title: Application of the ASLP technology to a novel platform for rapid and noise-free multiplexed SNP genotyping.
abstract: 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.
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21Program), KAIST, 291, Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Sung Chul
authors.initials: SC
authors.lastname: Shin
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21Program), KAIST, 291, Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Gahee
authors.initials: G
authors.lastname: Kim
authors.affiliation: Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea.
authors.forename: Hee-Bum
authors.initials: HB
authors.lastname: Yang
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21Program), KAIST, 291, Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Kwan Woo
authors.initials: KW
authors.lastname: Park
authors.affiliation: Department of Plant Science, Plant Genomics and Breeding Institute, and Research Institute for Agriculture and Life Sciences, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea.
authors.forename: Byoung-Cheorl
authors.initials: BC
authors.lastname: Kang
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21Program), KAIST, 291, Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: hgpark@kaist.ac.kr.
authors.forename: Hyun Gyu
authors.initials: HG
authors.lastname: Park
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D002212:Capsicum / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D005838:Genotype; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide
chemical_list: D018744:DNA, Plant
keywords: ASLP; Amplification of separated ligation-dependent probe; Multiplexed SNP genotyping; RecA-assisted hybridization; Universal amplification