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

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.

From Self-Organized to Extended Criticality

“…We notice that the role of the neuron cooperation is also recognized by Soares et al ( 2011 ) who propose the creation of coupled networks to make a significant progress toward the creation of intelligent systems.…”

Accelerating DNA Computing via PLP-qPCR Answer Read out to Solve Traveling Salesman Problems

“…Arcs were made to complement every combination of nodes with the exception of the respective 5 0 and 3 0 ends of A start and A end . Oligo sequences ( Table 2) designed for the PLP-mediated qPCR assay followed the protocol of Xiong and Frasch [23]. All oligonucleotides were purified by PAGE under denaturing conditions [12].…”

“…The core sequence contained the forward (P f 19-mer) and reverse (P r 21-mer) PCR primerbinding sequences, and a qPCR reporter-identifying sequence known as a TaqMan spacer for use with the TaqMan-MGB ® (Applied Biosystems) NED reporter dye (λ max = 580 nm). The PLPs specific for each ONP in the answer sequences were made from core and arm components by ligation as per Xiong and Frasch [23].…”

“…Preparation included the steps summarized in Figure 5 for the BC-PLP that was used to detect the ONP in which the sequence encoding node B immediately precedes that of node C in a tour. For the BC-ONP, the 5 0 -arm of the PLP complementary to the last 10 bases of sequence B (designated B 0 ), and the 3 0 -arm (designated C″) that were complementary to the first 10 bases of sequence C were created [23]. The hybridized PLP was circularized by ligase, after which the PLP was denatured from the target strand and exonuclease was added to eliminate any DNA that had not been circularized.…”

Novel Trends in the Traveling Salesman Problem

“…Together they are called exchange or local improvement heuristics. The exchange is considered to be a single move, from this point of view, such heuristics search the neighborhood of the current solution, that is, perform a local search and provide a locally optimal solution (k-optimal) to the problem [23].…”