Detection of single-nucleotide polymorphisms in the p53 gene by LDR/RCA in hydrogel microarrays

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.