What is oligonucleotide site-directed mutagenesis?
Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering. …
Which phage is used in oligonucleotide directed mutagenesis?
M13
An olignucleotide sequence complementary to the segment of interest, but containing an alteration at a selected site, is chemically synthesized. Next this is hybridized to a complementary wild-type target gene contained in a single-stranded phage such as M13.
How does Quick Change PCR work?
QuikChange™ works by using a pair of complementary primers with a mutation. In a round of PCR cycles these primers anneal to the template DNA, replicating the plasmid DNA with the mutation. The mutant DNA product has a strand break (nick) (Figure 1A).
Why is DPN1 important?
DpnI is specific for methylated and hemimethylated DNA. Since DNA isolated from most E. coli strains is dam methylated, it is susceptible to DpnI digestion. Hence, DpnI is frequently used after a PCR reaction to digest the methylated parental DNA template and select for the newly synthesized DNA containing mutations.
What is importance of M13 bacteriophage in site-directed mutagenesis?
The bacteriophage known as “M13” forms the basis of cloning systems designed to easily introduce mutations into genes inserted into the phage genome. It also has been used in various “phage display” methodologies and “combinatorial” DNA and peptide libraries.
What is Quick Change mutagenesis?
The PCR Quick Change or site directed mutagenesis is used to change DNA bases on a sequence of interest (maximum 5 bases). The most important step in this experiment is the design of the primers.
What does DpnI restriction enzyme do?
DpnI is a Type IIM restriction enzyme that specifically cleaves DNA containing methylated adenine (mA) in the recognition sequence GmA | TC, also referred to as the dam sequence since it is recognized by dam methylase. DpnII and MboI do not cleave adenomethylated sequences.
