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

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.

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.

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.

Existing sequence5′ggacgcaagc-------------aggacattga 3′3′cctgcgttcgac------------- tcctgtaact 5’

Desired sequence5′ggaTCcaagc-------------aggacattga 3′3′cctAGgttcgac------------- tcctgtaact 5′

PCR for Base Substitutions.

PCR for Terminal AdditionsPCR for Deletions

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.

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.

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.

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.

Primer Extension for Longer Additions.

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.

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

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

Thank you