Rapid and sensitive detection of point mutations and DNA polymorphisms in Factor IX Gene by using the Single Strand Conformation Polymorphism (SSCP)

The Factor IX gene coding for the coagulation factor IX and its deficiency causes Hemophilia B, or Christmas disease. It is an X-linked recessive disorder that affects 1:25000 male births. In the last few years the searches for mutations and sequence polymorphisms have been dramatically changed by the use of PCR and subsequently by direct sequencing of PCR products. In addition a number of prescreening methods have been developed such as single strand conformation polymorphism (SSCP). SSCP is the simplest and most used method of mutation detection. In this study we evaluated the sensitivity of SSCP in finding Factor IX gene mutations. We used peripheral blood from Iranian Hemophilia B patients. DNA were extracted by Phenol Chloroform and other methods. All coding and regulatory regions of the gene were amplified by 11 pairs of primers. SSCP was done by a modified Orita et al. method. The PCR fragments with altered mobility were sequenced. We found deletion, insertion, missense and nonesense mutations in 80% of our samples. Therefore SSCP was highly sensitive in our study