large-scale SNP genotyping is the number of individual biochemical reactions that must be performed. An efficient method that can be used to simultaneously amplify a set of genetic loci across a genome with high reliability can provide a valuable tool for large-scale SNP genotyping studies. In this paper we describe and characterize a method that addresses this goal. We have developed a strategy for reducing genome complexity by using degenerate oligonucleotide primer (DOP)-PCR and applied this strategy to SNP genotyping in three complex eukaryotic genomes; human, mouse, and Arabidopsis thaliana. Using a single DOP-PCR primer, SNP loci spread throughout a genome can be amplified and accurately genotyped directly from a DOP-PCR product mixture. DOP-PCRs are extremely reproducible. The DOP-PCR method is transferable to many species of interest. Finally, we describe an in silico approach that can effectively predict the SNP loci amplified in a given DOP-PCR, permitting the design of an efficient set of reactions for large-scale, genome-wide SNP studies.

[1]  M. Weiner,et al.  A microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension. , 2000, Genome research.

[2]  C. Nusbaum,et al.  Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. , 1998, Science.

[3]  N. Carter,et al.  Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. , 1992, Genomics.

[4]  G Bernardi,et al.  The compositional evolution of vertebrate genomes. , 2000, Gene.

[5]  N Risch,et al.  The Future of Genetic Studies of Complex Human Diseases , 1996, Science.

[6]  S. P. Fodor,et al.  Blocks of Limited Haplotype Diversity Revealed by High-Resolution Scanning of Human Chromosome 21 , 2001, Science.

[7]  P. Kwok,et al.  Methods for genotyping single nucleotide polymorphisms. , 2003, Annual review of genomics and human genetics.

[8]  J. T. Eppig,et al.  Maps from two interspecific backcross DNA panels available as a community genetic mapping resource , 1994, Mammalian Genome.

[9]  L. Kruglyak.  Prospects for whole-genome linkage disequilibrium mapping of common disease genes , 1999, Nature Genetics.

[10]  J. V. Moran,et al.  Initial sequencing and analysis of the human genome. , 2001, Nature.

[11]  C. Lister,et al.  Recombinant inbred lines for mapping RFLP and phenotypic markers in Arabidopsis thaliana , 1993 .

[12]  K. Lindblad-Toh,et al.  SBE-TAGS: an array-based method for efficient single-nucleotide polymorphism genotyping. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[13]  G. Bernardi,et al.  The compositional organization and the expression of the Arabidopsis genome , 2000, FEBS letters.

[14]  V G Cheung,et al.  Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[15]  A. Nekrutenko,et al.  Assessment of compositional heterogeneity within and between eukaryotic genomes. , 2000, Genome research.

[16]  D. Nickerson,et al.  Variation is the spice of life , 2001, Nature Genetics.

[17]  The Arabidopsis Genome Initiative.  Analysis of the genome sequence of the flowering plant Arabidopsis thaliana , 2000, Nature.

[18]  Lee M. Silver,et al.  Mouse Genetics: Concepts and Applications , 1995 .

[19]  A. Metspalu,et al.  Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology. , 2000, Genetic testing.