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Download Random Mutagenesis Why Mutagenesis?
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Why Mutagenesis? • Analysis of protein function June 09, 2009 Random Mutagenesis Site-directed vs. random Site-directed • Knowledge about sequence / structure • Insertion of single mutations possible • Number of mutants limited • Time-consuming Analysis of highly conserved postions Random • Less information about relevant positions • Saturating analysis impossible • Introduction of more than one mutation • Efficient screening system Generation of mutant libraries • Analysis of protein structure • Protein engineering – Analysis of structure-function relationship – Analysis of the catalytic center – Design of proteins with novel features How to insert mutations randomly • E. coli XL1red • UV irradiation • Chemical methods – Deamination – Alkylation – Base-Analog Mutagens • PCR based methods – DNA shuffling – Error prone PCR – Site directed random mutagenesis DNA Shuffling PCR based random mutagenesis • Slightly different genes coding for the same product • Homologous recombination of genes • Generation of gene libraries Recombination of sequence blocks • In vitro molecular evolution 1 DNA Shuffling Error Prone PCR Denaturization Parent sequence • Analysis of large protein regions • PCR conditions enhance mismatches DNase treatment PCR w/o primers • Saturating mutagenesis virtually impossible • Combination of mutations Random fragments Extension via polymerase Shuffled produkt Error Prone PCR Error Prone PCR lacks 3’→5’ exonuclease activity posseses intrinsic error rate Taq lacks 3’→5’ exonuclease activity posseses intrinsic error rate Taq dCTP, dTTP dCTP, dTTP ↑ dGTP, dATP dGTP, dATP ↓ Mg2+ Mg2+ Error Prone PCR Taq dCTP, dTTP ↑ dGTP, dATP ↓ Mg2+ ↑ promotes misincorporation Error Prone PCR lacks 3’→5’ exonuclease activity posseses intrinsic error rate Taq promotes misincorporation dCTP, dTTP ↑ stabilizes non-complementary base pairs Mg2+ ↑ dGTP, dATP ↓ Mn2+ lacks 3’→5’ exonuclease activity posseses intrinsic error rate promotes misincorporation stabilizes non-complementary base pairs reduces the specificity of the polymerase low annealing temperature template amount, cycle number 2 Site-directed Random Mutagenese Site-directed Random Mutagenese Restriction site • Analysis of conserved regions Restriction site • PCR with degenerated primers 5‘-CAC ATC AGA ACC NNN GTG TGG GTA AGA-3‘ • -NNN- represents 64 codons • 19 possible mutants per position Site-directed Random Mutagenese Restriction site Restriction site Site-directed Random Mutagenese Restriction site Restriction site NNN Site-directed Random Mutagenese Restriction site Restriction site Examples NNN 3 Example: DNA shuffling Example: Error Prone PCR Novel Enzymes • Enzyme engineering Ligand binding abilities • Conversion of antagonist to agonist • No hint, which AA involved • Combination of mutations necessary • Screening of mutant libraries DNA shuffling • Optimized function of Monooxygenase • Degradation of pollutants • Synthesis of chemicals Error prone PCR Vardar, G and Wood, TK Appl Environ Microbiol. 2004 Example: Error Prone PCR Example: Error Prone PCR • Sense primer: N-terminal domain Antisense primer: C-terminal domain 7 mM MgCl2, 0,5 mM MnCl2, 1 mM dTTP/dCTP, 0,2 mM dATP/dGTP • Screening of about 10 million mutants Efficiant screening system! • 13 atropine mutants identified All contained combinations of mutations Example: Site-directed random mutagenesis Glu Trp Pro Identification of essential receptor determinants • Mutation of specific AA • Single mutations requested • Saturating mutagenesis necessary Example: Site-directed random mutagenesis 1 primer • -NNN- 64 codons/20 AA Problems: • Coverage • Wild-type prefered Asn Site-directed random mutagenesis New primer design • Exclusion of wild-type • Specific for every AA 4 Example: Site-directed random mutagenesis Example: Site-directed random mutagenesis 1 primer • -NNN- 64 codons/20 AA 2 primers for Valine mutation • -WTK-NVK- 28 codons/19 AA 3 primers for Valine mutation • -VVG-WKS-NAT- 21 codons/19 AA 5
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