title: Development of a PCR/LDR/capillary electrophoresis assay with potential for the detection of a beta-thalassemia fetal mutation in maternal plasma.
abstract: OBJECTIVE
  Analysis of fetal DNA in maternal plasma has recently been introduced for non-invasive prenatal diagnosis. We have now investigated the feasibility of polymerase chain reaction (PCR)/ligase detection reaction (LDR)/capillary electrophoresis for the detection of fetal point mutations, such as the beta-thalassemia mutation, IVS2 654(C --> T), in maternal plasma DNA.
  
  
  METHODS
  The sensitivity of LDR/capillary electrophoresis was examined by quantifying the mutant PCR products in the presence of a vast excess of non-mutant competitor template, a situation that mimics the detection of rare fetal mutations in the presence of excess maternal DNA. PCR/LDR/capillary electrophoresis was applied to detect the mutation, IVS2 654(C --> T), in an experimental model at different sensitivity levels and from 10 maternal plasma samples.
  
  
  RESULTS
  Our results demonstrated that this approach to detect a low abundance IVS2 654(C --> T) mutation achieved a sensitivity of approximately 1:10,000. The approach was applied to maternal plasma DNA to detect the paternally inherited fetal IVS2 654(C --> T) mutation, and the results were equivalent to those obtained by PCR/reverse dot blot of amniotic fluid cell DNA.
  
  
  CONCLUSIONS
  PCR/LDR/capillary electrophoresis has a very high sensitivity that can distinguish low abundance single nucleotide differences and can detect paternally inherited fetal point mutations in maternal plasma.
authors.affiliation: Department of Obstetrics and Gynecology, Research Institute of Surgery, Daping Hospital, Third Military Medical University, 10 Changjiangzhilu, Daping, Yuzhong District, Chongqing 400042, People's Republic of China.
authors.forename: Ping
authors.initials: P
authors.lastname: Yi
authors.forename: Zhuqin
authors.initials: Z
authors.lastname: Chen
authors.forename: Lili
authors.initials: L
authors.lastname: Yu
authors.forename: Yingru
authors.initials: Y
authors.lastname: Zheng
authors.forename: Guodong
authors.initials: G
authors.lastname: Liu
authors.forename: Haichang
authors.initials: H
authors.lastname: Xie
authors.forename: Yuanguo
authors.initials: Y
authors.lastname: Zhou
authors.forename: Xiuhui
authors.initials: X
authors.lastname: Zheng
authors.forename: Jian
authors.initials: J
authors.lastname: Han
authors.forename: Li
authors.initials: L
authors.lastname: Li
mesh_terms: D000328:Adult; D004247:DNA / Q000097:blood / Q000737:chemistry; D011088:DNA Ligases; D019075:Electrophoresis, Capillary; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D055815:Young Adult; D055544:beta-Globins / Q000235:genetics; D017086:beta-Thalassemia / Q000175:diagnosis* / Q000235:genetics
chemical_list: D055544:beta-Globins; D004247:DNA; D011088:DNA Ligases