User:Bfigura/Sandbox-Kunkel

Sandbox Version of an article on the Kunkel Method, an expansion from Site-directed_mutagenesis. I'm using some content from there to start the article. It'll be expanded upon before creation.

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The Kunkel Method is a classic site-directed mutagenesis technique used in molecular biology to cause a mutation at a defined site in a circular DNA molecule, known as a plasmid. While it is not currently the most efficient method available, it is notable for being the first method that eliminated the need for phenotypic expression. The method was developed by TA Kunkel in 1985, and takes advantage of the fact that uracil (a base normally found in RNA, not DNA) will not translate properly if recombinantly introduced into a bacteria (such as E. coli).

Method

A plasmid containing the sequence to be mutated is transformed in an E. coli deficient in two genes, dUTPase & uracil deglycosidase (also known as an ung^— dut^— E. coli). As dUTPase contributes to breakdown of dUTP, a nucleotide that replaces dTTP in RNA, its removal results in an over abundance of dUTP within the cell. The seco, and deficiency in the latter would prevent the removal of dUTP from newly synthesized DNA. As the double-mutant E. coli replicates the up-taken plasmid, its enzymatic machinery incorporates the dUTP, resulting in a distinguishable copy. This copy is then extracted and incubated with an oligonucleotide containing the desired mutation, which attaches by base pair hydrogen bonding to the complementary wild-type gene sequence, as well as the Klenow enzyme, dNTPs, and DNA ligase. The reaction essentially replicates the dUTP-containing plasmid using as primer the oligonucleotide, giving a nearly identical copy. The essential differences being that the copy contains dTTP rather than dUTP, as well as the desired mutation. When the chimeric double-stranded plasmid, containing the dUTP, unmutated strand and the dTTP, mutated strand, is inserted into a normal, wild-type E. coli, the dUTP-containing strand is broken down, whereas the mutation-containing strand is replicated.