D L Blake1, N L Dean, C Knight, S L Tan, A Ao. 1. Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, 687 Pine Avenue West, Women's Pavilion F3.16, Montreal, Quebec, Canada, H3A 1A1.
Abstract
PURPOSE: Single-cell polymerase chain reaction (PCR) requires efficient amplification and accurate detection. We compare the accuracy of heteroduplex, fluorescent-fragment, and fluorescent single-strand conformation polymorphism (F-SSCP) analysis as detection systems for analysis of a PCR assay developed for preimplantation genetic diagnosis. METHODS: A single-cell, fluorescent multiplex PCR assay was developed for the cystic fibrosis delta F508 mutation and the short tandem repeat, D21S11. Detection systems were compared by analyzing blinded PCR products. RESULTS: Amplification rates for cystic fibrosis were 89% by heteroduplex and 91% by fragment analysis, while it was 72% for D21S11 by fragment analysis. No difference in allele dropout was detected for cystic fibrosis by any method (2%). Overall accuracy was high, > 97%, although SSCP was the least accurate. CONCLUSIONS: Heteroduplex and fragment analysis proved equal in the diagnosis of a single amplified locus. We determined that fragment analysis allows maximal accuracy of detection and permits analysis of a second loci, controlling for DNA contamination and allelic dropout.
PURPOSE: Single-cell polymerase chain reaction (PCR) requires efficient amplification and accurate detection. We compare the accuracy of heteroduplex, fluorescent-fragment, and fluorescent single-strand conformation polymorphism (F-SSCP) analysis as detection systems for analysis of a PCR assay developed for preimplantation genetic diagnosis. METHODS: A single-cell, fluorescent multiplex PCR assay was developed for the cystic fibrosisdelta F508 mutation and the short tandem repeat, D21S11. Detection systems were compared by analyzing blinded PCR products. RESULTS: Amplification rates for cystic fibrosis were 89% by heteroduplex and 91% by fragment analysis, while it was 72% for D21S11 by fragment analysis. No difference in allele dropout was detected for cystic fibrosis by any method (2%). Overall accuracy was high, > 97%, although SSCP was the least accurate. CONCLUSIONS: Heteroduplex and fragment analysis proved equal in the diagnosis of a single amplified locus. We determined that fragment analysis allows maximal accuracy of detection and permits analysis of a second loci, controlling for DNA contamination and allelic dropout.
Authors: A Kuliev; S Rechitsky; O Verlinsky; V Ivakhnenko; J Cieslak; S Evsikov; G Wolf; M Angastiniotis; G Kalakoutis; C Strom; Y Verlinsky Journal: J Assist Reprod Genet Date: 1999-04 Impact factor: 3.412
Authors: J C Dreesen; L J Jacobs; M Bras; J Herbergs; J C Dumoulin; J P Geraedts; J L Evers; H J Smeets Journal: Mol Hum Reprod Date: 2000-05 Impact factor: 4.025