title: T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids.
abstract: Body fluid DNA sequencing is a powerful noninvasive approach for the diagnosis of genetic defects, infectious agents and diseases. The success relies on the quantity and quality of the DNA samples. However, numerous clinical samples are either at low quantity or of poor quality due to various reasons. To overcome these problems, we have developed T oligo-primed polymerase chain reaction (TOP-PCR) for full-length nonselective amplification of minute quantity of DNA fragments. TOP-PCR adopts homogeneous "half adaptor" (HA), generated by annealing P oligo (carrying a phosphate group at the 5' end) and T oligo (carrying a T-tail at the 3' end), for efficient ligation to target DNA and subsequent PCR amplification primed by the T oligo alone. Using DNA samples from body fluids, we demonstrate that TOP-PCR recovers minute DNA fragments and maintains the DNA size profile, while enhancing the major molecular populations. Our results also showed that TOP-PCR is a superior method for detecting apoptosis and outperforms the method adopted by Illumina for DNA amplification.
authors.affiliation: Department of Biotechnology and Animal Science, National Ilan University, Yilan, Taiwan.
authors.forename: Yu-Shin
authors.initials: YS
authors.lastname: Nai
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors.forename: Tzu-Han
authors.initials: TH
authors.lastname: Chen
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors.forename: Yu-Feng
authors.initials: YF
authors.lastname: Huang
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei, Taiwan.|Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Mohit K
authors.initials: MK
authors.lastname: Midha
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors.forename: Hsin-Chieh
authors.initials: HC
authors.lastname: Shiau
authors.affiliation: Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
authors.forename: Chen-Yang
authors.initials: CY
authors.lastname: Shen
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei, Taiwan.
authors.forename: Chien-Jen
authors.initials: CJ
authors.lastname: Chen
authors.affiliation: Institute of Stem Cell and Translational Cancer Research, Chang Gung Memorial Hospital at Linkou and Chang Gung University, No. 5, Fu-Shin St., Kuei Shang, Taoyuan 333, Taiwan.|Department of Pediatrics, University of California in San Diego, CA USA.
authors.forename: Alice L
authors.initials: AL
authors.lastname: Yu
authors.affiliation: Genomics Research Center, Academia Sinica, Taipei, Taiwan.|Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.|Department of Life Sciences, College of Life Sciences, National Taiwan University, Taipei, Taiwan.
authors.forename: Kuo Ping
authors.initials: KP
authors.lastname: Chiu
mesh_terms: D001826:Body Fluids; D000073888:Cell-Free Nucleic Acids; D017931:DNA Primers; D006801:Humans; D011071:Poly T; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000073888:Cell-Free Nucleic Acids; D017931:DNA Primers; D011071:Poly T