Chapter 8
Mutagenesis of cloned DNA
To creating numerous mutations in a small DNA
sequence,
• mutagenesis with degenerate oligonucleotides
• a palindromic RE recognition sequence at the 3’
end of the oligonucleotide for the mutual priming
reaction ( 230C for 1 hr).
To synthesize a desired sequence,
• assembly of target sequences using mutually
priming long oligonucleotides
• use pairs of long oligonucleotides ( ﹥100
nucleotides)
• can generate desired sequences up to 400 bp in
a single step
• a 15-bp duplex is a conventional length for
mutual priming
Random mutagenesis by PCR (error-prone PCR,
EP-PCR),
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use low fidelity of Taq DNA polymerase
add Mn+2
increase Mg+2 concentration
use unequal dNTP concentrations
PCR cycles vs PCR doublings
Each PCR cycle generally increases the
amount of DNA by a factor of 1.7 to 1.9 until the
DNA concentration reaches a plateau (generally
5 to 50 ng/μl), and then stop increasing
altogether.
If mutagenesis of a library of sequences,
To avoid loss of complexity,
1. Four EP-PCR cycles are performed at a higher
template concentration.
2. 10% of the product is then transferred to a fresh
EP-PCR tubes as template, for another four
EP-PCR cycles.
3. Repeat the process, until the desired number of
doublings is achieved.
Linker-scanning mutagenesis of DNA
1. Bal31 and linkers to generate serious deletion
plasmids
2. Determine the deletion endpoints by sequencing
3. Ligation of backbone fragments, hybridized
oligonucleotides, and the deleted fragments.
Directed mutagenesis by PCR
(I ). Introduction of restriction endonuclease
sites by PCR
(II). Introduction of point mutations by PCR
(III). Introduction of a point mutation by
sequential PCR steps
Transpososome mutagenesis
(Epicentre Co.)
(I). Mutagenesis of target clone
(II). Random mutagenesis of bacteria in vivo
(to generate mutants)