title: PCR assay for the inversion causing severe Hemophilia A and its application.
abstract: 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.affiliation: Beijing Gene Diagnosis Laboratory, Beijing Red Cross Chaoyang Hospital, Beijing 100020, China.
authors.forename: J
authors.initials: J
authors.lastname: Liu
authors.forename: Q
authors.initials: Q
authors.lastname: Liu
authors.forename: Y
authors.initials: Y
authors.lastname: Liang
authors.forename: L
authors.initials: L
authors.lastname: Wang
authors.forename: G
authors.initials: G
authors.lastname: Nozary
authors.forename: B
authors.initials: B
authors.lastname: Xiao
authors.forename: Z
authors.initials: Z
authors.lastname: Zhu
authors.forename: Y
authors.initials: Y
authors.lastname: Zhou
authors.forename: L
authors.initials: L
authors.lastname: Liu
authors.forename: Y
authors.initials: Y
authors.lastname: Guan
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.forename: S S
authors.initials: SS
authors.lastname: Sommer
mesh_terms: D007446:Chromosome Inversion; D005169:Factor VIII / Q000235:genetics*; D005260:Female; D006467:Hemophilia A / Q000235:genetics*; D006579:Heterozygote; D006801:Humans; D008297:Male; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D005169:Factor VIII