Abstract
The invention of polymerase chain reaction (PCR) (1,2) provided a powerful tool to modify DNA sequences in genetic engineering. With numerous mutagenesis methods available, such as traditional sequential PCR (3), “megaprimer PCR”(4–7), marker-coupled PCR (8), and so on, introducing changes to DNA sequences has become less tedious and more efficient. Recently, marketed site-directed mutagenesis (SDM) kits, such as Transformer™ Site-Directed Mutagenesis Kit (Clontech, San Francisco, CA), and Altered Site® II in vitro Site-Directed Mutagenesis Systems (Promega, Madison, WI), even eliminate the necessity of subcloning the amplified fragment.
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
Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp Quant Biol. 51, 263–273.
Mullis, K. B. and Faloona, F. A. (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 155, 335–350.
Cormack, B. (1994) Introduction of a point mutatation by sequential PCR steps. Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R. E., Kingston, D. D., et al., eds.), John Wiley & Sons, New York, NY, pp. 8.5.7–8.5.9.
Sarkar, G. and Sommer, S. S. (1990) The “megaprimer” method of site-directed mutagenesis. Biotechniques 8, 404–407.
Barik, S. and Galinski, M. S. (1991) “Megaprimer” method of PCR: increased template concentration improves yield. Biotechniques 10, 489,490.
Aiyar, A. and Leis, J. (1993) Modification of the megaprimer method of PCR mutagenesis: improved amplification of the final product. Biotechniques 14, 366–369.
Barik, S. (1996) Site-directed mutagenesis in vitro by megaprimer PCR. Methods Mol. Biol. 57, 203–215.
Shen, T. J., Zhu, L. Q., and Sun, X. (1991) A marker-coupled method for site-directed mutagenesis. Gene 103, 73–77.
Wang, W. and Malcolm, B. A. (1999) Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. Biotechniques 26, 680–682.
Parikh, A. and Guengerich, F. P. (1998) Random mutagenesis by whole-plasmid PCR amplification. Biotechniques 24, 428–431.
Ellington, A. and Pollard Jr., J. D. P.(1998) Purification of oligonucleotides using denaturing polyacrylamide gel electrophoresis, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R. E., Kinston, R. E., et al., eds.), John Wiley & Sons, New York, NY, p. 2.12.1
Schunck, B., Kraft, W., and Truyen, U. (1995) A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces. J. Virol. Methods 55, 427–433.
Kuang, S., Wang, J., Huang, W., Zhang, Y., Lu, L., Cheng, Z., and Jin, L. (1998) Fluorescence-based semi-automated gene scan with microsatellite markers by multiplex PCR techniques. Chung Hua IHsueh I Chuan Hsueh Tsa Chih 15, 104–107.
Kim, Y. G. and Maas, S. (2000) Multiple site mutagenesis with high targeting efficiency in one cloning step. Biotechniques 28, 196–198.
Smith, A. M. and Klugman, K. P. (1997) “Megaprimer” method of PCR-based mutagenesis: the concentration of megaprimer is a critical factor. Biotechniques 22, 438, 442.
Datta, A. K. (1995) Efficient amplification using’ megaprimer’ by asymmetric polymerase chain reaction. Nucleic Acids Res. 23, 4530–4531.
Gorelov, V. N., Roher, H. D., and Goretzki, P. E. (1994) A method to increase the sensitivity of mutation specific oligonucleotide hybridization using asymmetric polymerase-chain reaction (PCR). Biochem Biophys Res Commun. 200, 365–369.
Wooddell, C. I. and Burgess, R. R. (1996) Use of asymmetric PCR to generate long primers and single-stranded DNA for incorporating cross-linking analogs into specific sites in a DNA probe. Genome Res. 6, 886–892.
Millican, D. S. and Bird, I. M. (1998) Preparation of single-stranded antisense cDNA probes by asymmetric PCR. Methods Mol Biol. 105, 337–350.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Other types of gels, in Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, Vol. I, pp. 6–49.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Humana Press Inc.
About this protocol
Cite this protocol
Wang, W., Malcolm, B.A. (2002). Two-Stage Polymerase Chain Reaction Protocol Allowing Introduction of Multiple Mutations, Deletions, and Insertions, Using QuikChangeTM Site-Directed Mutagenesis. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology™, vol 182. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-194-9:037
Download citation
DOI: https://doi.org/10.1385/1-59259-194-9:037
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-910-0
Online ISBN: 978-1-59259-194-7
eBook Packages: Springer Protocols