Comparison of conventional and real time PCR methods to determine of the ace I/D and angiotensinogen m235t polymorphisms

Anjiyotensin dönüştürücü enzim (ACE) ve Anjiyotensinojen (AGT) renin-anjiyotensin sisteminin anahtar bileşenleridir. ACE I/D ve AGT M235T polimorfizmleri genellikle konvansiyonel PZR tekniği ile saptanır. Fakat son zamanlarda hızlı genotiplendirme tekniğinin geliştirilmesi ile bu polimorfizmlerin belirlenmesi kolaylaşmıştır. Çalışmamızda ACE I/D ve AGT M235T polimorfizmleri konvansiyonel ve Eşzamanlı PZR teknikleri ile çalışıldı ve bu tekniklerin avantajları ve dezavantajları belirlendi. Bulgularımıza göre konvansiyonel PCR ile I/D polimorfizminin yanlış genotiplendirme oranı % 6 belirlendi. Bu nedenle hatalı genotiplendirmeden kaçınmak için tüm DD genotipli örneklere ikinci bir PCR daha uygulandı (İnsersiyon spesifik PCR). DNA örnekleri aynı zamanda Eşzamanlı PZR ile de analiz edildi. Son olarak konvansiyonel PZR ve Eşzamanlı PZR sonuçları karşılaştırıldı. Tüm genotipler Eşzamanlı PZR ile tek seferde doğru olarak saptandı. Ayrıca M235T polimorfizmi de PCR-RFLP ve Eşzamanlı PZR teknikleri ile analiz edildi. İki tekniğin sonuçları arasıda herhangi bir farklılık gözlemedi. Ancak PCR-RFLP yönteminde tamamlanmamış enzim kesimi gibi olası problemler yanlış genotiplendirmeye neden olabilmektedir. Özetle ACE I/D ve M235T polimorfizmlerinin hızlı genotipleme tekniği olan Eşzamanlı PZR ile belirlenmesi güvenilirlik ve zaman kaybı yönünden laboratuvar araştırmaları ve tanı için uygun bir seçenek sunmaktadır.

Ace I/D ve anjiyotensinojen m235t polimorfizmlerinin belirlenmesinde konvansiyonel pzr ve eşzamanlı PZR yöntemlerinin karşılaştırılması

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.

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