SITE DIRECTED MUTAGENESISSITE DIRECTED MUTAGENESIS

It’s a new in-vitro technique in which specific change in specific location can be brought about in a gene sequence of interest.

Mutagenesis gives us the capability of testing the role of any amino acid in a protein by replacing it with any of the other naturally occurring amino acids

•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

Basic Mechanism of Site-directed mutagenesis

CAGGTC

CAG

GCC

CAG

GCC

CAG

+ polymerase

+ primer

replication

GCC

CGG

Mutant

Thr

translation

Wild type

GTC

CAG

Val

translation

Only one amino acid changed

Wild type protein

Mutant protein

primer

(1)

(2)

(3)

(5)

(4)

(6)

Val → ThrSmith (1993) Ju

an

g R

H (

20

04

) B

Cb

asi

cs

Primer design for site-directed mutagenesisPrimer design for site-directed mutagenesis

Stratagene:

• 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.

5’

5’

3’

3’

*

*

TYPES OF SDMTYPES OF SDM

Oligo-nucleotide directed mutagenesis Cassette mutagenesis SDM using PCR Random chemical mutagenesis

Oligo-NT directed mutagenesisOligo-NT directed mutagenesis

SDM using PCRSDM using PCR

Cassette MutagenesisCassette Mutagenesis

• Used if the segment to be mutated lies between two close-spaced, unique, RE- cleaving sites.

• Intervening sequence is excised and replaced by chemically synthesised oligonucleotide containing the required mutation.

• Oligo-NT containing a mix. of substitutes at a particular site used to generate large type of mutants.

Random Chemical MutagenesisRandom Chemical Mutagenesis

Chemical mutagens used These chemical mutagen must act on ss-DNA Eg : Na bisulphite Mutated DNA is then used as template and copy

is generated.

Chemical Target

Na bisulphite

Nitrous acid

Formic acid

Hydrazine

dC dT

dC Du

dA dHX

Glycosyl bonds break.

Pyrimidine ring break.

Applications of SDMApplications of SDM• Is the basis of protein engineering.• To get mutated forms of enzyme gene :• to study the structure , function and

stability of an enzyme• Used to improve the effect of f(n)s of antibodies and

their affinity• Can be used to change the aa pattern of storage

proteins in grains so that deficiency of aa,if any can be removed.

Papers and useful websitesPapers and useful websites

Site-directed mutagenesis

• http://www.stratagene.com/

• http://www.invitrogen.com/

• Zheng et al. An efficient one-step site-directed and site-saturation

mutagenesis protocol. Nucleic Acids Res. 2004 Aug 10;32(14):e115.