Efficiency and accuracy of polymerase-chain-reaction assay for cystic fibrosis allele delta F508 in single cell.
Diagnosis of genetic disorders in the embryo before implantation, though possible by removal of one or two blastomeres at the eight-cell stage, is still experimental because the procedures of gene analysis of DNA from a single cell are not yet reliable enough for clinical application. We have evaluated the efficiency and accuracy of polymerase-chain-reaction (PCR) amplification of a single-copy gene, wild-type or cystic fibrosis delta F508 allele, on single sperm cells from a donor known to be a heterozygous carrier of the delta F508 mutation. DNA from single spermatozoa was decontaminated by restriction-enzyme treatment, then the region around the delta F508 site was amplified by nested PCR. The distribution of the wild-type and mutant alleles (59 [55%] and 48 [45%, respectively]) in the 107 single spermatozoa did not differ from that expected (50% each). 1 sample did not provide an amplified signal. To check that the two alleles would be amplified with equal efficiency when they were both present within a cell, we did PCR for 51 two-sperm samples. Again the distribution did not deviate from that expected (17 [33%] both wild-type; 21 [41%] one wild-type, one delta F508; 13 [26%] both delta F508 vs 25%; 50%; 25% expected). None of the 74 blanks in these experiments was contaminated. We conclude that our delta F508 single-cell assay is efficient and accurate and can be used for analysis of blastomere DNA to diagnose cystic fibrosis before embryo implantation.
Centre for Reproductive Medicine, Brussels Free University, Belgium.
J
J
Liu
W
W
Lissens
P
P
Devroey
A
A
Van Steirteghem
I
I
Liebaers
D000483:Alleles; D003550:Cystic Fibrosis / Q000175:diagnosis* / Q000235:genetics; D047108:Embryonic Development; D005260:Female; D006579:Heterozygote; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis; D013094:Spermatozoa