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 nucleo﹥tides)

• 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),

• 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)