Dr. Jonathan Southard, a biochemist in the Department of Chemistry, recently had a manuscript accepted for publication in the journal Biochemistry and Molecular Biology Education.
Protein Analysis Using Real-Time PCR Instrumentation: Incorporation in an Integrated, Inquiry-Based Project
Instrumentation for real-time PCR is used primarily for amplification and
quantitation of nucleic acids. The capability to measure fluorescence while
controlling temperature in multiple samples can also be applied to the analysis of
proteins. Conformational stability and changes in stability due to ligand binding
are easily assessed. Protein structure studies possible with a real-time PCR
instrument address core topics in biochemistry and have valuable high-
throughput applications in the fields of drug discovery and protein engineering.
Protein analysis using real-time PCR instrumentation has been incorporated in an
undergraduate laboratory project based on previously described projects.
Students express, purify, and characterize a protein. Based on literature
research and analysis using bioinformatics tools, they select a specific mutation
to investigate. They then attempt to express, purify, and characterize their
mutated protein. Thermal denaturation using a real-time PCR instrument is the
primary tool used to compare the wild-type and mutated proteins. Alternative
means for incorporation of protein analysis by real-time PCR instrumentation into
laboratory experiences and additional modes of analysis are also described.