Determination of the Rh factor: A practical illustrating the use of the polymerase chain reaction

Abstract

A practical experiment on the PCR is described that has been used over several years as part of an undergraduate biochemistry and molecular biology course for chemistry students. In the first experimental session, students prepare their own DNA samples from epithelial cells of the mouth and use them as templates in the PCR. In the second session, they analyze the amplified DNA by electrophoresis and determine their Rh factor.Keywords: Polymerase chain reaction (PCR), Taq polymerase, Rh factor, blood group.The theory of DNA structure and organization as well as the basics of DNA replication was reviewed in a lecture before the practical. Students were then provided with detailed information about the PCR and the Rh factor system as summarized below.The PCR-PCR allows selective amplification of a determined portion of DNA in vitro a million times or more. The technique was made possible by the discovery of Taq polymerase, the DNA polymerase that is used by the thermophilic bacterium Thermus aquaticus, which is stable at high temperatures and has become an essential research and diagnostic tool in the detection of infection disease organisms and detection of variations and mutations in human genes. Because the technique allows identification and amplification of tiny amounts of DNA, even old or damaged, PCR technology has proved of great value in criminal law and forensic DNA fingerprinting. In the practical reported in this article, PCR was used to determine the Rh factor of the students' own DNA, which is based on the diagnostic amplification of a DNA sequence present in Rh-positive individuals and absent in Rh-negative.The Rh System-In 1940, Landsteiner and Wiener showed that antibodies produced against the rhesus monkey red blood cells were able to agglutinate the red blood cells of about 85% of a human population. The antibodies produced were shown to be directed against a molecule that was called the rhesus (Rh) 1 antigen. Individuals possessing this molecule were called Rh positive and the remaining 15% Rh negative [1]. This was the basis for the human Rhesus blood group system, which more than 60 years after its discovery is, next to ABO, the most clinically significant in transfusion medicine [2].The Rh locus consists of two structural genes, D and CcEe, which code for the polypeptide chain D and the proteins C/c and E/e, respectively [3,4]. The presence or absence of gene D in the genome determines the genetic basis for the polymorphism of the Rh-positive/Rh-negative blood groups. Therefore, individuals are classified as Rh positive if they contain the main antigen D on the membrane of their red blood cells. The Rh system is, however, much more complex, and up to 47 Rh different antigens have been reported [2,5].The D antigen is highly immunogenic and induces an immune response in most D-negative persons when transfused with D-positive blood. For this reason, in most countries D typing is performed routinely on every blood donor and transfusion recipient so that D-negative patients receive exclusively D-n...

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