Abstract
The inverse polymerase chain reaction (IPCR) was the first extension of the conventional polymerase chain reaction to allow the amplification of unknown nucleotide sequences without recourse to conventional cloning. In the conventional polymerase chain reaction (PCR), synthetic oligonucleotides complementary to the ends of a known sequence are used to amplify the sequence (1,2). The primers are oriented with their 3′ ends facing each other, and the elongation of one primer creates a template for annealing the other primer. Repeated rounds of primer annealing, polymerization, and denaturation result in a geometric increase in the number of copies of the target sequence. However, regions outside the boundaries of the known sequence are inaccessible to direct amplification by PCR. Since DNA synthesis oriented toward a flanking region is not complemented by synthesis from the other direction, there is at most a linear increase in the number of copies of the flanking sequence.
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
Saiki, R. K., Scharf, S. J., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., et al. (1985) Enzymatic amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.
Saiki, R K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R. G., Horn, G. T., et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.
Burke, D. T., Carle, G. F., and Olson, M. V. (1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236, 806–812.
Hieter, P., Connelly, C., Shero, J., McCormick, M. K., Antonarakis, S., Pavav, W., et al. (1990) Yeast artificial chromosomes promises kept and pending, in Genetic and Physical Mapping, vol. 1 (Davies, K. E. and Tilghman, S. M., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 83–120.
Sternberg, N. (1990) Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs, Proc. Natl. Acad. Sci. USA 87, 103–107.
Pierce, J. C. and Sternberg, N. L. (1993) Using the bacteriophage PI system to clone high molecular weight (HMW) genomic DNA. Meth. Enzymol. (in press).
Ochman, H., Gerber, A. S., and Hartl, D. L. (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120, 621–623.
Triglia, T., Peterson, M. G., and Kemp, D. J. (1988) A procedure for in vitro amplification of DNA segments that he outside the boundaries of known sequences. Nucleic Acids Res. 16, 8186.
Silver, J. and Keerikatte, V. (1989) Novel use of polymerase chain reaction to amplify cellular DNA adjacent to an integrated provirus. J. Virol. 63, 1924–1928.
Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517.
Garza, D., Ajioka, J. W., Carulli, J. P., Jones, R. W., Johnson, D. H., and Hartl, D. L. (1989) Physical mapping of complex genomes. Nature 340, 577,578.
Gchman, H., Medhom, M. M., Garza, D., and Hartl, D. L. (1990) Amplification of flanking sequences by IPCR, in PCR Protocols: A Guide to Methods and Applications (Innis, M., Gelfand, D., Sninsky, J., and White, T., eds.), Academic, New York, pp. 219–227.
Silverman, G. A., Ye, R. D., Pollack, K. M., Sadler, J. E., and Korsmeyer, S. J. (1989) Use of yeast artificial chromosome clones for mapping and walking within human chromosome segment 18q21.3. Proc. Natl. Acad. Sci. USA 86, 7485–7489.
Silverman, G. A., Jockel, J. I., Domer, P. H., Mohr, R. M., Taillon-Miller, P., and Korsmeyer, S. J. (1991) Yeast artificial chromosome cloning of a two-megabasesize contig within chromosomal band 18q21 establishes physical linkage between BCL2 and plasminogen activator inhibitor type-2. Genomics 9, 219–228.
Huang, S., Hu, Y., Wu, C., and Holcenberg, J. (1990) A simple method for direct cloning cDNA sequence that flanks a region of known sequence from total RAN by applying the inverse polymerase chain reaction. Nucleic Acids Res. 18, 1922.
Breukel, C., Wijnen, J., Tops, C., Klift, H. V., Dauwerse, H., and Meera Khan, P. (1990) Vector-Alu PCR: a rapid step in mapping cosmids and YACs. Nucleic Acids Res. 18, 3097.
Ochman, H., Ayala, F. J., and Hartl, D. L. (1993) Use of the polymerase chain reaction to amplify segments outside the boundaries of known sequences. Meth. Enzymol. 218, 309–321.
Pfeifer, G. P., Steigerwald, S. D., Mueller, P. R., Wold, B., and Riggs, A. D. (1989) Genomic sequencing and methylation analysis by ligation mediated PCR. Science 246, 810–813.
Mueller, P. R. and Wold, B. (1989) In viva footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246, 780–786.
Fors, L., Saavedra, R. A., and Hood, L. (1990) Cloning of the shark PO promoter using a genomic walking technique based on the polymerase chain reaction. Nucleic Acids Res. 18, 2793–2799.
Riley, J., Butler, R., Ogilvie, D., Finniear, R., Jenner, D., Powell, S., et al. (1990) A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res. 18, 2887–2890.
Rosenthal, A. and Jones, D. S. C. (1990) Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. Nucleic Acids Res. 18, 3095,3096.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Hartl, D.L., Ochman, H. (1996). Inverse Polymerase Chain Reaction. In: Harwood, A.J. (eds) Basic DNA and RNA Protocols. Methods in Molecular Biology™, vol 58. Humana Press. https://doi.org/10.1385/0-89603-402-X:293
Download citation
DOI: https://doi.org/10.1385/0-89603-402-X:293
Publisher Name: Humana Press
Print ISBN: 978-0-89603-402-0
Online ISBN: 978-1-59259-251-7
eBook Packages: Springer Protocols