USE mutagenesis

목차

1. INTRODUCTION
2. MATERIALS
3. METHOD
4. TROUBLESHOOTING

본문내용

▶ INTRODUCTION
In USE method, two oligonucleotide primer are hybridized to the same strand of a denatured double-stranded recombinant plasmid. One primer (the mutagenic primer) introduces the desired mutation into the target sequences, whereas the second primer carries a mutation that destroys a unique restriction site in the plasmid. Both primers are elongated by bacteriophage T4 DNA polymerase and bacteriophage T4 DNA ligase.
The products of the first part of the method are wild-type molecules that were never used as templates for DNA syntheses primed by two oligonucleotide primers and heteroduplex molecules that have lost the unique restriction site and gained the desired mutation.
In the second phase of the method, the mixed population is incubated with the restriction enzyme that cleaves the unique site. The mixture of circular heteroduplex DNA and linear wild-type DNA is then used to transform a strain of E. coli that is deficient in repair of mismatched bases. The first round of replication generates a wild-type plasmid that carries the original restriction site and a mutated plasmid that does not.
DNA from the first set of transformants is recovered, digested once more with the same restriction enzyme to linearize the wild-type molecules, and then used to transform a standard laboratory strain of E. coli.

참고 자료

Sambrook and Russel. 2001. Molecular Cloning 3rd edition. vol 2 : 13.26-13.30