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) a...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 sp ecific for 5' and 3' flanking regions of F8A 1 respectively. A&B is specific fo r 5' and 3' flanking regions of F8A 2/F8A 3 respectively. LD PCR with 3 p rime rs 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 t o detect the gene inversion and discriminate carrier from wild type. A blind ana lysis of 53 DNA samples from HA families was carried out by the LD PCR and Sout hern blotting respectively. Two sets of the results were completely identical. T hey were 23 cases of inversion, 27 cases of wild type and 3 cases of carriers. T he sensitivity and specificity of LD PCR are both 100%. Three inversion hem izygotes 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 det ect the gene inversion and the carrier of inversion. Compared with Southern blot ting, this technique is simple, rapid, inexpensive, more sensitive, accurate and non isotopic.展开更多
文摘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 sp ecific for 5' and 3' flanking regions of F8A 1 respectively. A&B is specific fo r 5' and 3' flanking regions of F8A 2/F8A 3 respectively. LD PCR with 3 p rime rs 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 t o detect the gene inversion and discriminate carrier from wild type. A blind ana lysis of 53 DNA samples from HA families was carried out by the LD PCR and Sout hern blotting respectively. Two sets of the results were completely identical. T hey were 23 cases of inversion, 27 cases of wild type and 3 cases of carriers. T he sensitivity and specificity of LD PCR are both 100%. Three inversion hem izygotes 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 det ect the gene inversion and the carrier of inversion. Compared with Southern blot ting, this technique is simple, rapid, inexpensive, more sensitive, accurate and non isotopic.
