Padlock probe-mediated qRT-PCR for DNA computing answer determination

Fusheng Xiong, Wayne Frasch

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Padlock probe-mediated quantitative real time PCR (PLP-qRT-PCR) was adapted to quantify the abundance of sequential 10mer DNA sequences for use in DNA computing to identify optimal answers of traveling salesman problems. The protocol involves: (i) hybridization of a linear PLP with a target DNA sequence; (ii) PLP circularization through enzymatic ligation; and (iii) qRT-PCR amplification of the circularized PLP after removal of non-circularized templates. The linear PLP was designed to consist of two 10-mer sequence-detection arms at the 5′ and 3′ ends separated by a core sequence composed of universal PCR primers, and a qRT-PCR reporter binding site. Circularization of each PLP molecule is dependent upon hybridization with target sequence and high-fidelity ligation. Thus, the number of PLP circularized is determined by the abundance of target in solution. The amplification efficiency of the PLP was 98.7% within a 0.2 pg-20 ng linear detection range between thermal cycle threshold (C t value) and target content. The C t values derived from multiplex qRT-PCR upon three targets did not differ significantly from those obtained with singleplex assays. The protocol provides a highly sensitive and efficient means for the simultaneous quantification of multiple short nucleic acid sequences that has a wide range of applications in biotechnology.

Original languageEnglish (US)
Pages (from-to)947-959
Number of pages13
JournalNatural Computing
Volume10
Issue number2
DOIs
StatePublished - Jun 2011

Keywords

  • DNA computing
  • Ordered node pair abundance
  • Padlock probe
  • Quantitative real-time PCR
  • TaqMan chemistry
  • Traveling salesman problem

ASJC Scopus subject areas

  • Computer Science Applications

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