Site directed mutagenesis

Genetic engineering

1. SITE DIRECTED MUTAGENESIS Dr. SMT. ARUNIMA KARKUN (ASST. PROF.) G.D. RUNGTA COLLEGE OF SCIENCE AND TECHNOLOGY KOHKA KURUD ROAD BHILAI, 490023 1

2. INTRODUCTION HISTORY MUTATION DIRECTED MUTAGENESIS BASIC MECHANISM OF SITE DIRECTED MUTAGENESIS METHOD FOR SITE DIRECTED MUTATIONS THE SINGLE PRIMER METHOD CASETTEE MUTAGENESIS   PCR SITE-DIRECTED MUTAGENESIS APPLICATION OF SITE DIRECTED MUTAGENESIS SUMMARY CONCLUSION REFERENCES SITE DIRECTED MUTAGENESIS S Y N O P S I S 2

3. SITE DIRECTED MUTAGENASIS In molecular biology and genetics, mutations are changes in a genomic sequence. Mutations are caused by radiation, viruses, transposes and mutagenic chemicals, as well as errors that occur during meiosis or DNA replication. Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide directed mutagenesis, is a molecular biology technique often used in bio molecular engineering in which a mutation is created at a defined site in a DNA molecule. Site-directed mutagenesis is the technique for generating amino acid coding changes in the DNA(gene). Site-directed mutagenesis is incorporation of a desired amino acid (of one's choice) in place of a specific amino acid in a protein or a polypeptide. SITE DIRECTED MUTAGENESIS I N T R O D U C T I O N 3

4. IN 1791, SETH WRIGTHT first time study in mutations in sheep genome. IN 1910, MORGEN study in .Mutations in drosophila melangaster. IN 1927, H.J. MULLER give the CLB method for detection of mutation. IN 1971, CLYDE HUTCHISON AND MARSHALL EDGELL showed that it is possible to produce mutants with small fragments of phage φx174 and restriction nucleases. IN 1973, CHARLES WEISSMANN using N 4 -hydroxycytidine which induces transition of GC to AT .  IN 1978, MICHAEL SMITH site-directed mutagenesis by using oligonucleotides in a primer extension method with DNA polymerase. IN 1993,  KARY B. MULLIS who invented polymerase chain reaction. H I S T O R Y SITE DIRECTED MUTAGENESIS 4

5. In molecular biology and genetics, mutation s  are accidental changes in a genomic sequence of  DNA. Mutations can involve large sections of DNA becoming duplicated, usually through genetic recombination. Mutation is may be define as a change in the nucleic sequence (bases) of an organism’s genetic material (a change in the genetic material of an organism). Directed mutagenesis may be define as a change in the nucleic acid sequence (or genetic material) of an organism at a specific predetermined location. SITE DIRECTED MUTAGENESIS M U T A T I O N 5

6. Directed mutagenesis may define is a change in the nucleic acid sequence (or genetic material) of an organism at a specific predetermined location. Site-directed mutagenesis is the technique for generating amino acid coding changes in the DNA (gene). By this approach specific (site-directed) change (mutagenesis) can be made in the base (or bases) of the gene to produce a desired enzyme. A large amount of experimental procedures have been developed for directed mutagenesis of cloned genes. A synthetic oligonucleotide complimentary to the area of the gene of interest but has the desired nucleotide change. An oligonucleotide is a short piece of DNA usually 10-30 nucleotide long. Directed mutagenesis can be done using: M13,Plasmid DNA, PCR, Random primers, Degenerate primers, Nucleotide analogs, DNA shuffling SITE DIRECTED MUTAGENESIS D I R E C T E D M U T A G E N A S I S 6

7. B A S I C M E C H A N I S M OF SITE DIRECTED MUTAGENASIS Gene in plasmid with target site mutation Denature the plasmid and anneal the oligonucleotide. primers containing the desired mutation Using the non-strand-displacing action of PfuTurbo polymerase, extend and incorporate the mutagenic primers resulting in nicked circular strands Digest the methylated, nonmutated parental DNA template with Dpn I Transform the circular, nicked dsDNA into super-competent cells After transformation the supercompetent cells repair the nicks in the mutated plasmid SITE DIRECTED MUTAGENESIS 7 Fig 1. Basic mechanism of site directed mutagenesis

8. METHOD FOR SITE DIRECTED MUTAGENESIS THE SINGLE PRIMER METHOD In the technique of oligonucleotide-directed mutagenesis, the primer is a chemically synthesized oligonucleotide (7-20 nucleotides long). It is complementary to a position of a gene around the site to be mutated. But it contains mismatch of or the base to be mutated. The starting material is a single-stranded DN A (to be mutated) carried in an M 13 , phage vector. On mixing this DNA with primer ,the oligonucleotide hybridizes with the complementary sequences, except at the point of mismatched nucleotide. Hybridization ( despite a single base mismatch) is possible by mixing at low temperature with excess of primer, and in the presence of high salt concentration SITE DIRECTED MUTAGENESIS M E T H O D FOR SITE D I R E C T E D MUTATIONS 8 Fig.No.2-single primer method

