title: High specific genotyping method using short target probe and helper probe.
abstract: Differentiating 1-bp differences using real-time PCR often leads to false-positive results. Therefore, we developed a fluorescence melting curve analysis (FMCA) method with a short target probe and helper probe labeled with a fluorophore and quencher, respectively. This fluorophore and quencher were designed to be near each other when the probes were hybridized to template DNA. The target probe was designed with a shorter length to facilitate a dramatic shift in melting temperature (Tm) upon encountering mismatched hybridization. In FMCA, when the temperature approached the target probe Tm, the target probe would begin to denature from the template DNA, and at the target probe Tm, the fluorescence signal increased markedly. Here, we examined 1-bp differences using the developed method with mitochondrial DNA from Larimichthys polyactis and Larimichthys crocea. Application of this method permitted specific genotype identification for all cases with no cross-reactivity, even when both templates were added to the same tube.
authors.affiliation: Department of Bio-Nanotechnology, Hanyang University, Sangnok-gu, Ansan, Gyeonggi-do, South Korea.
authors.forename: Jeong Jin
authors.initials: JJ
authors.lastname: Ahn
authors.affiliation: Department of Bio-Nanotechnology, Hanyang University, Sangnok-gu, Ansan, Gyeonggi-do, South Korea.
authors.forename: Ha Jeong
authors.initials: HJ
authors.lastname: Song
authors.affiliation: Department of Bio-Nanotechnology, Hanyang University, Sangnok-gu, Ansan, Gyeonggi-do, South Korea.
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Hong
authors.affiliation: Department of Molecular and Life Science, Hanyang University, Sangnok-gu, Ansan, Gyeonggi-do, South Korea.
authors.forename: Gi Won
authors.initials: GW
authors.lastname: Kim
authors.affiliation: Department of Bio-Nanotechnology, Hanyang University, Sangnok-gu, Ansan, Gyeonggi-do, South Korea; Department of Molecular and Life Science, Hanyang University, Sangnok-gu, Ansan, Gyeonggi-do, South Korea. Electronic address: syhwang@hanyang.ac.kr.
authors.forename: Seung Yong
authors.initials: SY
authors.lastname: Hwang
mesh_terms: D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D015342:DNA Probes / Q000378:metabolism*; D005453:Fluorescence; D060005:Genotyping Techniques / Q000379:methods*; D009691:Nucleic Acid Denaturation; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D015342:DNA Probes
keywords: DNA melting temperature; Fluorescence melting curve analysis; Fluorescence resonance energy transfer; Genotyping; Real-time polymerase chain reaction