High-throughput genotyping of single nucleotide polymorphisms with high sensitivity.
The ability to analyze a large number of genetic markers consisting of single nucleotide polymorphisms (SNPs) may bring about significant advance in understanding human biology. Recent development of several high-throughput genotyping approaches has significantly facilitated large-scale SNP analysis. However, because of their relatively low sensitivity, application of these approaches, especially in studies involving a small amount of material, has been limited. In this chapter, detailed experimental procedures for a high-throughput and highly sensitive genotyping system are described. The system involves using computer program selected primers that are expected not to generate a significant amount of nonspecific products during PCR amplification. After PCR, a small aliquot of the PCR product is used as templates to generate single-stranded DNA (ssDNA). ssDNA sequences from different SNP loci are then resolved by hybridizing these sequences to the probes arrayed onto glass surface. The probes are designed in such a way that hybridizing to the ssDNA templates places their 3'-ends next to the polymorphic sites. Therefore, the probes can be labeled in an allele-specific way using fluorescently labeled dye terminators. The allelic states of the SNPs can then be determined by analyzing the amounts of different fluorescent colors incorporated to the corresponding probes. The genotyping system is highly accurate and capable of analyzing >1000 SNPs in individual haploid cells.
Li
Honghua
H
Wang
Hui-Yun
HY
Cui
Xiangfeng
X
Luo
Minjie
M
Hu
Guohong
G
Greenawalt
Danielle M
DM
Tereshchenko
Irina V
IV
Li
James Y
JY
Chu
Yi
Y
Gao
Richeng
R
D017931:DNA Primers; D015342:DNA Probes; D005838:Genotype; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015342:DNA Probes