Padlock probe-mediated qRT-PCR for DNA computing answer determination |
| |
Authors: | Fusheng Xiong Wayne D Frasch |
| |
Affiliation: | (1) Faculty of Biomedicine and Biotechnology, School of Life Sciences, Arizona State University, P.O. Box 874501, Tempe, AZ 85287-4501, USA; |
| |
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. |
| |
Keywords: | |
本文献已被 PubMed SpringerLink 等数据库收录! |
|