Transposon mutagenesis & site directed mutagenesis

MICROBIOLOGY

1. TRANSPOSON MUTAGENESIS & SITE DIRECTED MUTAGENESIS PRESENTED BY: MOUSAMI JARIA ST. GEORGE COLLEGE OF MANAGEMENT AND SCIENCE MSC MICROBIOLOGY SEMESTER 2

2. WHAT ARE TRANSPOSONS? Transposons or transposable elements(TEs) also known as “jumping genes” are DNA sequences that move from one location on the genome to another . TEs are found in almost all organisms ,(but are best understood in bacteria) and typically in large numbers. Types : Retro transposons , or class 1 TEs DNA transposons or class 2 TEs.

3. TRANSPOSON INSERTION IN A GENE

4. TRANSPOSON MUTAGENESIS Transposon mutagenesis is a biological process that allows genes to be transferred to a host organism’s chromosome, interrupting or modifying the function of gene on the chromosome and causing mutation. It is much more effective than chemical mutagenesis, with a higher frequency and a lower chance of killing the organism.

5. TRANSPOSON MEDIATED MUTAGENESIS

6. HISTORY It was first studied by Barbara McClintock in mid 20 th century. In early 1940s McClintock was studying the progeny of self pollinated maize plants. These plants were missing their telomeres. This research prompted the first discovery of a transposable element , from their transposon mutagenesis have been exploited as biological tool.

7. BARBARA McCLINTOCK

8. TRANSPOSON AS TOOLS FOR MUTAGENESIS A transposon used for mutagenesis should have following properties: It should transpose at a fairly high frequency It should not be very selective in its target sequence It should carry an easily selectable gene, such as one for resistance and one for antibiotic. Should have broad host range for transposition

9. TRANSPOSON MUTAGENESIS IN VIVO Transposon Tn5 is ideal for random mutagenesis of gram negative bacteria as it embodies all of its features. Tn5 transpose at a relatively high frequency but has no target specificity. It also carries a kanamycin resistance gene that is expressed in most gram negative bacteria. ADVANTAGE: The target organism does not have to be naturally competent.

10. DISADVANTAGE: The transposon must be introduced into the host on a suicide vector , which may give some residual false positive results for transposon insertion mutants if the suicide vector is capable of limited replication. It is not very effective and requires powerful positive selection techniques to isolate the mutants. If DNA sequence or specific plasmid is to be mutated , there is no target specificity to insertion mutants so transposon hops into chromosome most of the time. There is also possibilty of multiple transposon events.

11. TRANSPOSON MUTAGENESIS IN VITRO This technology is made possible by the fact that the transposase enzyme by itself performs the reactions of ‘’cut and paste phase’’ transposition reaction. In the procedure the target DNA is mixed with a donor DNA containing the trasnposon , and the purified tansposase is added allowing the transposon to insert into the target DNA in the test tube. Multiple transposases have been adapted for this process.

12. ADVANTAGES: It has the ability to reach high saturation levels of mutagenesis, which allows one to conduct annalysis of the target locus on either large or small scales. DISADVANTAGE: It has the prerequisite for preliminary information on the target sequence.

13. APPLICATIONS Virulence genes in viruses and bacteria can be discovered by disrupting genes and observing for a change in phenotype. Non essential genes can be discovered by inducing transposon mutagenesis in an organism with the help of PCR and ORF specific primer. Cancer causing genes can be identified by transposon mutagenesis and screening of mutants containing tumours .

14. SITE DIRECTED MUTAGENESIS Site directed mutagenesis(SDM) is an in vitro technique for introducing mutation or alteration into the targeted (known) DNA sequence. There are many reasons to make specific DNA alterations including: To study changes in protein activity that occur as a result of DNA manipulation. To select or screen for mutations that have desired property To introduce or remove restriction endonuclease sites or tags.

15. SCHEMATIC REPRESENTATION OF SITE DIRECTED MUTAGENESIS

16. DIFFERENT TECHNIQUES Three approaches are most commonly used for different techniques for site directed mutagenesis: Conventional PCR Nested PCR/ Primer Extension Inverse PCR

17. CONVENTIONAL PCR: In this method PCR primers are designed in a manner which contains mutation. The Taq DNA polymerase used in the conventional PCR does not have exonuclease activity hence it cannot identify mismatch during the amplification The major reccomendation for the conventional PCR based mutagenesis is to insert mutant bases up to several limit at 5’ end of the primer or in the middle of the primer . LIMITATION: It carries mutant as well as non mutant DNA due to the presence of template DNA , hence the yield is lower.

18. B. PRIMER EXTENSION/NESTED PCR : Two sets of primers are used in which a single set of primer is nested. The mutation is introduced to the primer at one end C. INVERSE PCR : The primers amplify the fragment other than the target sequence , hence it amplifies in the reverse orientation. The method is used for inserting mutation into the plasmid having the gene of our interest . The fidelity DNA polymerase is used to do the amplification as well as to linearise the circular plasmid DNA Many nucleotides can be deleted by using inverse PCR.

19. DELETION BY INVERSE PCR

20. IMPORTANCE The site directed mutagenesis is used to remove restriction sites. Restriction digestion is a process in which the DNA having the recognition site for a particular restriction endonuclease is cleaved into fragments. If any mutation is introduced at the recognition site of REase , it cannot cut it .This can be done by site directed mutagenesis. At molecular level, the properties of a molecular gene or proteins can be screened or studied with the help of site directed mutagenesis.

21. APPLICATIONS SDM helps to improve the quality of protein by removing harmful elements from it. The tool is used in study of a gene characteristics. Used in gene synthesis and gene editing technology. Used in cloning. It is also useful in the screening of single nucleotide polymorphisms(SNPs).

22. CONCLUSION SDM has its own importance in the field of gene editing and gene manipulation It facilitates improvement in the wild type genotype to produce a commercially important phenotype SDM has employed in the knockout mice construction and gene knockout studies.