Eastern Kentucky University
ABO Blood Group Genotyping by PCR-RFLP
Institution
Eastern Kentucky University
Faculty Advisor/ Mentor
Rebekah Waikel
Abstract
Blood typing typically involves mixing a blood sample with antibodies A and B and observing any antigen – antibody reactions (agglutination). Blood borne diseases, ranging from HIV to Hepatitis make blood typing in a classroom setting risky. The purpose of this research is to design a DNA based ABO blood typing protocol that does not require the use of blood. DNA can easily be obtained through a cheek swab. Blood type can be determined by the presence or absence of glycosyl transferase genes, whose protein products add carbohydrate antigens to proteins and lipids on the surface of erythrocytes. Analysis of the known gene sequences of the ABO glycosyl transferase genes revealed detectable sequence differences found in exons 6 and 7. We amplified exons 6 and 7 of the ABO genes via the Polymerase Chain Reaction (PCR). We then digested the amplimers with sequence specific restriction endonuclease and separated resulting DNA bands by agarose gel electrophoresis (RFLP). Based on predicted DNA band sizes, we have been able to successfully determine blood types through PCR-RFLP of genomic DNA. To our knowledge, this is the first PCR-RFLP experimental test for blood type. This genotyping scheme will be used in undergraduate laboratory courses.
ABO Blood Group Genotyping by PCR-RFLP
Blood typing typically involves mixing a blood sample with antibodies A and B and observing any antigen – antibody reactions (agglutination). Blood borne diseases, ranging from HIV to Hepatitis make blood typing in a classroom setting risky. The purpose of this research is to design a DNA based ABO blood typing protocol that does not require the use of blood. DNA can easily be obtained through a cheek swab. Blood type can be determined by the presence or absence of glycosyl transferase genes, whose protein products add carbohydrate antigens to proteins and lipids on the surface of erythrocytes. Analysis of the known gene sequences of the ABO glycosyl transferase genes revealed detectable sequence differences found in exons 6 and 7. We amplified exons 6 and 7 of the ABO genes via the Polymerase Chain Reaction (PCR). We then digested the amplimers with sequence specific restriction endonuclease and separated resulting DNA bands by agarose gel electrophoresis (RFLP). Based on predicted DNA band sizes, we have been able to successfully determine blood types through PCR-RFLP of genomic DNA. To our knowledge, this is the first PCR-RFLP experimental test for blood type. This genotyping scheme will be used in undergraduate laboratory courses.