SITE DIRECTED MUTAGENESIS.HARISPresentation Transcript
SITE 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 + polymerase + primer replication Mutant translation Wild type translation Only one amino acid changed Wild type protein Mutant protein primer (1) (2) (3) (5) (4) (6) Val -> Thr Smith (1993) Juang RH (2004) BCbasics CAG GTC CAG G C C CAG G C C CAG G C C C G G Thr GTC CAG Val
Primer design for site-directed mutagenesis
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 SDM
Oligo-nucleotide directed mutagenesis
SDM using PCR
Random chemical mutagenesis
Oligo-NT directed mutagenesis
SDM using PCR
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 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 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 websites
Zheng et al. An efficient one-step site-directed and site-saturation
mutagenesis protocol. Nucleic Acids Res. 2004 Aug 10;32(14):e115 .