Lecture GeneCloning Oligonucleotide-directed mutagenesis · Oligonucleotide-directed mutagenesis ... • Quikchange method Advantages/disadvantages with these ......

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?