9. The addition of 4-deoxyribonucleoside triphosphates and DNA polymerase( usually klenow fragment of E.Coli DNA polymerase) replication occur. The oligonucleotide primer is extended to form a complementary strand of the DNA. The ends of the newly synthesized DNA are sealed by the enzyme DNA ligase. The double-stranded DNA ( i,e. M phage molecule) containing the mismatched introduced by nucleotide into E .coli transformation . The infected E. Coli cells produce M 13 virus particles containing either the original wild type sequence or the mutant sequence. It is expected that half of the phage M 13 particles should carry wild type sequence while the other half mutant sequence (since the DNA replicate semiconservatively). The double-stranded DNAs of M 13 are isolated. Oligonucleotide -directed mutagenesis by using plasmid DNA (instead of M 13 ) is also in use. SITE DIRECTED MUTAGENESIS M E T H O D FOR SITE D I R E C T E D MUTATIONS 9

10. There are some variations in use in the oligonucleotide directed mutagenesis,. SITE DIRECTED MUTAGENESIS M E T H O D FOR SITE D I R E C T E D MUTATIONS 10 Fig.No.3- Variations in oligonucleotide-directed mutagenesis

11. 11 CASETTEE MUTAGENESIS In casettee mutagenesis a, synthetic double stranded oligonucleotide (a small DNA fragment i.e., casettee) containing the requisite/desired mutant sequence is used. Casettee mutagenesis is possible if the fragment of the gene to be mutated lies between two restriction enzyme cleavage sites. This intervening sequence can be cut and replaced by the synthetic Oligonucleotide (with mutation). The plasmid DNA is cut with restriction enzymes (such as EcoR1 and Hind111). SITE DIRECTED MUTAGENESIS M E T H O D FOR SITE D I R E C T E D MUTATIONS Fig.No.4- Casettee mutagenesis

12. 12 SITE DIRECTED MUTAGENESIS M E T H O D FOR SITE D I R E C T E D MUTATIONS Fig.No.5- PCR-based mutagenesis

13. The PCR-based mutagenesis technique commonly employed is depicted in First the target DNA (gene) is cloned on to a plasmid vector and distributed in to two reaction tubes. To each tube are added two primers ( oligonucleotides synthesized by using PCR). One primer ( A in tube 1 and C in tube 2) is complementary to a region in one strand of the cloned gene except for one nucleotide mismatch( i.e. the one targeted for a change). The other primer (B in tube 1 and D in tube2 ) is fully complementary of a sequence in the Other strand with in or adjacent to the cloned gene. The placement of primers for hybridization ( with the DNA strands) in each tube is done in opposite direction. The PCR technique is carried out for amplification of the DNA molecule. The products of PCR in the two reaction tubes are mixed. The DNA molecules undergo denaturation and renaturation. A Strand from one reaction tube (strand A) hybridizes with its complementary strand from other reaction tube (strand C). 13 SITE DIRECTED MUTAGENESIS M E T H O D FOR SITE D I R E C T E D MUTATIONS

14. Site-directed mutagenesis is used to generate mutations that may produce rationally designed protein that has improved or special properties Investigative tools  - specific mutations in DNA allow the function and properties of a DNA sequence or a protein to be investigated in a rational approach. Commercial applications  - proteins may be engineered to produce proteins that are tailored for a specific application. Example, commonly-used laundry detergents may contain subtilise in whose wild-type form has a methionine that can be oxidized by bleach, inactivating the protein in the process. This methionine may be replaced by alanine, thereby making the protein active in the presence of bleach. Site-directed mutagenesis has been widely used in the study of protein functions .  A P P L I C A T I O N SITE DIRECTED MUTAGENESIS 14

15. Any amino acid in a protein can be selectively replaced with another naturally occurring amino acid The replacements are made at the genetic level by modifying the codon to incorporate the new amino acid Characterizing the mutant enzyme that is obtained will provide information on the role of the amino acid that has been replaced The only unequivocal result from mutagenesis studies is when the mutation has no effect on the enzyme’s function. SITE DIRECTED MUTAGENESIS C O N C L U S I O N 15

16. Mutations are caused by radiation, viruses, transposes and mutagenic chemicals, as well as errors that occur during meiosis or DNA replication. Site-directed mutagenesis, also called site-specific mutagenesis or  oligo nucleotide-directed mutagenesis, is a molecular biology technique often used in bio molecular engineering in which a mutation is created at a defined site in a DNA molecule. Directed mutagenesis may define is a change in the nucleic acid sequence (or genetic material) of an organism at a specific predetermined location. Mutation, a change in the nucleic sequence (bases) of an organism’s genetic material (a change in the genetic material of an organism). SITE DIRECTED MUTAGENASIS SUMMARY 16

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