Comparison of conventional and real time pcr methods to determine of the ace i/d and angiotensinogen m235t polymorphisms
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Angiotensin converting enzyme (ACE) and angiotensinogen (AGT) are the key components of the renin-angiotensin system. The ACE I/D and AGT M235T polymorphisms are usually analyzed by conventional PCR. However, recently genotyping of I/D and M235T polymorphism are facilitated by development of rapid genotyping technique. In our study, the genotyping of ACE I/D and AGT M235T polymorphism was performed by conventional and Real Time PCR techniques and determine advantage and disadvantage of both techniques. According to our study, mistyped ratio of ACE I/D polymorphism at conventional PCR was found 6%. Therefore, to avoid mistyping, an additional PCR amplification was performed for the confirmation of all DD genotypes (insertion specific PCR). The DNA samples were also analyzed by Real Time PCR technique. Finally, conventional and Real Time PCR results were compared. As a result, all genotype were determined correct at single step Real Time PCR. We also analysed M235T polymorphism by PCR-RFLP and Real Time PCR techniques. There were no different result among two techniques. However, potentional problems such as incomplete enzyme digestion may cause false genotyping. Consequently, rapid genotyping of ACE I/D and M235T polymorphism offers an appropriate option for laborotory investigation and diagnosis in point of reliability and labor intensive.