Post on 01-Jun-2015
transcript
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.