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
文摘目的建立一种简便、快速的血友病A（hemophilia A，HA）基因突变筛查方法学，并用于直接基因诊断及携带者筛查。方法对于临床确诊的24例血友病A患者和家系女性成员，首先采用倒位PCR及PCR技术，检测是否为F脚基因第22、1内含子倒位或倒位携带者；对非倒位者则用PCR扩增FⅧ基因26个外显子，继而应用构象敏感凝胶电泳（conformation sensitive gel electrophoresis，CSGE）技术对37个扩增片段进行突变筛查，阳性者予以进一步测序验证；据突变类型，对非倒位HA先证者家系女性成员直接测序或行CSGE检测，以判读其是否为携带者。结果24个HA家系中，7例先证者FⅧ基因第22内含子倒位检测为阳性，未发现第1内含子倒位；13个有家族史及3个散发非倒位HA家系中均存在FⅧ基因不同外显子区的单碱基突变，1例未检出突变。本研究中，24个家系中基因分型诊断及携带者筛查阳性率、可诊断率分别为94．12％（16／17），100％（17／17）。结论PCR—CSGE检测HA单碱基突变高度敏感、特异；F脚基因第22、1内含子检测联合PCR—CSGE基因分型诊断技术及核苷酸测序可对所有的HA家系（含散发）进行直接基因诊断，理论上可筛查新突变并明确其突变类型。该法简便、快速、成本低，在HA直接基因诊断及携带者筛查中优势独特，应具重要应用价值。
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.
