Site Directed Mutagenesis

1. Site Directed Mutagenesis

2. What Is a Mutation? What Is a Mutation? What Is a Mutation? • Genetic information is encoded by the sequence of the nucleotide bases in DNA of the gene. The four nucleotides are: adenine (A), thymine (T), guanine (G), and cytosine (C), a mutation is a change in the order of these nucleotides. • A change in the order can cause the gene to encode for wrong proteins and inhibit the function of the gene or cause the gene to be virtually inactive. • Genetic information is encoded by the sequence of the nucleotide bases in DNA of the gene. The four nucleotides are: adenine (A), thymine (T), guanine (G), and cytosine (C), a mutation is a change in the order of these nucleotides. • A change in the order can cause the gene to encode for wrong proteins and inhibit the function of the gene or cause the gene to be virtually inactive. • Genetic information is encoded by the sequence of the nucleotide bases in DNA of the gene. The four nucleotides are: adenine (A), thymine (T), guanine (G), and cytosine (C), a mutation is a change in the order of these nucleotides. • A change in the order can cause the gene to encode for wrong proteins and inhibit the function of the gene or cause the gene to be virtually inactive.

3. Random mutagenesis • -> based on the process of natural evolution • - NO structural information required • NO understanding of the mechanism required • General Procedure: • Generation of genetic diversity • Random mutagenesis • Identification of successful variants • Screening and seletion

4. Site-directed Mutagenesis Site Directed Mutagenesis is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule known as a plasmid. Wild-type gene sequence must be known. Or Site Directed Mutagenesis is a powerful technique where site specific changes in DNA sequence are produced in vitro-for instance to change an amino acid residue into another by changing the codon sequence within the gene sequence.

5. CAG GTC CGG Val CAG GCC GCC CAG Thr GCC CAG GTC CAG Site-directed mutagenesis Wild type (5) translation (1) Wild type protein + primer Mutant (2) primer translation (6) + polymerase (3) Mutant protein (4) Only one amino acid changed replication Val → Thr Smith (1993)

6. Genetics Mutant phenotype Mutant allele DNA sequence Protein sequence

7. Why Site directed mutagenesis -> site-directed mutagenesis -> point mutations in particular known area result -> library of wild-type and mutated DNA (site-specific) not really a library -> just 2 species -> random mutagenesis -> point mutations in all areas within DNA of interest result -> library of wild-type and mutated DNA (random) a real library -> many variants -> screening !!! if methods efficient -> mostly mutated DNA

8. INVENTION Site Directed Mutagenesis using oligonucleotide was first described in 1978 by Michael Smith & shared Nobel Prize in chemistry in October 1993 with Kary B. Mullis who developed the PCR technique. Site –directed mutagenesis Requirements: ->Knowledge of sequence and preferable Structure (active site,….) ->Understanding of mechanism (knowledge about structure – function relationship)

9. Site Directed Mutagenesis • Cassette mutagenesis • Oligonucleotide directed mutagenesis • Using M13 DNA OR • Using Plasmid DNA 3. PCR amplified Oligonucleotide directed mutagenesis 4. Random mutagenesis • With Degenerate Oligonucleotide primers or • Using Nucleotide Analogues

10. CASSETTE MUTAGENESIS • Cleavage by a Restriction Enzyme (RE) at a particular site in the plasmid. • Ligation of an Oligonucleotide containing Mutation in the gene of interest to the plasmid. • RE that cuts at the plasmid and Oligonucleotide is same, permitting sticky ends of the plasmid & inserts to ligate to one another.

11. Site-directed mutagenesis methods – Oligonucleotide - directed method Oligonucleotides are… • Synthetic single-stranded fragments of DNA used for priming the mutated clones. • In order to work, the primers must meet the following criteria: • -must contain desired mutation. • -mutation should be in the middle of the primer. • -the GC content should be at a minimum of 40% and should terminate in one or more of C or G bases.

12. Oligonucleotide directed mutagenesis • Basic Methods • 1. Base pair substitution: Change of one nucleotide (A-> C) • 2. Insertion: Gaining one additional nucleotide • 3. Deletion: Loss of one nucleotide

14. Deletion Mutagenesis

15. Oligonucleotide directed mutagenesis with M13 DNA: 1.Synthesize an Oligonucleotide containing the changed sequence. Ex.---ATT---Wild type sequence (Codon for ILe) ---CTT---Desired Change (Leu) ---GAA---Mutagenic Oligonucleotide (MO) 2.Hybridize MO ss form of gene cloned into M13. 3.Synthesize second strand of DNA with KLENOW fragment &dNTPs. 4.Seal nick in new strand with T4 DNA ligase. 5.Introduce into E. Coli. 6.ss + phage isolated from plaques & screened by hybridization.

