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T Oligo-Primed Polymerase Chain Reaction (TOP-PCR): A Robust Method for the Amplification of Minute DNA Fragments in Body Fluids.

  • Author(s): Nai, Yu-Shin
  • Chen, Tzu-Han
  • Huang, Yu-Feng
  • Midha, Mohit K
  • Shiau, Hsin-Chieh
  • Shen, Chen-Yang
  • Chen, Chien-Jen
  • Yu, Alice L
  • Chiu, Kuo Ping
  • et al.

Published Web Location

https://doi.org/10.1038/srep40767
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.

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