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Real‐time PCR with molecular beacons provides a highly accurate assay for detection of Tay‐Sachs alleles in single cells
Author(s) -
Rice John E.,
Sanchez J. Aquiles,
Pierce Kenneth E.,
Wangh Lawrence J.
Publication year - 2002
Publication title -
prenatal diagnosis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.956
H-Index - 97
eISSN - 1097-0223
pISSN - 0197-3851
DOI - 10.1002/pd.500
Subject(s) - amplicon , molecular beacon , tay sachs disease , allele , microbiology and biotechnology , biology , genotype , lymphoblast , lysis , real time polymerase chain reaction , genetics , polymerase chain reaction , virology , dna , gene , cell culture , medicine , disease , pathology , oligonucleotide
The results presented here provide the first single‐cell genetic assay for Tay‐Sachs disease based on real‐time PCR. Individual lymphoblasts were lysed with an optimized lysis buffer and assayed using one pair of primers that amplifies both the wild type and 1278 + TATC Tay‐Sachs alleles. The resulting amplicons were detected in real time with two molecular beacons each with a different colored fluorochrome. The kinetics of amplicon accumulation generate objective criteria by which to evaluate the validity of each reaction. The assay had an overall utility of 95%, based on the detection of at least one signal in 235 of the 248 attempted tests and an efficiency of 97%, as 7 of the 235 samples were excluded from further analysis for objective quantitative reasons. The accuracy of the assay was 99.1%, because 228 of 230 samples gave signals consistent with the genotype of the cells. Only two of the 135 heterozygous samples were allele drop‐outs, a rate far lower than previously reported for single‐cell Tay‐Sachs assays using conventional methods of PCR. Copyright © 2002 John Wiley & Sons, Ltd.