Detection of multiple loci in single cell by primer extension preamplification and nest PCR
- Author:
Shu-Hui YUAN
1
;
Fan JIN
;
He-Feng HUANG
Author Information
1. The Affiliated Obstetrics and Gynecology Hospital, College of Medical Sciences, Zhejiang University, Hangzhou 310006, China.
- Publication Type:Journal Article
- From:
Journal of Zhejiang University. Medical sciences
2002;31(3):145-148
- CountryChina
- Language:Chinese
-
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.
