In vitro mutagenesis

Transcription

1 Core course BMS361N Genetic Engineering In vitro mutagenesis Prof. Narkunaraja Shanmugam Dept. Of Biomedical Science School of Basic Medical Sciences Bharathidasan University

2 In vitro mutagenesis Many methods are available Random mutagenesis Site directed mutagenesis Deletion mutagenesis Insertion mutagenesis Random mutagenesis is used generally to screen particular genetic functional analysis

3 Mutagenesis Mutagenesis One can clone gene to express it in a specific host organism to produce a large amount and very pure protein that can be used commercially. However physico chemical properties of these proteins (which are naturally occurring) are often not suited for particular task at extreme environment. Ex. Glucose oxidase, purified naturally occurring enzyme s optimal temperature is at 37oC but if one commercialize this, harsh handling, changing environment may inactivate. To over come this type of problem, molecular biologist came up with idea to create or modify or changing aa encodes an enzyme with desired properties so that which can be used at both extreme conditions or particular task.

4 By using a set of techniques what specifically change aa encoded by a cloned gene Protein with properties that are better suited than naturally occurring enzymes. Ex. By altering both substrate binding and maximal rate of conversion of the substrate to product. By changing the thermal tolerance or ph stability or both. reaction. By changing an enzyme so that a cofactor is no longer required for large scale By changing substrate binding site to increase its specificity, so that non-specific reactions and its products are reduced. By increasing resistance to cellular protease, so that protease cannot act on it thereby increase the yield of proteins. By altering the allosteric regulation to diminish the impact of feedback inhibition and increase the yield.

5 Mutants Deletions One can delete one nucleotide to few hundred nucleotides depending upon their experimental need. Insertions One can insert one nucleotide to few hundred nucleotide depending upon their experimental need.

6 Deletion Deletion If RE site present in site where one wants to make deletion, they can use RE to digest and remove part of the DNA fragment from the cloned DNA fragments DNA Polymerase Klenow fragments 35kDa C N N 3-5 exonuclease activity DNA polymerase exonuclease C 68kDa

7 Few nucleotides Deletion BamH1 BamH1 BamH1 BamH1 Bam H1 digest G CCTAG GATCC G G CCTAG GATCC G T4 DNA ligase Bam H1 GGATCC CCTAGG GGATCC CCTAGG N C

8 Few nucleotides Deletion Removing few nucleotide Kpn I Bal 31 enzyme which chew few nucleotides Ligase

9 Deletion BamH1 BamH1 Bam H1 digest BamH1 BamH1 BamH1 BamH1 T4 DNA ligase T4 DNA ligase Mutated fragment Protein Translation Bam H1 GGATCC CCTAGG Mutated Protein

10 Few nucleotides Deletion GGATCC CCTAGG GGATCC CCTAG G T4 DNA ligase GGATCC CCTAGG GGATCC CCTAG G

11 Removing few nucleotide Kpn1 GGTAC C C CCATG Bal 31 enzyme which chew few nucleotides T4 DNA ligase Ligation Protein Translation Mutated Protein

12 Insertion BamH1 BamH1 G CCTAG GATCC G GATCC G G CCTAG

13 Insertion BamHI G GATCC CCTAG G T4 DNA ligase GATCC G G CCTAG Mutated Protein

14 Site directed mutagenesis By many method one can achieve this mutagenesis. Main mechanism is Oligonucleotides can be synthesized and used to make mutant DNA. This mutant DNA used to express mutant protein and then analyzed the mutant induced effect by comparing wild type protein.

15 Site directed mutagenesis Site Directed mutagenesis Site directed mutagenesis: A single nucleotide (point mutation) change can make various functions Ex. Single nucleotide can make» non-sense mutant» Sense mutant» Frame shift

16 The Good, the Bad, and the Silent Mutations can serve the organism in three ways: The Good : A mutation can cause a trait that enhances the organism s function: Mutation in the sickle cell gene provides resistance to malaria. The Bad : The Silent: A mutation can cause a trait that is harmful, sometimes fatal to the organism: Huntington s disease, a symptom of a gene mutation, is a degenerative disease of the nervous system. A mutation can simply cause no difference in the function of the organism. Campbell, Biology, 5 th edition, p. 255

17 Oligonucleotide-directed mutagenesis

18 Oligonucleotide-directed mutagenesis Plasmid based T4 DNA ligase P GGA GCT AAT TTA ATG CCT CGA ATA AAT TAG Klenow enzyme GGA GCT AAT TTA ATG CCT CGA ATA AAT TAG Transform to E.coli 50% 50% 5 P M13 + strand GGA GCT AAT TTA ATG CCT CGA ATA AAT TAG M13 + strand GGA GCT AAT TTA ATG M13 + strand CCTCGA ATAAATTAG Gly ala asn leu met GGA GCT AAT TTA ATG CCT CGA TTA AAT TAG CCT CGA ATA AAT TAG Gly ala tyr leu met Select the mutant by high stringency hybridization using mutant Oligo as probe. Because of some reason only 1-5% of mutated phage can be recovered.

19 Oligonucleotide-directed mutagenesis

20 Oligonucleotide-directed mutagenesis Site Directed mutagenesis T4 DNA ligase A TACTGCG ACGTCCGTTT ATGACGCATGCAGGCAA

21 TACTGCG A ACGTCCGTTT ATGACGCATGCAGGCAA Replicative Form RF

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24 By hybridization method one can isolate mutated Plasmid.

25 Colony Hybridization A way to screen plasmid-based genome libraries for a DNA fragment of interest Host bacteria containing a plasmid-based library of DNA fragments are plated on a petri dish and allowed to grow overnight to form colonies Replica of dish made with a nitrocellulose disk

26 Disk is treated with base or heated to convert dsdna to ssdna and incubated with probes Colonies that bind probe (with P-32) hold the fragment of interest

27 Specific sequences are detectable by hybridization

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30 Confirmation by DNA sequencing AAA Lys ATA Ile

31 Modified Oligo-directed mutageneis Oligo-directed mutagenesis is modified in a number of ways to enrich number of mutant phage. One of the method is: Use of different E.coli strains one of the E.coli strain has 2 defective DNA metabolism enzymes (1) defective form of dutpase (dut - ) (2) defective form of Uracil N-glycosylase (ung - ) Because of these defective enzymes incorporation of UTP residue cannot be remove. So the genomic DNA and vectors transformed into this E.coli strains have UTPs in the place of TTPs. By make use of these properties one can enrich mutant vectors.

32 Modified Oligo-directed mutageneis Transform to E.coli dut ung M13 vector RF U U U U U U U U U U U U U U U U Phage M13 + strand CCTCGA ATAAATTAG T U T U T U T T T T T U U U U U U U U U U T T T T T T U U U T T T4 DNA ligase Transform into Wild type E.coli (dut + ung + M13 vector RF U U U U U U U U U U U U U U U U

33 Modified Oligo-directed mutageneis Using Ds Plasmid Target Heat v Amp S Tet r Grow it on Tetracycline plate NaOH V V DNA synthesis Transform Mutated Target Amp r Tet s Grow it on ampicilin plate

34 PCR based Oligonucleotide directed mutagenesis v v v v v v v v nick nick Circular with nick on it Circular with cut on it

35 PCR based Mutation x x 1F x 2F x 2R 1R 2F PCR with 2F x 2R 1F x PCR with 1R 1R x x x x x

36 Core course BMS361N Genetic Engineering The End Prof. Narkunaraja Shanmugam Dept. Of Biomedical Science School of Basic Medical Sciences Bharathidasan University