Case-Study Investigation of Equine Maternity via PCR-RFLP: A Biochemistry Laboratory Experiment.
Abstract: A simple and robust biochemistry laboratory experiment is described that uses restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR) products to verify the identity of a potentially valuable horse. During the first laboratory period, students purify DNA from equine samples and amplify two loci of mitochondrial DNA. During the second laboratory period, students digest PCR products with restriction enzymes and analyze the fragment sizes through agarose gel electrophoresis. An optional step of validating DNA extracts through realtime PCR can expand the experiment to three weeks. This experiment, which has an engaging and versatile scenario, provides students with exposure to key principles and techniques of molecular biology, bioinformatics, and evolution in a forensic context.
Publication Date: 2013-12-24 PubMed ID: 24363455PubMed Central: PMC3867210DOI: 10.1021/ed300740rGoogle Scholar: Lookup
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- Journal Article
Summary
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The research article describes a straightforward and compelling biochemistry lab experiment using the PCR-RFLP technique to confirm the identity of a possibly valuable horse. It offers students the experience to understand core principles and techniques of molecular biology, bioinformatics, and evolution in a forensic context.
Introduction and Background
- This research paper discusses a biochemistry laboratory experiment aimed at verifying the identity of a horse through the analysis of mitochondrial DNA. The potential animal of interest is considered to be valuable and, consequently, has generated considerable attention.
- The study utilizes two main techniques – the Polymerase Chain Reaction (PCR) and the Restriction Fragment Length Polymorphism (RFLP). PCR is employed to amplify segments of DNA making it easier to analyze, whereas the RFLP technique is used to identify variation in DNA sequences.
- Given the applied nature of the experiment, it offers significant educational value for students in molecular biology, bioinformatics, and evolution.
Methodology and Experiment Design
- The experiment, spanning two or optionally three lab periods, begins with students purifying DNA from equine samples and amplifying two loci of the mitochondrial DNA. This process is vital in extracting genetic material for analysis.
- In the subsequent laboratory period, students digest PCR products (the amplified DNA) using restriction enzymes that slice these DNA strands at specific sites. This leads to the creation of different segments of DNA that can be identified through a method known as agarose gel electrophoresis.
- With an additional step in the experiment, they can validate the DNA extracts through real-time PCR, extending the experiment to three weeks. Real-time PCR allows for the monitoring of the PCR amplification as it progresses.
Learning Outcome and Benefits
- Through this experiment, students get a hands-on experience of applying key principles and techniques often used in a real-world forensic context.
- It also allows students to understand and experience the bioinformatics approach of identifying and studying animals or humans based on their DNA.
- The practical exercise reinforces the understanding of key genetic concepts, such as the use of mitochondrial DNA in identifying animals, and the function and use of PCR and RFLP in analyzing and typifying genetic material.
Conclusion
- The experiment described in the research article provides an engaging and versatile platform for students to learn, understand, and apply key principles and techniques of molecular biology, bioinformatics, and evolution in a forensic context.
- The use of these techniques, particularly PCR-RFLP, proves to be a sound method of verifying the identity of a potentially valuable horse, highlighting its implications in the real world.
Cite This Article
APA
Millard JT, Chuang E, Lucas JS, Nagy EE, Davis GT.
(2013).
Case-Study Investigation of Equine Maternity via PCR-RFLP: A Biochemistry Laboratory Experiment.
J Chem Educ, 90(11).
https://doi.org/10.1021/ed300740r Publication
Researcher Affiliations
- Department of Chemistry, Colby College, Waterville, Maine 04901, United States.
- Department of Chemistry, Colby College, Waterville, Maine 04901, United States.
- Department of Chemistry, Colby College, Waterville, Maine 04901, United States.
- Department of Chemistry, Colby College, Waterville, Maine 04901, United States.
- Department of Chemistry, Colby College, Waterville, Maine 04901, United States.
Grant Funding
- P20 GM103423 / NIGMS NIH HHS
- P20 RR016463 / NCRR NIH HHS
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Citations
This article has been cited 1 times.- Johanson KE, Watt TJ. Inquiry-based experiments for large-scale introduction to PCR and restriction enzyme digests.. Biochem Mol Biol Educ 2015 Nov-Dec;43(6):441-8.
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