title: Highly sensitive and selective colorimetric genotyping of single-nucleotide polymorphisms based on enzyme-amplified ligation on magnetic beads.
abstract: Herein we report a new strategy for highly sensitive and selective colorimatric assay for genotyping of single-nucleotide polymorphisms (SNPs). It is based on the use of a specific gap ligation reaction, horseradish peroxidase (HRP) for signal amplification, and magnetic beads for the easy separation of the ligated product. Briefly, oligonucleotide capture probe functionalized magnetic beads are first hybridized to a target DNA. Biotinylated oligonucleotide detection probes are then allowed to hybridize to the already captured target DNA. A subsequent ligation at the mutation point joins the two probes together. The introduction of streptavidin-conjugated HRP and a simple magnetic separation allow colorimetric genotyping of SNPs. The assay is able to discriminate one copy of mutant in 1000 copies of wild-type KRAS oncogene at 30 picomolar. The detection limit of the assay is further improved to 1 femtomolar by incorporating a ligation chain reaction amplification step, offering an excellent opportunity for the development of a simple and highly sensitive diagnostic tool.
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Chen
authors.forename: Andrew
authors.initials: A
authors.lastname: Ying
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Gao
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D001710:Biotin / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D059346:Magnets / Q000737:chemistry; D009154:Mutation; D015345:Oligonucleotide Probes / Q000737:chemistry; D020641:Polymorphism, Single Nucleotide; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000302:isolation & purification; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: C117307:KRAS protein, human; D015345:Oligonucleotide Probes; D011518:Proto-Oncogene Proteins; D001710:Biotin; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins