Padlock probe-mediated qRT-PCR for DNA computing answer determination
Source: Natural Computing, Volume 10, Number 2, June 2011 , pp. 947-959(13)
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 (Ct value) and target content. The Ct 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.
Document Type: Research Article
Affiliations: 1: Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, P.O. Box 874501, Tempe, AZ, 85287-4501, USA 2: Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, P.O. Box 874501, Tempe, AZ, 85287-4501, USA, Email: email@example.com
Publication date: June 1, 2011