Genome complexity reduction for genome-wide single nucleotide polymorphism analysis

Millions of single nucleotide polymorphisms (SNPs) have been identified in the human genome, and more are cataloged every day. The challenge now is to use these SNPs to discover the genetic risk factors underlying common and complex diseases. Efficient, large-scale genotyping methods are one necessary component of this endeavor. Current SNP genotyping techniques all rely on an initial PCR amplification of each SNP locus. Individual or low-level multiplexed PCR reactions are sufficient for genotyping a few to a few hundred different SNPs, but genome-wide linkage and association studies in humans will require thousands to tens of thousands of different SNPs, each typed on a few thousand individuals. To efficiently reach this goal, PCR techniques capable of amplifying a few hundred loci per reaction are needed. To meet this need we investigated the use of PCR-based genome complexity reduction methods for SNP genotyping. We discovered that degenerate oligonucleotide primed PCR (DOP-PCR) is capable of amplifying a specific fraction of a genome in a highly reproducible manner. The genomic sequences amplified are determined by the oligonucleotide primer's nondegenerate, 8-12 nucleotide, 3' end sequence. The amplified complexity can be varied from one to over 10,000 loci by changing the DOP-PCR primer's length and specific sequence. We collected SNPs from a human DOP-PCR that amplifies roughly 600 loci, and demonstrated that about half of the SNPs tested could be genotyped directly from the DOP-PCR product mixture, using the allele specific oligonucleotide hybridization genotyping technique.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2002

Author dc:creator

• Jordan, Barbara M. (Barbara Marie), 1975-

Advisor dc:contributor.advisor

• David E. Housman.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/8319

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/8319