Site directed mutagenesis
From DrugPedia: A Wikipedia for Drug discovery
INTRODUCTION
An important milestone in modern molecular biology came in 1978 with the first description of site directed mutagenesis. Much of the present day knowledge of biomolecules originates from the study of spontaneous or induced mutants. In the past, their isolation and characterization had to await the identification of phenotypically deviant host organisms. The advent of site directed mutagenesis offered us a change from passive to a more active mode of study. It made feasible the prospect of artificially introducing changes in DNA, selecting them in the absence of phenotypic expression and studying the effects of these deliberate alterations in DNA either in vitro or after reintroduction in vivo. So central has this technique become to all of biochemistry and molecular biology that Michael Smith, its pioneer, shared the Nobel Prize in chemistry in October, 1993 with Kary Mullis who developed PCR.
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[edit] WHAT IS SITE DIRECTED MUTAGENESIS
Site directed mutagenesis is a technique in which a mutation is created at a defined site in a DNA molecule. It requires:
• Complete sequence of the gene to be mutated to be known.
• Single stranded DNA molecule containing the gene to be mutated to be available preferably cloned in M13 vector.
• A synthetic primer with the desired mutation at the desired site.
Complete sequencing of the gene is essential to identify a potential region for mutation. Once the precise base change has been identified, an oligonucleotide is designed that is complementary to part of the gene but has one base difference. This difference is designed to alter a particular codon, which after translation, gives rise to a different amino acid and hence may alter the properties of protein. The oligonucleotide and the single stranded DNA are annealed and DNA polymerase is added together with the dNTPs. The primer for the reaction is the 3’end of the oligonucleotide. The DNA polymerase produces a new strand complementary to the existing one but incorporating the oligonucleotide with the base mutation. The subsequent cloning of the recombinant heteroduplex produces multiple copies, whose sequences are either that of original wild type DNA or that containing the mutated base. The frequency with which mutated clones arise, may be low. Therefore in order to pick up mutants, the clones can be screened by nucleic acid hybridization with 32P- labeled oligonucleotide as probe. Under suitable stringent conditions i.e. temperature and cation concentration, a positive signal will be obtained only with mutant clones. The other way to conduct mutagenesis involves the use of two primers i.e. one mutagenic primer and one selection primer. Mutagenic primer encodes the mutation to be inserted in the target gene and the other primer encodes a mutation that enhances the antibiotic resistance determinant on the vector by conferring resistance to another antibiotic as well. After extending the two primers to yield an intact circular DNA molecule, the mutated plasmid is transformed into E.coli and selection made for the enhanced antibiotic resistance. In theory, cloning should yield equal number of mutant and non-mutated progeny, but in practice, mutants are counter selected. The major reason for this low yield is that the methyl directed mismatch repair system of E.coli favors the repair of non methylated DNA. In the cell, newly synthesized DNA strands that have not yet been methylated are preferentially repaired at the position of the mismatch, thereby eliminating the mutation. The problem associated with the mismatch repair system can be overcome by using host strains carrying mutL, mutS or mutH mutations which prevent the methyl directed repair of mismatches. A second problem arises from the fact that progeny phage originate from both plus and minus strands. Although minus strands are preferred as templates, the yield of mutants can be considerably increased if phage progeny is selected such that it uniquely originates from replication of mutant strand. In 1987, Kunkel et al introduced an improvement to the technique that eliminated the need for phenotypic selection. The plasmid to be mutated would be transformed into an E.coli deficient in two genes, dUTPase and uracil diglycosidase. The former would prevent the breakdown of dUTP, a nucleotide that replaces dTTPs in RNA, resulting in an abundance of the molecule, and deficiency in the latter, would prevent the removal of dUTP from newly synthesized DNA. As the double mutant E.coli replicates the up taken plasmid, its enzymatic machinery incorporates the dUTP resulting in a distinguishable copy. This copy is then extracted and incubated with an oligonucleotide containing the desired mutation which attaches by base pair hydrogen bonding to the complementary wild type gene sequence, as well as the Klenow enzyme, dNTPs, and DNA ligase. The reaction essentially replicates the dUTP-containing plasmid the oligonucleotide as primer, giving a nearly identical copy. The essential differences being that the copy contains dTTPs rather than dUTPs, as well as the desired mutation. When the chimeric double stranded plasmid, containing the dUTP ,unmutated strand and the dTTPs, mutated strand, is inserted into a normal wild type E.coli, the dUTP-containing strand is broken down , where as the mutation containing strand is replicated. Oligonucleotides not only form a convenient tool to introduce single mutations in cloned DNA, but can also be used for
• Multiple point mutagenesis
• Insertion mutagenesis
• Deletion mutagenesis
As long as stable hybrids are formed with single stranded wild type DNA, priming of in vitro DNA synthesis can occur, ultimately giving rise to clones corresponding to the inserted or deleted sequences.
