Site-directed mutagenesis by overlap extension using the polymerase chain reaction

Gene. 1989 Apr 15;77 (1):51-9 2744487 [Cited: 762]
Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
[My paper] S N Ho , H D Hunt , R M Horton , J K Pullen , L R Pease
Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.
Mesh-terms: Amino Acid Sequence; Animals; DNA, Recombinant; DNA-Directed DNA Polymerase; Exons; Gene Amplification; Genes, MHC Class I;Genes, Synthetic; Genetic Engineering, methods; Mice; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides, chemical synthesis; Oligodeoxyribonucleotides, genetics; Support, Non-U.S. Gov't; Support, U.S. Gov't, P.H.S.; Taq Polymerase;
[Pubmed] [Scholar] [EndNote] [BibTex] http://lib.bioinfo.pl/pmid:2744487

Overlap extension polymerase chain reaction

From Wikipedia, the free encyclopedia

The overlap extension polymerase chain reaction (OE-PCR) technique was first published in 1988 by Higuchi et al. and describes a process based on the polymerase chain reaction used to a) insert mutations at specific points in a sequence, further than ~55 nucleotides from either end and/or b) produce polynucleotides from smaller fragments.<sup>[1]

Full Details Here

http://en.wikipedia.org/wiki/Overlap_extension_polymerase_chain_reaction</sup>

Mutagenesis by PCR Protocols (CSH)

	Rapid and Efficient Site-directed Mutagenesis by the Single-tube Megaprimer PCR Method Joseph Sambrook and David W. Russell CSH Protocols; 2006; doi:10.1101/pdb.prot3467 [Extract] [Full text]
	Site-specific Mutagenesis by Overlap Extension Joseph Sambrook and David W. Russell CSH Protocols; 2006; doi:10.1101/pdb.prot3468 [Extract] [Full text]
	In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with DpnI Joseph Sambrook and David W. Russell CSH Protocols; 2006; doi:10.1101/pdb.prot3813 [Extract] [Full text]
	Genetic Engineering with PCR Joseph Sambrook and David W. Russell CSH Protocols; 2006; doi:10.1101/pdb.prot3836 [Extract] [Full text]
	Mutagenic PCR R. Craig Cadwell and Gerald F. Joyce CSH Protocols; 2006; doi:10.1101/pdb.prot4143 [Extract] [Full text]
	Rapid PCR Site-Directed Mutagenesis Gina L. Costa and Michael P. Weiner CSH Protocols; 2006; doi:10.1101/pdb.prot4144 [Extract] [Full text]
	PCR-Mediated Gene Disruption: One-Step Method David C. Amberg, Daniel J. Burke, and Jeffrey N. Strathern CSH Protocols; 2006; doi:10.1101/pdb.prot4169 [Extract] [Full text]

Purification for PCR CSH Protocols

	Purification of Genomic DNA from Whole Blood: A Solution-Based Method Craig Smith, Paul Otto, Rex Bitner, and Gary Shiels CSH Protocols; 2006; doi:10.1101/pdb.prot4096 [Extract] [Full text]
	A Silica Membrane-Based Method for the Isolation of Genomic DNA from Tissues and Cultured Cells Craig Smith, Paul Otto, Rex Bitner, and Gary Shiels CSH Protocols; 2006; doi:10.1101/pdb.prot4097 [Extract] [Full text]
	DNA IQ Isolation of Genomic DNA from Stains and Buccal Swabs Craig Smith, Paul Otto, Rex Bitner, and Gary Shiels CSH Protocols; 2006; doi:10.1101/pdb.prot4098 [Extract] [Full text]

Polymerase Chain Reaction PCR

PCR

Polymerase Chain Reaction

1) Add the following to a microfuge tube:
10 ul reaction buffer
1 ul 15 uM forward primer
1 ul 15 uM reverse primer
1 ul template DNA
5 ul 2 mM dNTP
8 ul 25 mM MgCl2 or MgSO4 (volume variable)
water (to make up......

Full Details Here

http://www.biochem.ucl.ac.uk/bsm/nmr/protocols/protocols/PCR.html

Detection of Alu by PCR

A Human DNA Fingerprinting Lab Protocol

1994 Cold Spring Harbor Laboratory
DNA Learning Center

In this experiment, polymerase chain reaction (PCR) is used to amplify a nucleotide sequence from chromosome 8 to look for an insertion of a short DNA sequence called Alu within the tissue plasminogen activator (TPA) gene. Although the DNA from different individuals is more alike than different, there are many regions of the human chromosomes t..

Full Details Here

http://www.accessexcellence.com/AE/AEPC/DNA/detection.html

Calculating Concentrations for PCR

Molecular Biology Techniques Manual

Third Edition

Edited by:

Vernon E Coyne, M Diane James, Sharon J Reid and Edward P Rybicki

Primers

Nucleotides

http://www.mcb.uct.ac.za//pcrconcn.htm

Protocol for PCR with Taq DNA Polymerase

Protocol for PCR with Taq DNA Polymerase

Avoiding Contamination

PCR allows the production of more than 10 million copies of a target DNA sequence from only a few molecules. The sensitivity of this technique means that the sample should not be contaminated with any other DNA or previously amplified products (amplicons) that may reside in the laboratory environment.

Full Protocol Here

http://www.fermentas.com/techinfo/pcr/dnaamplprotocol.htm

PCR Amplification of DNA

PCR Amplification of DNA

Materials:

	sterile water
	10X amplification buffer with 15mM MgCl2
	10 mM dNTP
	50 μM oligonucleotide primer 1
	50 μM oligonucleotide primer 2
	5 unit/μl Taq Polymerase
	template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl
	mineral oil (for thermocyclers without a heated lid 1. Combine the following for each reaction (on ice) in a 0.2 or 0.5 ml tube:  10X PCR buffer 10 μl Primer 1 1 μl Primer 2  1 μl dNTP  2 μl template DNA and water 85.5 μl Taq Polymerase 0.5 μl 2. Prepare a control reaction with no template DNA and an additional 10 μl of sterile water. 3. If the thermocycler does not have a heated lid, add 70-100 μl mineral oil (or 2 drops of silicone oil) to each reaction. 4. Place tubes in a thermal cycler preheated to 94 degrees C. 5. Run the following program:

94 degrees C 1 min

55 degrees C 1 min or annealing temperature appropriate for particular primer pair

72 degrees C 1 min (if product is <500 bp), 3 min (if product is >500 bp)

for 30 cycles.

Program a final extension at 72 degrees C for 7 min.

Interactions of Blood Cell Constituents

Interactions of Blood Cell Constituents: Experimental investigation and Computational Modeling by Discrete Particle Dynamics Algorithm
Microvascular Research, In Press, Accepted Manuscript, Available online 12 October 2007
N. Filipovic, D. Ravnic, M. Kojic, S.J. Mentzer, S. Haber and A. Tsuda

Computer model to forecast gyrodactylid infections on fish hosts

Gyroscope: an individual-based computer model to forecast gyrodactylid infections on fish hosts
International Journal for Parasitology, In Press, Accepted Manuscript, Available online 14 October 2007
C. van Oosterhout, R. Potter, H. Wright and J. Cable

Multivariate Analysis

Identifying functional imaging markers of mild cognitive impairment in early Alzheimer’s and Parkinson’s disease using multivariate analysis
Clinical Neuroscience Research, Volume 6, Issue 6, November 2007, Pages 367-373
Chaorui Huang, Paul Mattis and Per Julin

Ethical issues in withholding and withdrawing life-prolonging medical treatment

Ethical issues in withholding and withdrawing life-prolonging medical treatment in the ICU
Current Anaesthesia & Critical Care, In Press, Uncorrected Proof, Available online 11 October 2007
Margot Heaney, Carole Foot, William D. Freeman and John Fraser

Care of the brain-dead organ donor

Care of the brain-dead organ donor
Current Anaesthesia & Critical Care, In Press, Uncorrected Proof, Available online 11 October 2007
Konstantinos Linos, John Fraser, William D. Freeman and Carole Foot

Orthotopic liver transplantation

Orthotopic liver transplantation in Jehovah's Witnesses
Current Anaesthesia & Critical Care, In Press, Uncorrected Proof, Available online 15 October 2007
Lucy Yang and Zorica Jankovic