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SITE DIRECTED MUTAGENESIS.HARIS
 

SITE DIRECTED MUTAGENESIS.HARIS

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  • 定點突變 是使用人工合成的引子,先與目標基因雜合 (2) ,而此引子的核苷酸序列與目標基因互補,但其上有一個核苷酸變異 (GTC->G C C) 。然後補滿雙股核酸 (3) ,再把此混成基因轉殖進入宿主,則宿主菌的複製系統將會因複製而產生兩種質體 (4) ,其一為原來的野生型,轉譯可得原來的蛋白質 (5) ;另一則為突變型,轉譯則得到突變的蛋白質,但只改變了一個指定位置的胺基酸 (6) 。

SITE DIRECTED MUTAGENESIS.HARIS SITE DIRECTED MUTAGENESIS.HARIS Presentation Transcript

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  • 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
    • 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 SDM
    • Oligo-nucleotide directed mutagenesis
    • Cassette mutagenesis
    • SDM using PCR
    • Random chemical mutagenesis
  • Oligo-NT directed mutagenesis
  • SDM using PCR
  • Cassette 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 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
            • 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 .
  • Thank You haris.p mac ernakulam