[edit] CASSETTE MUTAGENESIS
Sometimes the need arises to substitute every possible amino acid at a specific site of protein. Using site directed mutagenesis it is possible to change two or three nucleotides for every possible amino acid substitution that is made at a site of interest. But this generates a requirement for 19 different mutagenic oligonucleotides assuming only one codon will be used for each substitution. This problem can be solved by cassette mutagenesis. Cassette mutagenesis involves replacing a fragment of gene with different fragments containing the desired codon changes. It is a simple method for which the efficiency of mutagenesis is close to 100%.
== SITE DIRECTED MUTAGENESIS USING PCR ==
Major disadvantage of all of the primer extension methods is that they require a single stranded template. The reason for this has been the need for separating the complementary strands to prevent reannealing. Use of PCR in site directed mutagenesis accomplishes strand separation \by using a denaturing step to separate the complementary strands and allowing efficient polymerization of the PCR primers. PCR site directed mutagenesis thus allows site specific mutations to be incorporated in virtually any double stranded DNA; linear or circular, eliminating the need for M13 based vectors or single stranded rescue.
Several features should be noted concerning site directed mutagenesis using PCR:
[edit] Main Features
• First, the procedure is not restricted to single base changes; by selecting appropriate primers it is possible to make insertions as well as deletions.
• Second, Taq polymerase copies DNA with low fidelity and there is a significant risk of extraneous mutations being introduced during amplification reaction. This problem can be minimized by
1. Use of high fidelity thermostable polymerase.
2. High template concentration in PCR reaction mixture approx. 1000 fold over conventional PCR conditions.
3. Fewer than 10 cycles of amplification.
The basic PCR mutagenesis system involves the use of two primary PCR reactions to produce two overlapping DNA fragments both bearing the same mutation in the overlap region. The technique is termed as overlap extension PCR. The overlap in sequence allows the fragments to hybridize. One of the two possible hybrids is extended by DNA polymerase to produce duplex fragments. The other hybrid with a 5’-hydroxyl group cannot act as substrate for polymerase, is effectively lost from the reaction mixture. Thus the overlapped and extended product will now contain the directed mutation. Although the requirement of four primers and three PCR reactions limit the general applicability of the technique. Various modifications of the basic procedure are now developed. In the Gene TailorTM method, the target DNA is methylated invitro before the mutagenesis step and overlapping primers are used. Once again, linear amplicons are produced that carry the desired mutation but in this case, they are transformed directly into E.coli. The host cell repair enzymes circularize the linear mutated DNA while the Mcr BC endonuclease digests the methylated template DNA leaving only unmethylated, mutated product.
[edit] QUICK CHANGE SITE DIRECTED MUTAGENESIS
The method described above enable defined mutations to be introduced at defined locations with in a gene and are of particular value in determining structure-function relationships. However ifthe objective of a study is to select mutants with altered or improved characteriste\ics or to construct a randomly mutagenized DNA library for a gene, then a better approach is to mutate the gene at random and then positively select those with desired properties. It is well known that the PCR is error prone and that there is high probability of base changes in amplicons. However, even the relatively low fidelity Taq. Polymerase is too accurate to be of value in generating mutanty libraries. Increase in erroe rates can be obtained by number of ways:
. To introduce a small amount of Mn2+ in place of normal Mg2+ . To include an excess of Dgtp and dTTP relative to other two nucleotides . By using nucleoside triphosphate analogs . By addition of Ditp . By shuffling of amplified fragments.
In general, Poll type DNA polymerase such as Taq. Polymerase and the amplified fragments are inserted into an appropriate vector to generate a mutated plasmid library.
[edit] Applications of Site Directed Mutagenesis
Site directed mutagenesis allows a nucleic acid sequence (and hence an encoded protein sequence) to be altered either randomly or in a predefined way. Its principal applications are:
• The investigation of the nucleic acid or protein structure/function; • The investigation of cellular pathways (for example, biochemical or signaling); • Synthesis of proteins with novel properties (Protein Engineering); • Construction of randomly mutagenized DNA library;and • Identification of critical residues for protein ligand interaction.
One of the most exciting aspects of site directed mutagenesis is that it permits the design, development and isolation of proteins with improved operating characteristics and even completely novel proteins. Any study on structure- function relationship can now easily be carried out using site directed mutagenesis.The principle of protein engineering is that the gene is mutated, either at a discrete site or at random and then made for a protein variant with a desired property. The improved variant can be subjected to further rounds of mutagenesis and selection, a process known as Directed Evolution. The paradigm for this approach is the enzyme Subtilisin. Every property of this serine protease has been altered including its rate of catalysis, substrate specificity, pH rate profile, and stability to oxidative, thermal and alkaline inactivation. Variations also have been produced that favor aminolysis(synthesis) over hydrolysisin aqueous solvents. Subtilisin is now being used in soap industry and in food and leather industries. Engineered Subtilisin of improved bleach resistance and wash performances are now used in many brands of washing powders.
