This describes about the basic principle behind the site directed mutagenesis by pcr............

1. Site Directed
Mutagenesis By PCR
Submitted By, Submitted To,
POORANACHITHRA M DR.P.S. SUDHAKAR
GANDHI,
Ist M.Th. Biotechnology, Assistant Professor,
Dept. Of Biotechnology Dept. Of Biotechnology,
Bharathidasan Institute Of Technology, Bharathidasan Institute Of Technology,
Anna University, Trichy-620024. Anna University, Trichy-620024

2. Site Directed Mutagenesis
Site-directed mutagenesis is a molecular biology method that is used
to make specific and intentional changes to the DNA sequence of
a gene and any gene products.
Also called as site-specific mutagenesis or oligonucleotide-directed
mutagenesis.
This can be done by using oligonucleotides in a primer extension
method with DNA polymerase (in PCR), developed by Michael
Smith who was awarded a Nobel Prize in 1993 for this contribution.

3. Principle Behind PCR Based SDM
The principle of site-directed mutagenesis is that a mismatched
oligonucleotide is extended, incorporating the "mutation" into a strand
of DNA that can be cloned.
Here the synthetic primer contains the desired mutation and is
complementary to the template DNA around the mutation site so it
can hybridize with the DNA in the gene of interest
The single-strand primer is then extended using a DNA polymerase,
which copies the rest of the gene.

4. Different Approaches Of PCR based SDM
Site-directed is typically performed using PCR in 2different ways,
1.normal PCR with mutated primer
2. primer extension method
Primers designed with mutations can introduce small sequence changes
 primer extension can be used to achieve longer mutant regions.

5. Existing sequence
5′ggacgcaagc-------------aggacattga 3′
3′cctgcgttcgac------------- tcctgtaact 5’
Desired sequence
5′ggaTCcaagc-------------aggacattga 3′
3′cctAGgttcgac------------- tcctgtaact 5′
PCR for Base Substitutions.

6. PCR for Terminal AdditionsPCR for Deletions

7. Primer Extension for an Insertion:
 Primers B and C contain the complementary
sequence that will be inserted (indicated by
the blue line).
The first round of PCR uses two reactions
with primer pairs A/B (1) and C/D (2).
The two resulting PCR products are mixed
together with primer pair A/D for a second
round of PCR.

8. The overlapping regions of the two,
first-round PCR products allow the
strands to hybridize and the second
round of PCR creates the final,
fulllength product with the desired
insertion.

9. Primer Extension for Délétions:
Primers B and C are located on either side
of the sequence to be deleted and contain
sequence from both sides of the deletion
(indicated by black or gray additions that
match the black or gray original sequence).
This sequence will allow them to overlap
with the other fragment after the first round
of PCR. The first round of PCR uses primer
pairs A/B and C/D.

10. The two resulting PCR products are mixed
together with the primer pair A/D for a
second round of PCR.
The overlapping regions of these two, first-
round PCR products allow the strands to
hybridize and the second round of PCR
creates the final, full-length product with the
desired area deleted.

11. Primer Extension for Longer Additions.

12. Applications
Site-directed mutagenesis is used to generate mutations that may
produce rationally designed protein that has improved or special properties
(i.e. Protein engineering)
This method of altering the sequence allows researchers to investigate the
impact of sequence changes, such as single nucleotide polymorphisms (SNPs),
or to insert or delete a sequence element, such as a ligand binding site or
restriction site.

13. With PCR based methods it is hard to replicate the mutated DNA,
in order for replication to occur super competent cells must be used
and are expensive!
Screening can be tedious, usually requires sequencing to confirm if
mutation occurred.
Limitations

14. Reference
Site-directed mutagenesis - From Wikipedia, the free encyclopedia
PCR-Based Site-Directed Mutagenesis - Atsushi Shimada
Site-directed mutagenesis - Albert Jeltsch, Thomas Lanio
A simple method for site-directed mutagenesis using the polymerase chain reaction -
Anne Hemsley, Norman Arnheim, Michael Dennis Toney, Gino Cortopassi and David
J.Galas
Ultramer™ Oligonucleotides - Adam Clore PhD, Brian Reinertson, and Scott Rose PhD

15. Thank you