Abstract. Taq Polymerase Is Essential In Polymerase Chain

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ABSTRACT Taq Polymerase is essential in Polymerase Chain Reaction(PCR) experiments to obtain a PCR amplification of an unknown gene. The unknown gene is then ligated into a vector plasmid, which is placed in a bacterium Escherichia Coli to transform and multiply. Ultimately, identification and characterization of the unknown gene is done using electrophoreses and gel imaging. Cloning techniques such as the one performed have been used for many years to isolate genes from a variety of species. Isolation of unknown genes serves many purposes. Nucleic acid sequence of the unknown gene can be derived which can be compared to other known gene sequences and the function of the gene can be derived. The clone made can be used to study the…show more content…
The active monomer form of Taq polymerase has a molecular mass of 94 kD with a full length of 832 amino acids. (2) Mainly, Taq polymerase catalyzes the incorporation of dNTPs into DNA and contains a polymerization dependent 5’-3’ DNA polymerase activity. (1) Although there are many advantages towards the use of Taq polymerase in the amplification reaction, Taq polymerase does not possess 3’ to 5’ exonuclease (proofreading) activity. Taq polymerase is purified and tested using SDS-PAGE gel electrophoresis to separate and identify the purity of the sample. The recombinant protein is purified from an E. coli strain containing the pTTQ18 plasmid that contains the Taq DNA polymerase I gene that is cloned downstream Ptac promoter. After purification through a “salting out” method containing (NH4)2SO4, a small sample of Taq polymerase will be used to be identified through electrophoresis. After enrichment of the protein, the sample of Taq polymerase will be saved to be used in further experiments involving PCR amplification, cloning, and DNA sequencing. Polymerase Chain Reaction is a method developed in the 1980s by Kary Mullis. The main objective to PCR is the ability of the DNA polymerase, in our case Taq polymerase, to synthesize a new strand of DNA that is complementary to the unknown gene offered in the template
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