Direct comparison of detection systems used for the development of single-cell genetic tests in preimplantation genetic diagnosis.
OBJECTIVE
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.
Blake
D L
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
N L
NL
Knight
C
C
Tan
S L
SL
Ao
A
A
D003550:Cystic Fibrosis / Q000175:diagnosis* / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D008297:Male; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*; D011296:Prenatal Diagnosis / Q000379:methods*; D016037:Single-Blind Method
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA