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

    1. 2. SITE DIRECTED MUTAGENESIS <ul><li>It’s a new in-vitro technique in which specific change in specific location can be brought about in a gene sequence of interest. </li></ul>
    2. 3. <ul><li>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 </li></ul>
    3. 4. <ul><li>any amino acid in a protein can be selectively replaced with another amino acid </li></ul><ul><li>the replacements are made at the genetic level by modifying the codon to incorporate the new amino acid </li></ul>
    4. 5. 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
    5. 7. Primer design for site-directed mutagenesis <ul><li>Stratagene: </li></ul><ul><li>Primers must contain the mutation. </li></ul><ul><li>The mutation should be in the middle of the primer. </li></ul><ul><li>Primers should be 25-45 nucleotides long and have a GC content of at least 40%. </li></ul><ul><li>The melting temperature (Tm) should be ≥ 78˚C. </li></ul><ul><li>The 3’-end of the primer has to end on an C or a G. </li></ul>5’ 5’ 3’ 3’ * *
    6. 8. TYPES OF SDM <ul><li>Oligo-nucleotide directed mutagenesis </li></ul><ul><li>Cassette mutagenesis </li></ul><ul><li>SDM using PCR </li></ul><ul><li>Random chemical mutagenesis </li></ul>
    7. 9. Oligo-NT directed mutagenesis
    8. 10. SDM using PCR
    9. 11. Cassette Mutagenesis <ul><li>Used if the segment to be mutated lies between two close-spaced, unique, RE- cleaving sites. </li></ul><ul><li>Intervening sequence is excised and replaced by chemically synthesised oligonucleotide containing the required mutation. </li></ul><ul><li>Oligo-NT containing a mix. of substitutes at a particular site used to generate large type of mutants. </li></ul>
    10. 12. Random Chemical Mutagenesis <ul><li>Chemical mutagens used </li></ul><ul><li>These chemical mutagen must act on ss-DNA </li></ul><ul><li>Eg : Na bisulphite </li></ul><ul><li>Mutated DNA is then used as template and copy is generated. </li></ul>
    11. 13. Chemical Target Na bisulphite Nitrous acid Formic acid Hydrazine dC dT dC Du dA dHX Glycosyl bonds break. Pyrimidine ring break.
    12. 14. Applications of SDM <ul><li>Is the basis of protein engineering. </li></ul><ul><li>To get mutated forms of enzyme gene : </li></ul><ul><li>to study the structure , function and stability of an enzyme </li></ul><ul><li>Used to improve the effect of f(n)s of antibodies and their affinity </li></ul><ul><li>Can be used to change the aa pattern of storage proteins in grains so that deficiency of aa,if any can be removed. </li></ul>
    13. 15. Papers and useful websites <ul><ul><ul><ul><ul><li>Site-directed mutagenesis </li></ul></ul></ul></ul></ul><ul><li>http://www.stratagene.com/ </li></ul><ul><li>http://www.invitrogen.com/ </li></ul><ul><li>Zheng et al. An efficient one-step site-directed and site-saturation </li></ul><ul><li>mutagenesis protocol. Nucleic Acids Res. 2004 Aug 10;32(14):e115 . </li></ul>
    14. 16. Thank You haris.p mac ernakulam

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