Parallel single nucleotide polymorphism genotyping by surface invasive cleavage with universal detection.
Large-scale investigations of sequence variation within the human species will provide information about the basis of heritable variation in disease susceptibility and human migration. The surface invader assay (an adaptation of the invasive cleavage reaction to an array format) is capable of exquisitely sensitive and specific detection of genetic variation. It is shown here that this genotyping technology can be multiplexed in a DNA array format, permitting the parallel analysis of a panel of single nucleotide polymorphisms (SNPs) directly from an unamplified genomic DNA target. In addition, a "universal" mode of detection was developed that makes use of a mixture of degenerate templates for DNA ligation to the surface-bound cleaved oligonucleotides and thereby makes this strategy amenable to any desired SNP site or combination of SNP sites, without regard to their particular DNA sequences. This approach was demonstrated on a proof-of-principle scale using small DNA arrays to genotype 6 SNP markers in the PTPN1 gene and 10 mutations in the cystic fibrosis transmembrane conductance regulator gene. This ability to analyze many different genetic variations in parallel, directly from unamplified human genomic DNA samples, lays the groundwork for the development of high-density arrays able to analyze hundreds of thousands or even millions of SNPs.
Chen
Yan
Y
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706, USA.
Shortreed
Michael R
MR
Olivier
Michael
M
Smith
Lloyd M
LM
D001483:Base Sequence; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000235:genetics
C505032:CFTR protein, human; D017931:DNA Primers; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases