caa a22 4500 445843101 CHVBK 20180317145350.0 cr unu---uuuuu 170323e20110601xx s 000 0 eng 10.1007/s11047-010-9227-8 doi (NATIONALLICENCE)springer-10.1007/s11047-010-9227-8 Padlock probe-mediated qRT-PCR for DNA computing answer determination [Elektronische Daten] [Fusheng Xiong, Wayne Frasch] 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 0.2pg-20ng 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. Springer Science+Business Media B.V., 2010 DNA computing nationallicence Padlock probe nationallicence Quantitative real-time PCR nationallicence TaqMan chemistry nationallicence Ordered node pair abundance nationallicence Traveling salesman problem nationallicence Xiong Fusheng Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, P.O. Box 874501, 85287-4501, Tempe, AZ, USA aut Frasch Wayne Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, P.O. Box 874501, 85287-4501, Tempe, AZ, USA aut Natural Computing Springer Netherlands 10/2(2011-06-01), 947-959 1567-7818 10:2<947 2011 10 11047 https://doi.org/10.1007/s11047-010-9227-8 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11047-010-9227-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Xiong Fusheng Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, P.O. Box 874501, 85287-4501, Tempe, AZ, USA aut NATIONALLICENCE 700 1- Frasch Wayne Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, P.O. Box 874501, 85287-4501, Tempe, AZ, USA aut NATIONALLICENCE 773 0- Natural Computing Springer Netherlands 10/2(2011-06-01), 947-959 1567-7818 10:2<947 2011 10 11047 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer