Site-directed mutagenesis by inverse PCR
Overview
Inverse PCR is a method for the rapid in vitro amplification of DNA sequences that flank a region
of known sequence. The method uses the polymerase chain reaction (PCR), but it has the primers
oriented in the reverse direction of the reference sequences. Important application of inverse PCR is
site-directed mutagenesis of plasmid construct (Fig. 1) (Hemsley et al., 1989). The method is based on
the amplification of the entire plasmid using primers that include the desired changes (Fig. 2). Sitedirected mutagenesis by inverse PCR is rapid, simple in its execution, and requires only minute
amounts of plasmid template DNA. It is significant that there are no special requirements for
appropriately placed restriction sites in the sequence to be manipulated. The generality of the method
should make it useful for the direct alteration of most cloned genes.
Figure. 2. Various mutations by
inverse PCR.
Figure. 1. Overview of site-directed mutagenesis
by inverse PCR.
Procedure
Primer phosphorylation
1. Prepare following reaction mixture:
Primer A (100 pmole/μl [100 μM])
Primer B (100 pmole/μl [100 μM])
10mM rATP
10×Buffer for T4 kinase
T4 Polynucleotide Kinase (5~20U/μl)
Total Volume
7 μl
7 μl
2 μl
2 μl
2 μl
20 μl
2. Gently and thoroughly mix each reaction mixture by pipetting.
3. Centrifuge the reaction mixture and incubate each reaction at 37°C for 1 hr.
3. Incubate at 95°C for 5 min.
4. Add 50μl water (10 pmol/μl phosphorylated primer mix).
Inverse PCR
1. Prepare the PCR reaction mixture as follows:
PCR grade water
10×Buffer for DNA polymerase
2 mM dNTPs
Phosphorylated primer mix (10 pmol/μl each)
Plasmid Template DNA (50 ng/μl)
High fidelity DNA polymerase
Total Volume
36.5 μl
5 μl
5 μl
1.5 μl
1 μl
1 μl
50 μl
2. Perform PCR reaction as follows:
94°C
2 min.
98°C
10 sec.
x X cycle *2
68°C
X min.*1
4°C
Hold
*1
Extension time should be 1 min/kb of plasmid length (e.g., a 5 kb-size plasmid: 5 min.).
*2
Cycle number should be 1 cycle/kb of plasmid length (e.g., a 5 kb-size plasmid: 5 cycles).
DpnI digestion of the template plasmid
Note: Dpn I digests methylated DNA, such as plasmid DNA from typical E. coli cell lines
1. Add 2 μl of DpnI restriction enzyme (10 U/μl) to 50 μl PCR reaction mixture.
2. Gently and thoroughly mix each reaction mixture by pipetting.
3. Spin down the reaction mixture and incubate at 37°C for 1 hr to digest the template plasmid.
Self-ligation of the PCR Product
1. Prepare the ligation reaction mixture using a fresh tube as follows:
DpnI- treated PCR product
2 μl
PCR grade water
5 μl
Ligation high
5 μl
T4 Polynucleotide Kinase
1.5 μl
Total Volume
15 μl
2. Gently and thoroughly mix each reaction mixture by pipetting.
3. Centrifuge the reaction mixture and incubate each reaction solution at 16°C for 1 hr.
Transformation
1. Gently thaw the competent cells (100 μl) on ice. Standard E. coli strains can be used as the
transformation host.
2. Add 10 μl of reaction mixture from previous step to the competent cells, swirl gently and
incubate for 30 min on ice.
3. Incubate for 30 sec at 42°C and then place on ice for 2 min.
4. Add 900 μl of LB medium and incubate at 37°C for 30 min.
5. Plate appropriate volumes (10-200 μl) of transformed E. coli solution on agar plates containing
the appropriate antibiotic for the plasmid vector.
6. Incubate the plates at 37°C for ~16 hr.
Analysis of Transformants
4-8 clones should be checked by sequence.
(Modified from Instruction manual KOD -Plus- Mutagenesis Kit 0811(TOYOBO))
References
1. Hemsley, A., Arnheim, N., Toney, M.D., Cortopassi, G., and Galas, D.J. A simple method for sitedirected mutagenesis using the polymerase chain reaction. Nucleic Acid Res., 17, 6545-6551, 1989.
2. Instruction manual KOD -Plus- Mutagenesis Kit 0811 (TOYOBO)
http://www.toyobo.co.jp/e/seihin/xr/lifescience/support/manual/SMK-101.pdf