Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification.
An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM-ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n+1 primers are used for an n-plex PCR amplification. The specificity of ALM-ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM-ASA. The results for genotyping 70 different genome samples by the 17-plex ALM-ASA were completely consistent with those obtained by both Sanger's sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) analysis. ALM-ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM-ASA with a well-developed automation device for sample processing.
Wang
Wei-peng
WP
Huadong Research Institute for Medicine and Biotechnics, Nanjing 210002, People's Republic of China.
Ni
Kun-yi
KY
Zhou
Guo-hua
GH
D000483:Alleles; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016678:Genome / Q000235:genetics / Q000502:physiology; D005838:Genotype; D006801:Humans; D008099:Liver / Q000201:enzymology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013997:Time Factors
D019389:Cytochrome P-450 CYP2D6; D011088:DNA Ligases