Direct comparison of detection systems used for the development of single-cell genetic tests in preimplantation genetic diagnosis.

Autor: Blake DL; Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, 687 Pine Avenue West, Women's Pavilion F3.16, Montreal, Quebec, Canada, H3A 1A1., Dean NL, Knight C, Tan SL, Ao A
Jazyk: angličtina
Zdroj: Journal of assisted reproduction and genetics [J Assist Reprod Genet] 2001 Oct; Vol. 18 (10), pp. 557-65.
DOI: 10.1023/a:1011958008240
Abstrakt: 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.
Databáze: MEDLINE