Inverse PCR

cDNA Cloning
  • Sheng-He Huang
Part of the Methods in Molecular Biology™ book series (MIMB, volume 192)


Since the first report on cDNA cloning in 1972 (1), this technology has been developed into a powerful and universal tool in isolation, characterization, and analysis of both eukaryotic and prokaryotic genes. But the conventional methods of cDNA cloning require much effort to generate a library that is packaged in phage or plasmid and then survey a large number of recombinant phages or plasmids. There are three major limitations in those methods. First, substantial amount (at least 1 μg) of purified mRNA is needed as starting material to generate libraries of sufficient diversity (2). Second, the intrinsic difficulty of multiple sequential enzymatic reactions required for cDNA cloning often leads to low yields and truncated clones (3). Finally, screening of a library with hybridization technique is time-consuming.


Recombinant Phage Inverse Polymerase Chain Reaction Select Restriction Enzyme Thermal Cycler Block Flank Unknown Sequence 
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  1. 1.
    Verma, I. M., Temple, G. F., Fan, H., and Baltimore, D. (1972) In vitro synthesis of double-stranded DNA complimentary to rabbit reticulocyte 10S RNA. Nature 235, 163–169.CrossRefGoogle Scholar
  2. 2.
    Akowitz, A. and Mamuelidis, L. (1989) A novel cDNA/PCR strategy for efficient cloning of small amounts of undefined RNA. Gene 81, 295–306.PubMedCrossRefGoogle Scholar
  3. 3.
    Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T., and Arai, K. (1987) High-efficiency cloning of full-length cDNA; Construction and screening of cDNA expression libraries for mammalian cells. Meth. Enzymol. 154, 3–28.PubMedCrossRefGoogle Scholar
  4. 4.
    Brenner, C. A., Tam, A. W., Nelson, P. A., Engleman, E. G., Suzuki, N., Fry, K. E., and Larrick, J. W. (1989) Message amplification Phenotyping (MAPPing): a technique to simultaneously measure multiple mRNAs from small numbers of cells. BioTechniques 7, 1096–1103.PubMedGoogle Scholar
  5. 5.
    Frohman, M. A. (1990) RACE: Rapid amplification of cDNA ends, in: PCR Protocols: A Guide to Methods and Applications (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds.), Academic, San Diego, CA, pp 28–38.Google Scholar
  6. 6.
    Shyamala, V. and Ames, G. F.-L. (1989) Genome walking by single-specific-primer polymerase chain reaction: SSP-PCR. Gene 84, 1–8.PubMedCrossRefGoogle Scholar
  7. 7.
    Huang, S.-H., Jong, A. Y., Yang, W., and Holcenberg, J. (1993) Amplification of gene ends from gene libraries by PCR with single-sided specificity. Meth. Mol. Biol. 15, 357–363.Google Scholar
  8. 8.
    Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–625.PubMedGoogle Scholar
  9. 9.
    Triglia, T., Peterson, M. G., and Kemp, D. J. (1988) A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucl. Acids Res. 16, 8186.PubMedCrossRefGoogle Scholar
  10. 10.
    Huang, S.-H., Hu, Y. Y., Wu, C.-H. and Holcenberg, J. (1990) A simple method for direct cloning cDNA sequence that flanks a region of known sequence from total RNA by applying the inverse polymerase chain reaction. Nucl. Acids Res. 18, 1922.PubMedCrossRefGoogle Scholar
  11. 11.
    Delort, J., Dumas, J. B., Darmon, M. C., and Mallet, J. (1989) An efficient strategy for cloning ′5′ extremities of rare transcrips permits isolation of multiple 5′-untranslated regions of rat tryptophan hydroxylase mRNA. Nucl. Acids Res. 17, 6439–6448.PubMedCrossRefGoogle Scholar
  12. 12.
    Cusi, M. G., Cioe′, L., and Rovera, G. (1992) PCR amplification of GC-rich templates containing palindromic sequences using initial alkali denaturation. BioTechniques 12, 502–504.PubMedGoogle Scholar
  13. 13.
    Lau, E. C., Li, Z.-Q., and Slavkin, S. C. (1993) Preparation of denatured plasmid templates for PCR amplification. BioTechniques 14, 378.PubMedGoogle Scholar
  14. 14.
    Green, I. R. and Sargan, D. R. (1991) Sequence of the cDNA encoding ovine tumor necrosis factor-α: problems with cloning by inverse PCR. Gene 109, 203–210.PubMedCrossRefGoogle Scholar
  15. 15.
    Zilberberg, N. and Gurevitz, M. (1993) Rapid Isolation of full length cDNA clones by &quote;Inverse PCR:&quote; purification of a scorpion cDNA family encoding α-neurotoxins. Analyt. Biochem. 209, 203–205.PubMedCrossRefGoogle Scholar
  16. 16.
    Austin, C. A., Sng, J.-H., Patel, S., and Fisher, L. M. (1993) Novel HeLa topoisomerase II is the IIβ isoform: complete coding sequence and homology with other type II topoisomerases. Biochim. Biophys. Acta 1172, 283–291.PubMedGoogle Scholar
  17. 17.
    Delidow, B. C., Lynch, J. P., Peluso, J. J., and White, B. A.(1993) Polymerase Chain Reaction: Basic Protocols. Meth. Mol. Biol. 15, 1–29.Google Scholar
  18. 18.
    Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Basic Methods in Molecular Biology, Elsevier Science, New York.Google Scholar
  19. 19.
    Kru, M. S. and Berger, S. L. (1987) First strand cDNA synthesis primed by oligo(dT). Meth. Enzymol. 152, 316–325.CrossRefGoogle Scholar
  20. 20.
    Promega (1996) Protocols and Applications 3rd ed., pp. 179–190.Google Scholar
  21. 21.
    Sambrook, J., Fritch, E. F., and Maniatis, T. (1989) Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory Press, New York.Google Scholar
  22. 22.
    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 amplification of DNA with ather-mostable DNA polymerase. Science 239, 487–491.PubMedCrossRefGoogle Scholar
  23. 23.
    Moon, I. S. and Krause, M. O. (1991) Common RNA polymerase I, II, and III upstream elements in mouse 7SK gene locus revealed by the inverse polymerase chain reaction. DNA Cell Biol. 10, 23–32.PubMedCrossRefGoogle Scholar
  24. 24.
    Strobel, S. A. and Dervan, P. B. (1990) Site-specific cleavage of a yeast chromosome by oligonucleotide-directed triple-helix formation. Science 249, 73–75.PubMedCrossRefGoogle Scholar
  25. 25.
    Dreyer, G. B. and Dervan, P. B. (1985) Sequence-specific cleavage of single-stranded DNA: Oligodeoxynucleotide-EDTA.Fe(II). Proc. Natl. Acad. Sci. USA 82, 968–972.PubMedCrossRefGoogle Scholar
  26. 26.
    Zhang, H., Scholl, R., Browse, J., and Somerville, C. (1988) Double strand DNA sequencing as a choice for DNA sequencing. Nucl. Acids Res. 16, 1220.Google Scholar
  27. 27.
    Sugino, A., Goodman, H. M., Heynecker, H. L., Shine, J., Boyer, H. W., and Cozzarelli, N. R. (1977) Interaction of bacteriophage T4 RNA and DNA ligases in joining of duplex DNA at base-paired ends. J. Biol. Chem. 252, 3987.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Sheng-He Huang
    • 1
  1. 1.Department of PediatricsUniversity of Southern CaliforniaLos Angeles

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