Site Directed Mutagenesis

Site Directed Mutagenesis - BCHM464, Spring 2011...

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BCHM464, Spring 2011 Mutagenesis & Transformation Site Directed Mutagenesis of phoA and Transformation of Mutant pJR-1U into Mph44 cells by Electroporation I. Introduction to Mutagenesis During this exercise, you will introduce specific nucleotide changes into the sequence of the phoA gene, which is contained in plasmid pJR-1U. In the next few weeks, you will prepare plasmid DNA from several different transformants, and analyze them to see whether they contain the desired mutation in the phoA gene. Cells containing the mutated plasmid can then be used as a source of the mutant enzyme. The Polymerase Chain Reaction (PCR) has allowed development of a wide variety of uses. PCR has gained solid ground in scientific areas and has opened up the analysis of cellular and molecular processes to those outside the field of molecular biology. In addition to its powerful ability to amplify specific DNA sequences, applications of PCR include DNA sequencing, detection of unknown mutations, screening of gene libraries, and production of mutant proteins to name a few. A commonly used protocol for site-directed mutagenesis (figure at right) is based on the use of double-stranded DNA and two complementary primers containing the desired mutation. The three stage process (mutant strand synthesis, Dpn I digestion, and transformation) results in amplification of the entire plasmid and incorporation of the mutation in the mutagenic primer. The amplification reaction mixture includes two complementary primers containing the desired mutation sequence, double stranded template DNA, a mixture of dNTPs, and a thermostable polymerase in reaction buffer. The reaction mixture is cycled through a series of temperature conditions during which the thermostable polymerase extends the primers by incorporation of the dNTPs. Each cycle of PCR consists of three defined sets of conditions termed cycles: 1) “denaturation” (double-stranded DNA melted at 95 C exposing regions of single-stranded template DNA to the mutagenic primers); 2) “annealing” (the mutagenic primers hybridize with their complementary sites at 40-60 C), and 3) “extension” (DNA synthesis by the polymerase is carried out at an optimal temperature). The amplification step is an automated process in which a thermal cycler programmed with the proper temperature cycling parameters is used to repeat the cycles reproducibly and accurately. Because the mutagenic primers and dNTPs are in excess, the reaction can be repeatedly cycled,
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Site Directed Mutagenesis - BCHM464, Spring 2011...

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