title: PCR-free digital minisatellite tandem repeat genotyping.
abstract: We demonstrated a proof-of-concept for novel minisatellite tandem repeat typing, called PCR-free digital VNTR (variable number tandem repeat) typing, which is composed of three steps: a ligation reaction instead of PCR thermal cycling, magnetic bead-based solid-phase capture for purification, and an elongated sample stacking microcapillary electrophoresis (μCE) for sensitive digital coding of repeat number. We designed a 16-bp fluorescently labeled ligation probe which is complementary to a repeat unit of a biotinylated synthetic template mimicking the human D1S80 VNTR locus and is randomly hybridized with the minisatellite tandem repeats. A quick isothermal ligation reaction was followed to link the adjacent ligation probes on the DNA templates, and then the ligated products were purified by streptavidin-coated magnetic beads. After a denaturing step, a large amount of ligated products whose size difference was equivalent to the repeat unit were released and recovered. Through the elongated sample stacking μCE separation on a microdevice, the fluorescence signal of the ligated products was generated in the electropherogram and the peak number was directly counted which was exactly matched with the repeat number of VNTR locus. We could successfully identify the minisatellite tandem repeat number with only 5 fmol of DNA template in 30 min.
authors.affiliation: Department of Chemical and Biomolecular Engineering (BK21 program) and Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
authors.forename: Yuchao
authors.initials: Y
authors.lastname: Chen
authors.forename: Tae Seok
authors.initials: TS
authors.lastname: Seo
mesh_terms: D015342:DNA Probes / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000295:instrumentation* / Q000379:methods*; D005838:Genotype; D006801:Humans; D046210:Microfluidic Analytical Techniques; D008904:Miniaturization; D018598:Minisatellite Repeats; D009693:Nucleic Acid Hybridization / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes