OBJECTIVE: To develop a new technique based on long distance polymerase chain reaction (LD-PCR) to replace Southern blotting method to detect Factor VIII (FVIII) gene inversion leading to severe Hemophilia A (HA) and carrier. METHODS: Four primers P, Q, A&B were designed and synthesized. P&Q is specific for 5' and 3' flanking regions of F8A1 respectively. A&B is specific for 5' and 3' flanking regions of F8A2/F8A3 respectively. LD-PCR with 3 primers and 3 temprature was set up, optimized and used to detect the inversion. RESULTS: The LD-PCR with primers P, Q, A&B, P, Q&B and P, Q&A can be used to detect the gene inversion and discriminate carrier from wild type. A blind analysis of 53 DNA samples from HA families was carried out by the LD-PCR and Southern blotting respectively. Two sets of the results were completely identical. They were 23 cases of inversion, 27 cases of wild type and 3 cases of carriers. The sensitivity and specificity of LD-PCR are both 100%. Three inversion hemizygotes and 4 female carriers were identified from 5 HA families by the LD-PCR technology. CONCLUSIONS: The LD-PCR with primer P, Q&B or P, Q, A&B can be used to detect the gene inversion and the carrier of inversion. Compared with Southern blotting, this technique is simple, rapid, inexpensive, more sensitive, accurate and non-isotopic.
OBJECTIVE: To develop a new technique based on long distance polymerase chain reaction (LD-PCR) to replace Southern blotting method to detect Factor VIII (FVIII) gene inversion leading to severe Hemophilia A (HA) and carrier. METHODS: Four primers P, Q, A&B were designed and synthesized. P&Q is specific for 5' and 3' flanking regions of F8A1 respectively. A&B is specific for 5' and 3' flanking regions of F8A2/F8A3 respectively. LD-PCR with 3 primers and 3 temprature was set up, optimized and used to detect the inversion. RESULTS: The LD-PCR with primers P, Q, A&B, P, Q&B and P, Q&A can be used to detect the gene inversion and discriminate carrier from wild type. A blind analysis of 53 DNA samples from HA families was carried out by the LD-PCR and Southern blotting respectively. Two sets of the results were completely identical. They were 23 cases of inversion, 27 cases of wild type and 3 cases of carriers. The sensitivity and specificity of LD-PCR are both 100%. Three inversion hemizygotes and 4 female carriers were identified from 5 HA families by the LD-PCR technology. CONCLUSIONS: The LD-PCR with primer P, Q&B or P, Q, A&B can be used to detect the gene inversion and the carrier of inversion. Compared with Southern blotting, this technique is simple, rapid, inexpensive, more sensitive, accurate and non-isotopic.
Authors: M V Ragni; O Ojeifo; J Feng; J Yan; K A Hill; S S Sommer; M N Trucco; D J Brambilla Journal: Haemophilia Date: 2009-06-26 Impact factor: 4.287