Single Primer-Mediated Polymerase Chain Reaction

  • Rene L. Myers
  • Ing-Ming Chiu
Part of the Methods in Molecular Biology book series (MIMB, volume 31)

Abstract

The polymerase chain reaction (PCR) is a powerful method for amplification of DNA segments between two regions of known sequences (1, 2). In standard PCR, sequences bound by two unique oligonucleotide primers can be exponentially amplified. However, it is a prerequisite to know the DNA sequence at both ends of the region of interest. To amplify regions of unknown sequence, methods such as inverted PCR (3), Alu PCR (4), and rapid amplification of cDNA ends (RACE) (5), also known as anchored PCR (6) or one‐sided PCR (7), have been developed. These methods require several enzymatic manipulations of DNA that are either tedious or only suitable for certain conditions. While using standard PCR for in vitro amplification of 5′ untranslated cDNA sequence of acidic fibroblast growth factor (aFGF) mRNA, we observed amplification of a second specific fragment in addition to the expected fragment. Sequence analysis showed that the additional fragment resulted from binding of the 5′ primer to a region that has an imperfect match to the actual primer binding site. Comparing the sequences of the primer (HBGF 1201) and the binding site revealed that only eight out of 32 nucleotides matched (Fig. 1A). Those eight nucleotides cluster in the 19 nucleotides at the 3′ end of the primer and therefore provided priming that allowed for a specific amplified product. With this observation, we reasoned that a single primer could be used, binding both to the complementary sequence

Keywords

Microwave Phenol EDTA Leukemia Serotonin 

Further Reading

  1. Kwok, S., and Higuchi, R. (1989) Avoiding false positives with PCR. Nature 339, 237–238.PubMedCrossRefGoogle Scholar
  2. White, B. A. ed. (1993) PCR Protocols: Current Methods and Applications; Methods in Molecular Biology, vol. 15, Humana, Totowa, NJ.Google Scholar

References

  1. 1.
    Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Primer directed enzymatic amplifica-tion of DNA with a thermo-stable DNA polymerase. Science 239, 487–491.PubMedCrossRefGoogle Scholar
  2. 2.
    Erlich, H. A., Gelfand, D., and Sninsky, J. J. (1991) Recent advances in the polymerase chain reaction. Science 252, 1643–1651.PubMedCrossRefGoogle Scholar
  3. 3.
    Triglia, T., Peterson, M. G., and Kemp, D. J. (1988) A procedure for in vitro amplification of DNA segments that lie outside the boundries of known sequences. Nucleic Acids Res. 16, 8186.PubMedCrossRefGoogle Scholar
  4. 4.
    Nelson, D. L., Ledbetter, S. A., Corbo, L., Victoria, M. F., Ramirez-Sohs, R., Webster, T. D., Ledbetter, D. H., and Caskey, C. T. (1989) Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources. Proc, Natl. Acad. Sci. USA 86, 6686–6690.CrossRefGoogle Scholar
  5. 5.
    Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Rapid production of full-length cDNA from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc. Natl. Acad. Sci. USA 85, 8998–9002.PubMedCrossRefGoogle Scholar
  6. 6.
    Loh, E. Y., Elliot, J. F., Cwirla, S., Lanier, L. L., and Davis, M. M. (1989) Polymerase chain reaction with single-sided specificity: analysis of T cell receptor 8 chain. Science 243, 217–220.PubMedCrossRefGoogle Scholar
  7. 7.
    Ohara, O., Dorit, R. L., and Gilbert, W. (1989) One-sided polymerase chain reaction: the amplification of cDNA. Proc. Natl. Acad. Sci. USA 86, 5673–5677.PubMedCrossRefGoogle Scholar
  8. 8.
    Wang, W.-P,, Myers, R. L., and Chiu, I.-M. (1991) Single primer-mediated polymerase chain reaction: application in cloning of two different 5′-untranslated sequences of acidic fibroblast growth factor mRNA. DNA Cell Biol. 10, 771–777.PubMedCrossRefGoogle Scholar
  9. 9.
    Parks, C. L., Chang, L.-S., and Shenk, T. (1991) A polymerase chain reaction mediated by a single primer-cloning of genomic sequences adjacent to a sero-tonin receptor protein coding region. Nucleic Acids Res 19, 7155–7160.PubMedCrossRefGoogle Scholar
  10. 10.
    Birnboim, H. C. and Doly, J. A. (1979) Rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513–1523.PubMedCrossRefGoogle Scholar
  11. 11.
    Marchuk, D., Drumm, M., Saulino, A., and Collins, F S. (1991) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19, 1154.Google Scholar
  12. 12.
    Gussow, D and Clackson, T. (1989) Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res. 17, 4000.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1994

Authors and Affiliations

  • Rene L. Myers
    • 1
  • Ing-Ming Chiu
    • 1
  1. 1.Departments of Molecular Genetics and Internal MedicineThe Ohio State University, Davis Medical Research CenterColumbus

Personalised recommendations