Comparison of the optimized conditions for genotyping of ACE ID polymorphism using conventional and direct blood PCR
Keywords: ACE ID polymorphism, step down PCR, direct blood PCR.
AbstractACE ID polymorphism is inevitable for genetic epidemiology of several cardiovascular and non cardiovascular diseases due to its direct influence on ACE activity level. In the present work, conditions were optimized for its analysis using conventional and direct blood PCR (DB PCR). Blood samples from nine normotensive male donors preserved in EDTA and lithium-heparin coated vacuatainers separately were used directly as template for DB PCR. Genomic DNA was isolated from each vacuatainer for the
conventional PCR and DB PCR also. Conditions were optimized by adjusting the suitable annealing temperature, amount of MgCl2 (in case of conventional PCR) and amount of blood used as DNA template for DB PCR. In case of DNA from EDTA treated blood, maximum amplification of target
sequence occurred at 53oC with 2 mM concentration of MgCl2 in all samples. However, when DNA from lithium heparin treated blood was used as template, 6 out of 9 samples gave amplification results with 4
mM concentration of MgCl2 at the same temperature. When 1 ìl genomic DNA from EDTA and lithium heparin treated blood was used as DNA template in DB PCR, all samples gave maximum yield at 53oC.
DB PCR successfully amplified the target region when 1 ìl blood treated with EDTA and 0.5 ìl lithium heparin treated blood was used per 50 microliter reaction mixture at 51oC as annealing temperature. It
can be concluded from the study that EDTA treated blood is more suitable for conventional and DB PCR.
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