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

5. 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.

6. TRANSPOSON MEDIATED
MUTAGENESIS

7. HISTORY
 It was first studied by Barbara McClintock in
mid 20th 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.

8. BARBARA
MCCLINTOCK

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

10. 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.

11. 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.

12. 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.

13. 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.

15. 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.

16. 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:
1. To study changes in protein activity that occur
as a result of DNA manipulation.
2. To select or screen for mutations that have
desired property
3. To introduce or remove restriction
endonuclease sites or tags.

17. SCHEMATIC REPRESENTATION OF
SITE DIRECTED MUTAGENESIS

18. DIFFERENT TECHNIQUES
 Three approaches are most commonly used for
different techniques for site directed
mutagenesis:
A. Conventional PCR
B. Nested PCR/ Primer Extension
C. Inverse PCR

19. A. 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.

20. 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.

21. DELETION BY INVERSE
PCR

22. 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.

23. 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).

24. 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.