Effects of heparin on thalassemia genes analysis by PCR

Abstract

EDTA-anticoagulated blood is usually recommended for use in routine analysis of thalassemia genes. However, collection of blood from the fetus or newborns is usually done using heparin because of its more potent anti-coagulant activity. However, since heparin can inhibit Taq polymerase enzyme used in PCR, the use of heparinized blood for PCR analysis may be affected. ACD, another blood anti-coagulant with a preservative property may be used as an alternative anti-coagulant. In this study, the effects of EDTA, heparin and ACD anti-coagulants on PCR analysis of a – thalassemia 1 (SEA deletion) and b^E – globin gene were compared. a – thalassemia 1 was detected using gap – PCR and b^E – globin gene was identified by allele specific PCR assay. It was found that both EDTA and ACD anti-coagulants had no effect on the efficiency of PCR analysis of the two thalassemia genes. However, substantial reduction in the efficiency of PCR analysis was observed with the use of heparin as an anti-coagulant at a concentration over 25 IU/ml. Amplification efficiency was improved when heparinized blood DNA was diluted prior to PCR analysis. The result from this study should prove useful for development of a guideline of blood collection for routine PCR analysis of halassemia.