site directed mutagenesis

SITE-DIRECTED MUTAGENESIS ON DOUBLE STRANDED DNA

Advances in molecular biology and DNA synthesis techniques have accelerated progress in the study of protein and its function. In the past, it had been accomplished either by chemically modifying individual amino acids or by inducing mutations in the gene coding for the protein under study1. However, there were problems in that chemical modification of a protein is not specific and several amino acids may be altered1. It was not always possible to produce the proper mutation in the desired gene function1. Later, these difficulties have been overcome with the technique called site-directed mutagenesis.

Such technique was first developed in the mid 1980’s. It is a widely used technique for introducing mutations into a particular DNA sequence, often with the goal of creating a point mutation in the corresponding amino acid sequence but leaving the overall sequence undisturbed2. Such a technique uses synthetic oligonucleotides and the whole process, in summary, is shown in fig 1.

Fig 1: Summary of an Oligonucleotide

directed site-directed mutagenesis1.

The DNA carries a gene of interest and is

purified and annealed to a synthetic

oligonucleotide that contains the desired

change in sequence, Here, an A:T base

pair is converted to a G:C base pair.

An oligonucleotide of about 20 residues that contains the required sequence change is synthesized1. It is now allowed to bind to a single-stranded copy of the complete gene1 (fig 2 below). DNA polymerase is added to the gene-primer complex1. The polymerase extends to the primer and replicates the remainder of the target gene to produce a new gene copy with the desired mutation. If the gene is attached to a single-stranded DNA bacteriophage (like M13 phage), it can be introduced into a host bacterium and cloned using the techniques1. This will yield large quantities of the mutant protein1.

Fig 2: Site-directed mutagenesis in details1.

A synthetic oligonucleotide is used to add a

specific mutation to a gene. See above

paragraph for description for this process.

The most common used approach is to use an oligonucleotide complementary to part of a single-stranded DNA template but containing an internal mismatch to direct the mutation3. This approach can also be used to construct multiple mutations, insertions and deletions3. An alternative strategy is to replace the region to be mutated by a fragment obtained by ligation of a number of synthetic oligonucleotides and purified either by HPLC (high-performance liquid chromatography) or by polyacrylamide gel electrophoresis3. Site-directed mutagenesis can be done on a lot of places (i.e. DNA, plasmid) and uses mismatched oligonucleotides (fig 3), single-stranded vectors (fig 4), double-stranded vectors (DNA) and chemical analysis3. However, site-directed mutagenesis on double stranded DNA will be emphasised here.

Fig 3: Mutagenesis using mismatched oligonucleotide3.

A mismatched oligonucleotide (C to a T) is annealed to a single-stranded DNA template (M13 in this case), extended with the Klenow fragment of DNA polymerase I and ligated with T4 DNA ligase. After transformation of E.coli, mutant and wild type progeny molecules result.

Fig 4: Simple strategies for M13 mutagenesis3.

The simplest ...

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Fig 3: Mutagenesis using mismatched oligonucleotide3.

Fig 4: Simple strategies for M13 mutagenesis3.

The simplest approach to M13 mutagenesis is ‘single priming’, where a mutagenic primer is annealed to the single-stranded template, extended briefly with Klenow fragment and used to transfect an E.coli host. This mismatch may be removed in vivo by the action of 5’-3’ exonucleases but may be overcome in several different ways. After extending ‘all-the-way-round’ the template, the ends of the new strand may be ligated. In the ‘double priming’ technique a second primer 5’ to the mutagenic primer is used to protect the mismatch after extension and ligation. In the ‘gapped duplex’ technique, mutagenesis is carried out in a single-stranded region formed by annealing the template with a restriction fragment from the vector. Again the 5’ end of the oligonucleotide is protected after extension and ligation.

Denatured double-stranded DNA can be sequenced directly by the Dideoxy method using oligonucleotide or by the chemical method4,5. In constructing plasmids, the plasmid is normally converted to a single or partially double-stranded form3. It is first nicked in the presence of ethidium bromide with a restriction endonuclease or DNAase I6,7. Next it is digested to completion with exonuclease II8. The ‘double priming’ technique for DNA mutagenesis helps to remove the need to isolate single-stranded DNA9. The double-stranded DNA is denatured by boiling and then annealed with a mutagenic primer and a second primer located on the 5’ side of the mutagenic primer3. After a brief extension reaction with Klenow fragment, the mixture is used directly to transform E.coli cells3. A lot of methods are used to construct ‘gapped-duplex’ plasmid DNA for site-directed mutagenesis. One method is to nick the plasmid at a restriction site near the target sequence followed by limited digestion with exonuclease III to produce the ‘gapped duplex’10. Another method is to linearize the plasmid near the target site and expose the target by limited digestion using exonuclease III3. After extension from the mutated oligonucleotides and ligation, the resulting double-stranded DNA is used to transform an E.coli host11. Above are the steps in which site-directed mutagenesis is carried out on double-stranded DNA.

As techniques in molecular biology improve, more advanced methods for site-directed mutagenesis are available. One of them is the Quik-Change method that requires two complementary oligonucleotide primers flanking the desired mutated nucleotide on both sense and anti-sense strands2, 12. The primer must contain one to several base-pair changes within the desired region and carried out using polymerase chain (PCR) reaction2, 12. The most recent technique is to use four primers instead of 1-2 primers for site-directed mutagenesis13. Two primers are within the ori region while the other two within the mutant region13. With this approach, site-directed mutagenesis can be obtained easily and successfully by circularising two fragments of PCR products together13. This method also allows carrying mutations more efficiently than that with the traditional mutagenesis methods and single inverted PCR method13. In addition, mutations such as insertions and deletions can be performed with this method13. Moreover, by halving a plasmid into two parts, this method can perform relatively large-sized plasmids and rapidly generating chimera using two different proteins’ cDNA cloned in the same vector13. Fig 5 shows the whole process of such method.

