site directed mutagenesis .pptx

Detailed explanation about site directed mutagenesis, history, mechanisms, applications and disadvantages.

1. SITE DIRECTED MUTAGENESIS By Sushmitha

2. Mutation alteration in the nucleotide sequence of the genome of an organism , virus , or extrachromosomal DNA . Types Germline/ somatic mutation - gametes/ body cell Chromosomal alteration- chromosome structure Point mutation- single nucleotide Frameshift mutation - shift in reading frame

3. Site directed mutagenesis Molecular biology method- make specific&intentional changes DNA seq. Investigating stuct& bio activity of DNA,RNA,protein. Imp lab techniq for creating DNA libs by introducing mutations into DNA seq. Numerous methods r there- but low cost of oligonucleotide ,artificial gene syn is used as alternative method. Other names Site specific mutagenesis Oligonucleotide directed mutagenesis

4. History Early attempts at mutation- radiation or chemical mutagens-non specific , generating random mutation. Nucleotides &other chemicals were used to generate localized point mutation -aminopurine,nitroguanidine,bisulfate. 1974-Charles Weissmann- N 4 -hydroxycytidine - GC to AT. 1971-Clyde Hutchison& Marshall Edgell -produce mutants with phage ϕX174 and restriction nucleases . 1978-Hutchison& Michael smith - primer extension method. 1993 Michael Smith & Kary B Mullis invent PCR - Nobel prize in chemistry.

5. Mechanism Requires synthesis of short primer. Synthetic primer- desired mutations &complementary to template strand. Mutation may be single/multiple base change ,deletion,addition. Single strand primer-extended using DNA polymerase- copies rest of genes. Gene copied contain mutated site- introduced through vector into host cell &cloned. Mutants are selected by dna sequencing to check. Due to low yield of this method ,several other methods have been found.

6. Mechanism

7. Other methods Kunkel’s method Cassette mutagenesis PCR method Whole plasmid In vivo method CRISPR method

8. Applications Investigative tools -specific mutation in seq allows the function & property of DNA& protein seq to be investigated. Eg . Phosphorylation investigated-Particular serine(Phosphate acceptor) to alanine(Phosphate non acceptor) Comercial application : laundry detergents. Subtilisin wild type has methionine can be oxidised by bleach-protein activity 🔻 methionine 🔄alanine making resistant to oxidation in presence of bleach-protein activity 🔺 Oligonucleotide rate🔻 possible to synthesis mutated complete gene.

9. Disadvantages Long amplification protocol - 25cycles ( b/w 4 to 8hrs ) . Low yield of amplified DNA Tandem primer insertion at or near the mutated site often occurs. Large insertion and deletion are problematic. 5’ 5’ 3’ 5’ 5’ 3’ 3’ 3’ Template strand Site to be mutated Primer Mutated strand with tandem primer