Asymmetric PCR for good quality ssDNA generation towards DNA aptamer production

Aptamers are ssDNA or RNA that binds to wide variety of target molecules with high affinity and specificity produced by systematic evolution of ligands by exponential enrichment (SELEX). Compared to RNA aptamer, DNA aptamer is much more stable, favourable to be used in many applications. The most critical step in DNA SELEX experiment is the conversion of dsDNA to ssDNA. The purpose of this study was to develop an economic and efficient approach of generating ssDNA by using asymmetric PCR. Our results showed that primer ratio (sense primer:antisense primer) of 20:1 and sense primer amount of 10 to 100 pmol, up to 20 PCR cycles using 20 ng of initial template, in combination with polyacrylamide gel electrophoresis, were the optimal conditions for generating good quality and quantity of ssDNA. The generation of ssDNA via this approach can greatly enhance the success rate of DNA aptamer generation.

[1]  Native polyacrylamide gel electrophoresis. , 1996, Methods in molecular biology.

[2]  I. Chiu,et al.  Single primer-mediated polymerase chain reaction: application in cloning of two different 5'-untranslated sequences of acidic fibroblast growth factor mRNA. , 1991, DNA and cell biology.

[3]  M. Uhlén,et al.  Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. , 1989, Nucleic acids research.

[4]  M. Avci-Adali,et al.  Upgrading SELEX Technology by Using Lambda Exonuclease Digestion for Single-Stranded DNA Generation , 2009, Molecules.

[5]  J. Kinet,et al.  High-affinity oligonucleotide ligands to human IgE inhibit binding to Fc epsilon receptor I. , 1996, Journal of immunology.

[6]  D. Bartel,et al.  PCR product with strands of unequal length. , 1995, Nucleic acids research.

[7]  H. Ochman,et al.  Production of single-stranded DNA templates by exonuclease digestion following the polymerase chain reaction. , 1989, Nucleic acids research.

[8]  S. Gurr.  PCR Protocols-A Guide to Methods and Applications: Edited by M A Innis, D H Gelfand, J J Sninsky and T J White. pp 482. Academic Press, London 1990. $39.95 , 1991 .

[9]  J. SantaLucia,et al.  A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[10]  R E Rhoads,et al.  Optimization of the annealing temperature for DNA amplification in vitro. , 1990, Nucleic acids research.

[11]  S K Poddar,et al.  Symmetric vs asymmetric PCR and molecular beacon probe in the detection of a target gene of adenovirus. , 2000, Molecular and cellular probes.

[12]  C. Tuerk.  Using the SELEX combinatorial chemistry process to find high affinity nucleic acid ligands to target molecules. , 1997, Methods in molecular biology.

[13]  S. Gopinath,et al.  Conditions optimized for the preparation of single-stranded DNA (ssDNA) employing lambda exonuclease digestion in generating DNA aptamer , 2011 .

[14]  S. Wölfl,et al.  Efficient preparation of single-stranded DNA for in vitro selection , 1997, Molecular biotechnology.

[15]  H A Erlich,et al.  Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[16]  K. Kousoulas,et al.  Efficient production of single-stranded DNA as long as 2 kb for sequencing of PCR-amplified DNA. , 1992, BioTechniques.

[17]  D. Bell,et al.  Excessive cycling converts PCR products to random-length higher molecular weight fragments. , 1991, Nucleic acids research.

[18]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.