Application of the ASLP technology to a novel platform for rapid and noise-free multiplexed SNP genotyping

作者：Shin Sung Chul; Kim Gahee; Yang Hee Bum; Park Kwan Woo; Kang Byoung Cheorl; Park Hyun Gyu^*

来源：Biosensors and Bioelectronics, 2014, 54: 687-694.

DOI：10.1016/j.bios.2013.10.071

• Amplification of separated ligation-dependent probe
• ASLP
• RecA-assisted hybridization
• Multiplexed SNP genotyping
• Universal amplification

A novel multiplexing method, which relies on universal amplification of separated ligation-dependent probes (ASLP), has been developed to genotype single-nucleotide polymorphisms (SNPs). The ASLP technique employs two allele-specific oligonucleotides (ASO), modified with universal forward primer sequences at the 5'-end and a common locus-specific oligonucleotide (LSO) extended with a universal separation (US) sequence at the 3'-end. In the process, allele-specific ligation first takes place when target genomic DNA is hybridized by perfectly matching the ASO together with the LSO. A separation probe, which consists of a universal reverse primer sequence labeled with biotin at the 5'-end and complementary sequence of US at the 3'-end, is then applied to the resulting ligation product. During the extension reaction of the separation probe, the ligated probes dissociate from target genomic DNA in the form of a double-stranded DNA and are separated from the reaction mixture, which includes genomic DNA and unligated probes, by simply using streptavidin-coated magnetic beads. PCR amplification of the separated ligation products is then carried out by using universal primers and the PCR products are hybridized on a DNA microarray using the RecA protein. The advantageous features of the new method were demonstrated by using it to genotype 15 SNP markers for cultivar identification of pepper in a convenient and correct manner.

• 出版日期2014-4-15