Detection of Single-Nucleotide Polymorphisms in the p53 Gene by LDR/RCA in Hydrogel Microarrays

摘要

To find single-nucleotide polymorphisms (SNPs) in the human genome,three modern technologies of molecular genetic analysis were combined:the ligase detection reaction (LDR),rolling circle amplification (RCA),and immobilized microarray of gel elements (IMAGE).SNPs were detected in target DNA by selective ligation of allele-specific nucleotides in microarrays.The ligation product was assayed in microarray gel pads by RCA.Two variants of microarray analysis were compared.One included selective ligation of short oligonu-cleotides immobilized in a microarray with subsequent amplification with a preformed circular probe (a common circle).The probe was especially designed for human genome research.The other variant employed immobilized allele-specific padlock probes,which could be circularized as a result of selective ligation.Codon 72 SNP of the human p53 gene was used as a model.RCA in microarrays proved to be a quantitative assay and,in combination with LDR,allowed efficient discrimination of alleles.The principles and prospects of LDR/RCA in microarrays are discussed.