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.
Similar content being viewed by others
References
Adleman L (1994) Molecular computation of solutions to combinatorial problems. Science 266(5187):1021–1024
Burgos J, Ramirez C, Tenorio R, Sastre I, Bullido M (2002) Influence of reagents formulation on real-time PCR parameters. Mol Cell Probes 16(4):257–260
Bustin S, Benes V, Nolan T, Pfaffl M (2005) Quantitative real-time RT-PCR—a perspective. J Mol Endocrinol 34(3):597–601
Henco K, Harders I, Wiese U, Riesner D (1994) Temperature gradient gel electrophoresis (TGGE) for the detection of polymorphic DNA and RNA. Methods Mol Biol 31:211–228
Ibrahim Z, Rose J, Suyama A, Khalid M (2008) Experimental implementation and analysis of a DNA computing readout method based on real-time PCR with TaqMan probes. Nat Comput 7(2):277–286
Lee J, Shin S, Sirk J, Park T, Zhang B (2003) Temperature gradient-based DNA computing for graph problems with weighted edges. Lect Notes Comput Sci 2568:73–84
Lee J, Shin S, Park T, Zhang B (2004) Solving traveling salesman problems with DNA molecules encoding numerical values. Biosystems 78(1–3):39–47
Lievens B, Brouwer M, Vanachter A, Levesque C, Cammue B, Thomma B (2003) Design and development of a DNA array for rapid detection and identification of multiple tomato vascular wilt pathogens. FEMS Microbiol Lett 223(1):113–122
Lipton R (1995) DNA solution of hard computational problems. Science 268(5210):542–545
Nilsson M, Malmgren H, Samiotaki M, Kwiatkowski M, Chowdhary U (1994) Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265(5181):4–5
Riesner D, Steger G, Wiese U, Wulfert M, Heibey M, Henco K (1992) Temperature-gradient gel electrophoresis for the detection of polymorphic DNA and for quantitative polymerase chain reaction. Electrophoresis 13(9–10):632–636
Spetzler D, Xiong F, Frasch W (2008) Heuristic solution to a 10-city asymmetric traveling salesman problem using probabilistic DNA computing. Lect Notes Comput Sci 4848:152–160
Szemes M, Bonants P, Weerdt M, Baner J, Landegren U, Schoen C (2005) Diagnostic application of padlock probes–multiplex detection of plant pathogens using universal microarrays. Nucleic Acids Res 33(8):e70
Tanaka F, Kameda A, Yamamoto M, Ohuchi A (2005) Design of nucleic acid sequences for DNA computing based on a thermodynamic approach. Nucleic Acids Res 33(3):903–911
Valasek M, Repa J (2005) The power of real-time PCR. Adv Physiol Educ 29(3):151–159
Ward L, Bej A (2006) Detection of vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes. Appl Environ Microbiol 72(3):2031–2042
Xiong F, Spetzler D, Frasch W (2009) Solving the fully-connected 15-city TSP using probabilistic DNA computing. Integr Biol 1(3):275–280
Yamamoto M, Kameda A, Matsuura N, Shiba T, Kawazoe Y, Ohuchi A (2002) A separation method for DNA computing based on concentration control. New Gener Comput 20(3):256–261
Acknowledgment
This work was supported by grants from AFOSR (FA95500710219) to W.D.F.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Xiong, F., Frasch, W.D. Padlock probe-mediated qRT-PCR for DNA computing answer determination. Nat Comput 10, 947–959 (2011). https://doi.org/10.1007/s11047-010-9227-8
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11047-010-9227-8