SITE-DIRECTED MUTAGENESIS.pptx

Site-Site directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene.

1. SITE-DIRECTED MUTAGENESIS Presented By … Fizza Mehwish Department of BiotechnologY University of Science & Technology, Bannu

2. Contents Mutagenesis Types of Mutagenesis 1. Random Mutagenesis 2. Site-directed Mutagenesis History Types Of Site-directed Mutagenesis 1. Oligonucleotide Site-directed Mutagenesis 2. Cassette Mutagenesis 3. PCR Site-directed Mutagenesis Applications

3. Mutagenesis Mutagenesis is a process by which the genetic information of an organism is changed by the production of a mutation. A mutation is  a change in a DNA sequence . Mutations can result from DNA copying mistakes made during cell division, exposure to ionizing radiation, exposure to chemicals called mutagens, or infection by viruses.

4. Types of Mutagenesis Random Mutagenesis.. When an organism exposed to physical or chemical mutagen, mutations are induced randomly In all genes of the organism. Hence, this process of generating mutations is known as Random Mutagenesis. Site-Directed Mutagenesis.. Site-directed mutagenesis is  a molecular biology method  that is used to make specific and intentional changes to the DNA sequence of a gene.

5. History Site-directed mutagenesis was achieved in 1974 in the laboratory of Charles Weissmann using a nucleotide analogue N4-hydroxycytidine , which induces transition of GC to AT . Hutchison later produced with his collaborator  Michael Smith  in 1978 a more flexible approach to site-directed mutagenesis by using  oligonucleotides  in a primer extension method with DNA polymerase .

6. Types of Site-directed Mutagenesis

7. Oligonucleotide site-directed Mutagenesis The synthetic primer contain the desired mutation and is complementary to the template DNA around the mutation site so it can hybridize with the DNA in the gene of interest. The single stranded primer ( 10-25 nucleotides ) is then extended using the DNA polymerase which copies rest of gene. The gene thus copied contains the mutated site, and is then introduced in a host cell as a vector (plasmid) and then cloned. Finally mutated is then selected by screening and selection method .

8. Cassette Mutagenesis Cassette mutagenesis is a type of site-directed mutagenesis that uses a short, double-stranded oligonucleotide sequence to replace a fragment of target DNA. It uses complementary restriction enzyme digest ends on the target DNA and gene cassette to achieve specificity.  It is different from method that use single nucleotide mutation, in that a single gene cassette can contain multiple mutations. Cassette Mutagenesis also does not involve Primer extension by DNA polymerase. In Cassette Mutagenesis, a synthetic double stranded oligonucleotide ‘Cassette’ containing the desired mutations is docked between two restriction enzyme sites on a Plasmid vector.

9. PCR SITE-DIRECTED MUTAGENESIS PCR Site-directed Mutagenesis method, thus allow specific mutations to be incorporated in any double stranded plasmid, eliminating the need of single stranded rescue. PCR in Site-directed Mutagenesis Accomplishes strand Separation by using a denaturing step to separate the complimentary strands and allowing efficient polymerization of PCR mutated primers. The following reactions take place during PCR-based site-directed mutagenesis: In the  denaturation step , high temperature breaks the hydrogen bonds between the double-stranded DNA template, separating the strands. During the  annealing step , the mutagenic oligonucleotides bind to complementary nucleobases on one strand. The other oligonucleotides bind to the complementary nucleobases on the opposite strand.

10. Continue….. 3 . I n the  extension step , new daughter strands are synthesized by the enzyme DNA polymerase, extending the primers. 4. At the end of the extension step , the resulting two complementary daughter strands possess the mutations derived from the mutagenic primers and serve as templates in the subsequent PCR cycle, in addition to the original template

11. Steps of PCR based Site-directed Mutagenesis

12. Applications Desired Products Production : Site-directed Mutagenesis is used to generate mutations that produce our desired protein or protein having desired function. Investigative tools : Specific mutations in DNA allow the function and properties of a DNA sequence to be figure out. Research: Allow researchers to study the impact of change or mutation within a generation.

13. Commercial Application Proteins may be engineered to produce mutant forms that are tailored for a specific application. For example, commonly used laundry detergent may contain Subtilisin , whose wild type form has a methionine that can be oxidized by bleach, hence, reducing the activity of the protein in this process. The methionine may be replaced by alanine or other residues , making it resistant to Oxidization thereby keeping the protein active in the presence of bleach .

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