Polymerase Chain Reaction Mutagenesis and Automated DNA Sequencing

 

Melanie L. Sims, Victor V. Tryon, Barry T. Nall , and Christy MacKinnon

Tested Studies in Laboratory Teaching, 1999, Volume 20

Abstract

This activity combines the Polymerase Chain Reaction, in vitro mutagenesis, and automated DNA sequencing. A model experimental system, a pEMBLYe30 plasmid containing the yeast cytochrome c gene, is utilized to analyze the relationship between genetic information and protein structure. This activity engages students in a pre-lab written activity that reviews the genetic code, types of amino acids, and DNA replication. Using the known sequence of the cytochrome c gene, students design an oligonucleotide mutagenic primer. The Megaprimer PCR protocol for in vitro mutagenesis is conducted by either the students or by the instructor as a demonstration. Concepts of DNA sequencing are discussed; examples of automated DNA sequencing chromatograms(and other support material)are available from the authors’ web site. Additional investigations (suitable for student research projects) involving cloning and transformation of the plasmid vector also are described, as are in vivo assays of cytochrome c function, and spectral analysis of cytochrome c structure.

Keywords:  PCR, mutagenesis, DNA sequencing, polymerase chain reaction

Florida State University (1998)