Amplification of a Hemoglobin Gene by PCR (IND-7)

The polymerase chain reaction (PCR) is one of the most powerful techniques used in molecular biology . With this method, a few ng of DNA can be amplified millions of times in a test tube in a few hours. The PCR has been used extensively in studies of gene structure and function. The method is also becoming increasingly important in DNA typing procedures such as DNA fingerprinting and in the identification and characterization of mutations that cause human diseases. This exercise is designed to illustrate the PCR in the teaching laboratory. In the experiment, students use PCR to amplify a rabbit ß-globin gene. The template for this reaction is a plasmid that contains this globin sequence. Following PCR amplification, the product of the reaction is analyzed on an agarose gel as shown on the below. The experiment was designed so that the PCR can be done manually provided that three standard water baths (45°C, 74°C, and 94°C) or three hot plates are available. The amplified globin DNA band can clearly be seen following staining of the agarose gels with methylene blue. The amplification reaction requires approximately 1.5 hours. Sufficient materials are provided so that 8 groups of students can perform the experiment.

The following materials are needed but not included: Three water baths (or an authorized DNA Thermal Cycler), electrophoresis equipment and Electrophoresis Package 3/4.