Fig 5: Diagram of steps of site-directed

mutagenesis for ColE1-type ori plasmid13.

Four primers were synthesized for this

Method, of which a pair of tail-to-tail

Primers is complementary coding for a

part of ColE1-type ori region. Two halves

of a plasmid were amplified by

independent PCR using primers 1 and 3

and primers 2 and 4. After gel purification,

blunting and phosphorylation, two PCR-

amplified fragments were joined by ligase.

Only the circularising ligation products

which have intact ColE1-type ori region

and desired mutant can amplify in E.coli

* Indicates mutant side.

Site-directed mutagenesis has many different uses, both in basic research and in applied biology14. Not only it can help someone to understand the role of a particular residue in the folding or function of a polypeptide, it can help in the analysis of the specific functions of minute parts of virtually every protein of interest14, such as the analyses and isolation of DNA methyltransferase SssI recently15 (fig 6 below). It is also being used to modify the structure of clinically useful proteins to increase their effectiveness or reduce unwanted side effects14. For example, thrombin in the blood can act either to promote or inhibit the formation of a blood clot, which depends on situations14. It is engineered to produce a thrombin molecule that retains its anticoagulant activity while losing nearly all of its procoagulant activity14. This engineered protein shows promise as an anti-clotting agent in the treatment of strokes and heart attacks14. In the most recent application, software, known as SiteFind, is built on the basis of the underlying principle of site-directed mutagenesis2. Such a software tool has introduced a restriction site as a marker for successful site-directed mutagenesis2.

Fig 6: Construction of plasmids to produce M.SssI and its mutants15

(a) Generation of the NcoI site: 1, the 5’-terminal fragment of sssI; 2, the 5’-terminal fragment of truncated sssI with the GCA (Ala) codon inserted between the initiator ATG and second (AGC) codons; 3. a fragment of modified sssI with an additional 36-bp sequence inserted between the initiator ATG and second (AGC) codons. The initiator ATG codon is framed; the NcoI site is shown in a dashed frame; the additional nucleotide sequences of the sssI operon are in bold.

(b) Schemes of (I) pCAL7, (2) pCAL7/ad, and (3) pCAL7/nH.

(c) Construction of mutant plasmids. 1,. Plasmid producing M. SssI; 2-4, plasmids producing the S300P, S300G and V188A mutants, respectively.

In summary, site-directed mutagenesis is one of the powerful methods in molecular biology to obtain or remove the gene (and hence the protein and the final product or cell) that one need. With advances in more powerful techniques for site-directed mutagenesis, it is anticipated that such technique will help in more molecular biology future discoveries and findings.

References

1 M.P. Lansing, P.H. John & A.K. Donald (2005). Microbiology (6th edition) (International

edition). The McGraw Hill companies.

2 M.E. Paul & C. Liu (2005). SiteFind: A software tool for introducing a restriction site as a

marker for successful site-directed mutagenesis. BMC Molecular Biology. 6 (22): 1-8.

3 C. Paul (1986). Site-directed mutagenesis. Biochem. J. 237: 1-7.

4 M. Smith, D.W. Leung, S. Gilliam, C.R. Astell, D.L. Montgomery & B.D. Hall (1979).

Sequence of the gene for iso-l-cytochrome c in saccharomyces cerevisiae. Cell. 16: 753-761.

5 A.M. Maxam & W. Gilbert (1977). A new method for sequencing DNA. Proc. Natl. Acad. Sci.

U.S.A. 74: 560-564.

6 L. Greenfield, L. Simpson & D. Kaplan (1975). Conversion of closed circular DNA molecules

to single-nicked molecules by digestion with DNAase I in the presence of ethidium bromide.

Biochim. Biophys. Acta. 407: 365-375.

7 R.C. Parker, R.M. Watson & J. Vinograd (1977). Mapping of closed circular DNAs by

cleavage with restriction endonucleases and calibration by agarose gel electrophoresis. Proc.

Natl. Acad. Sci. U.S.A. 74: 851-855.

8 R.B. Wallace, P.F. Johnson, S. Tanaka, M. Schold, K. Itakura & J. Abelson (1980). Directed

deletion of a yeast transfer RNA intervening sequence. Science. 209: 1396-1400.

9 M. Schold, A. Colombero, A.A. Reyes & R.B. Wallace (1984). Oligonucleotide-directed

mutagenesis using plasmid DNA templates and two primers. DNA. 3: 469-477..

10 G. Dalbadie-McFarland, L.W. Cohen, A.D. Riggs, K. Itakura & J.H. Richards (1982).

Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein

function. Proc. Natl. Acad. Sci. U.S.A. 79: 6409-6413.

11 V.A. Efimov, O.V. Mirskikh, O.G. Chakhmakcheva & Yu. A. Ovchinikov (1985). Convenient

modification of the method for oligonucleotide-directed in vitro mutagenesis of cloned DNA.

FEBS Lett. 181: 407-411.

12 C. Papworth, J.C. Bauer, J. Braman & D.A. Wright (1996). QuikChange site-directed

mutagenesis. Strategies. 9: 3-4..

13 C.J. Jin, X. Cai, H. Ma, Y. Xue, J.H. Yao & X.B. Yao (2007). Analytical Biochemistry. 363:

151-153.

14 K. Gerald (1999). Cell and Molecular Biology: Concepts and Experiments (2nd edition). John

Wiley & Sons Inc.

15 M.V. Daril, O.V. Kirsanova, V.L. Drutsa, S.N. Kochetkov & E.S. Gromova (2007). Molecular

Biology. 41 (1): 110-117.

site directed mutagenesis

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