Real-time PCR with molecular beacons provides a highly accurate assay for detection of Tay-Sachs alleles in single cells.
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.
Rice
John E
JE
Department of Biology, Brandeis University, Waltham, MA 02454-9110, USA.
Sanchez
J Aquiles
JA
Pierce
Kenneth E
KE
Wangh
Lawrence J
LJ
D000328:Adult; D000483:Alleles; D001681:Biological Assay / Q000379:methods; D002460:Cell Line; D015342:DNA Probes / Q000737:chemistry; D005260:Female; D005838:Genotype; D006801:Humans; D008214:Lymphocytes / Q000166:cytology; D011247:Pregnancy; D019836:Preimplantation Diagnosis / Q000379:methods*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013661:Tay-Sachs Disease / Q000175:diagnosis / Q000235:genetics*
D015342:DNA Probes