Abstract
Since the first report on complementary DNA (cDNA) cloning in 1972 (1), the technology has been developed into a powerful and universal tool in the isolation, characterization, and analysis of both eukaryotic and prokaryotic genes. However, the conventional methods of cDNA cloning require much effort to generate a cDNA library and then screen for a large number of recombinant phages or plasmid clones. There are three major limitations in these methods. First, a substantial amount of purified mRNA (at least 1 μg) 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/or truncated clones (3). Finally, screening of a library with hybridization technique is time-consuming.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
Akowitz, A. and Mamuelidis, L. (1989) A novel cDNA/PCR strategy for efficient cloning of small amounts of undefined RNA. Gene 81, 295–306.
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. Methods Enzymol. 154, 3–28.
Brenner, C. A., Tam, A. W., Nelson, P. A., Engleman, E. G., Suzuki, N., Fry, K. E., et al. (1989) Message amplification phenotyping (MAPPing): a technique to simultaneously measure multiple mRNAs from small numbers of cells. BioTechniques 7, 1096–1103.
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.
Shyamala, V. and Ames, G. F.-L. (1989) Genome walking by single-specific-primer polymerase chain reaction: SSP-PCR. Gene 84, 1–8.
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. Methods Mol. Biol. 15, 357–363.
Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–625.
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. Nucleic Acids Res. 16, 8186.
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. Nucleic Acids Res. 18, 1922.
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. Nucleic Acids Res. 17, 6439–6448.
Cusi, M. G., Cioé, L., and Rovera, G. (1992) PCR amplification of GC-rich templates containing palindromic sequences using initial alkali denaturation. BioTechniques 12, 502–504.
Lau, E. C, Li, Z.-Q., and Slavkin, S. C. (1993) Preparation of denatured plasmid templates for PCR amplification. BioTechniques 14, 378.
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.
Zilberberg, N. and Gurevitz, M. (1993) Rapid isolation of full_length cDNA clones by “inverse PCR”: purification of a scorpion cDNA family encoding α-neurotoxins. Anal. Biochem. 209, 203–205.
Austin, C. A., Sng, J.-H., Patel, S., and Fisher, L. M. (1993) Novel HeLa topo-isomerase II is the IIβ isoform: complete coding sequence and homology with other type II topoisomerases. Biochim. Biophys. Acta 1172, 283–291.
Delidow, B. C, Lynch, J. P., Peluso, J. J., and White, B. A. (1993) Polymerase chain reaction: basic protocols. Methods Mol. Biol. 15, 1–29.
Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) Basic Methods in Molecular Biology, Elsevier Science, New York.
Kru, M. S. and Berger, S. L. (1987) First strand cDNA synthesis primed by oligo(dT). Methods Enzymol. 152, 316–325.
Promega (1991) Protocols and Applications, 2nd ed., Promega, pp. 199–238.
Sambrook, J., Fritch, E. F., and Maniatis, T. (1989) Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.
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.
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.
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.
Zhang, H., Scholl, R., Browse, J., and Somerville, C. (1988) Double strand DNA sequencing as a choice for DNA sequencing. Nucleic Acids Res. 16, 1220.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Huang, SH., Chen, S.H.M., Jong, A.Y. (2003). Use of Inverse PCR to Clone cDNA Ends. In: Ying, SY. (eds) Generation of cDNA Libraries. Methods in Molecular Biology™, vol 221. Humana Press. https://doi.org/10.1385/1-59259-359-3:51
Download citation
DOI: https://doi.org/10.1385/1-59259-359-3:51
Publisher Name: Humana Press
Print ISBN: 978-1-58829-066-3
Online ISBN: 978-1-59259-359-0
eBook Packages: Springer Protocols