Lecture GeneCloning Oligonucleotide-directed mutagenesis · Oligonucleotide-directed mutagenesis...
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Oligonucleotide-directed mutagenesis Different types of mutations • Site specific • Random Design of primers for mutagenesis Controls Trouble shooting
Transcript of Lecture GeneCloning Oligonucleotide-directed mutagenesis · Oligonucleotide-directed mutagenesis...
Oligonucleotide-directedmutagenesis
Different types of mutations• Site specific• RandomDesign of primers for mutagenesisControlsTrouble shooting
Different types of mutagenesis
• Site specific mutagenesis• Random mutagenesis• Sticky-feet directed mutagenes
5’ ATGGCCCATCACTGGGGGTACGGCAAACACAACGGACCTGAGCACTGGCATAAGGACTTCCCCATTGCCAAGGGAGAGCGCCAGTCCCCTGTTGACATCGACACTCATACAGCCAAGTATGACCCTTCCCTGAAGCCCCTGTCTGTTTCCTATGATCAAGCAACTTCCCTGAGGATCCTCAACAATGGTCATGCTTTCAACGTGGAGTTTGATGACTCTCAGGACAAAGCAGTGCTCAAGGGAGGACCCCTGGATGGCACTTACAGATTGATTCAGTTTCACTTTCACTGGGGTTCACTTGATGGACAAGGTTCAGAGCATACTGTGGATAAAAAGAAATATGCTGCAGAACTTCACTTGGTTCACTGGAACACCAAATATGGGGATTTTGGGAAAGCTGTGCAGCAACCTGATGGACTGGCCGTTCTAGGTATTTTTTTGAAGGTTGGCAGCGCTAAACCGGGCCTTCAGAAAGTTGTTGATGTGCTGGATTCCATTAAAACAAAGGGCAAGAGTGCTGACTTCACTAACTTCGATCCTCGTGCCTCCTTCCTGAATCCCTGGATTACTGGACCTACCCAGGCTCACTGACCACCCCTCCTCTTCTGGAATGTGTGACCTGGATTGTGCTCAAGGAACCCATCAGCGTCAGCAGCGAGCAGGTGTTGAAATTCCGTAAACTTAACTTCAATGGGGAGGGTGAACCCGAAGAACTGATGGTGGACAACTGGCGCCCAGCTCAGCCACTGAAGAACAGGCAAATCAAAGCTTCCTTCAAATAA-3´
Genen för humant karboanhydras
Genetiska koden
Directed mutagenesis
Directed mutagenesis
Site specifik mutagenesis
Figure 11.21 page 201
Site specific mutagenesis
Figure 11.23, p203
Different methods of mutagenesis
• Kunkel method• Quikchange method
Advantages/disadvantages with thesemethods?
Variants of mutagenesis techniques
Fig 11.22, p 202
Design of oligonucleotides
• Length• GC content• Possibility for primer-dimer formation• Tm-value• Bases in 3`- and 5´-end
Vectors and bacterial strains
• M13• Plasmids• phagemides• Genotypes for mutagenesis
Random design
• Changing the gene randomly• Select for a variant with specific properties
e.g high stability• Problem of selection
(Error-prone) PCR
5’
5’
5’5’
5’5’
3’
3’
3’5’ 3’
5’3’
3’5’
3’
5’
3’3’
5’
3’
5’
95 C
50-65 C
72 C
Phage display technique
• Variants are cloned in a viral vector
• The gene is expressed as a fusionsprotein on the surface of the virus
• Select bindning properties at different [denaturant]
• Connection genotype-phenotype
Ref: Introduction to protein structure, Bränden & Tooze
Phage display technique
Fig 12.17, p 221
Detection of mutants
• DNA sequencing• Restriktion cleavage (silent mutations)• Screening for ”disturbed function”
Troubleshooting and controls
• Transformation control (are the bacteriacompetent?)
• Fresh mixture, buffers, reagents• How pure is the template?• Suitable bacterial strain?
