title: [Detection of multiple loci in single cell by primer extension preamplification and nest PCR]
abstract: OBJECTIVE: To investigate the feasibility of multiple loci detection in single cell by primer extension preamplification (PEP) followed by nest PCR. METHODS: Using PEP, the whole genomic DNA in single lymphocyte or single blastomere was amplified. In addition, CD17, nt-28 and linked ATTTT repeat for beta-thalassemia, F508 and linked GATT repeat for cystic fibrosis, DMD exon 17 and 48 for Duchenne muscular dystrophy, short tandem repeats of D18S51, D21S11 and D21S1411, and sex-determination gene SRY of the Y chromosome were all detected using nest-PCR from a small aliquot of the PEP reaction. RESULTS: The rate of successful single lymphocyte amplification was 89.5%(false positive 0.48%false negative 2.5%). The rate of successful single blastomere amplification was 85.56%(false positive 3%). CONCLUSION: The PEP technique followed by nest PCR analysis of single cell is very useful for simultaneous detection of multiple gene loci. It may be applicable for preimplantation genetic diagnosis.
authors.affiliation: The Affiliated Obstetrics and Gynecology Hospital, College of Medical Sciences, Zhejiang University, Hangzhou 310006, China.
authors.forename: Shu-Hui
authors.initials: SH
authors.lastname: Yuan
authors.forename: Fan
authors.initials: F
authors.lastname: Jin
authors.forename: He-Feng
authors.initials: HF
authors.lastname: Huang