16. Oligonucleotide Directed Mutagenesis Using Plasmids Increases the chances of obtaining the desired mutation in the target gene • Co selection technique • Antibiotic resistance gene restored and a second one eliminated • Approx 90% of Ampr and Tets clones have target mutated in directed fashion

17. 5’ * 3’ 3’ 5’ * Primer design for PCR-Amplified Oligonucleotide-directed mutagenesis • Both primers must contain the mutation. • The mutation should be in the middle of the primer. • Primers should be 25-45 nucleotides long and have a GC content of at least 40%. • The melting temperature (Tm) should be ≥ 78˚C. • The 3’-end of the primer has to end on an C or a G.

18. Site-directed mutagenesis methods -PCR based • Perform two separate PCR rx. With two sets of primers. • Each primer set introduces the desired nucleotide change but produces linear PCR products with different ends. • Denaturation, mixing & ligation of PCR products produces mutated circular DNA that can be introduced into E. Coli by transformation.

19. Random Mutagenesis Using Degenerate Oligonucleotide Primers • Make primers at sites to be mutated which are degenerate at one or more sites • Random changes in nucleotide sequences of the primer are introduced during chemical synthesis of a pool of degenerate oligonucleotides. • Must express mutant genes & screen gene products to find the improved version of the PROTEIN.

20. Random Mutagenesis With Nucleotide Analogue • Cut clone to side of target gene • Digest with ExoIII • Synthesize with DNAP I, dNTPs and a dNTP analog.Ex.N4-hydroxy cytosine is an analog of cytosine. • Presence in DNA causes an A-T base pair transition mutation when the DNA is replicated by the host. Ex. • ---AAA---(Lys) to ---GAA---(Glu) • ---TTT---…………---CTT--- • Analog causes replication errors when transformed into E. coli

21. Uses of Site Directed Mutagenesis • 1.Site Directed Mutagenesis is also used to ‘engineer’ commercially important Proteins for many different purposes, for example • Improve stability • Change specificity • Reduce toxicity • 2.It can be used to produce proteins which can ‘improve’ laundry detergents • SUBTILISIN, a bacterial serine-protease that can breakdown most proteins which soil clothing & a major additive to modern ‘BIO’ detergents. Before it became commercially useful, the recombinant protein was improved in order that it remained active in the presence of Bleach. Bleach caused the oxidation of a methionine residue (position 22) & the enzyme lost 90% of its activity. By replacing this amino acid with alanine, the enzyme was no longer sensitive to oxidation & found best in terms of ACTIVITY &STABILITY.

22. 3. Site Directed Mutagenesis enabled new approaches to drug designing – particularly in order to improve FUNCTION. 4.Random Mutagenesisis is used to construct large &diverse clone libraries of mutated DNA fragments.

23. FUTURE ASPECTS • Attempts are being made to produce biotechnically a mutated haemoglobin which may give us a new means of replacing blood. • By mutating proteins in the immune system researchers have come a long way towards constructing antibodies that can neutralize cancer cells. • Future also holds possibilities of gene therapy, curing hereditary diseases by specifically correcting mutated code words in the genetic material. • Site directed mutagenesis of plant proteins is opening up the possibility of producing of producing crops that can make more efficient use of atmospheric carbon dioxide during photosynthesis.

24. Conclusion: • any amino acid in a protein can be selectively replaced with another 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

25. REFERENCES: Old R.W. and Primrose S.B. (2001), Principles Of Gene Manipulation, Blackwell Scientific Publications, Page (132-138) Brown T.A. (2001) Gene cloning and DNA analysis – An Introduction Blackwell Publication Page – (236 – 241) Watson J.D., Gilman M, Zoller M and Witkowski(1992) Recombinant DNA, Scientific American Books, Page (191-207) En. Wikipedia. Org/wiki /site directed mutagenesis 32K WWW. Che.Utoledo. Edu / nadarajah / WebPages / mutagene. Htm.14K Glick, Molecular Biotechnology Page (171 – 183)