Detection of cystic fibrosis alleles from single cells using molecular beacons and a novel method of asymmetric real-time PCR.
We present a method for rapid and accurate identification of the normal and DeltaF508 alleles of the cystic fibrosis (CF) gene in single human cells that utilizes LATE (linear after the exponential)-PCR, a newly invented form of asymmetric PCR. Detection of the single-stranded amplicon is carried out in real time, using allele-specific molecular beacons. The LATE-PCR method permits controlled abrupt transition from exponential to linear amplification and thereby enhances the fluorescent signals and reduces variability between replicate samples relative to those obtained using typical real-time PCR. Of 239 single lymphoblasts generating amplification signals, 227 (95%) exhibited signals that met objective quantitative criteria required for diagnosis. Among these samples, 222 were genotyped correctly, for an assay accuracy of 98%. The small number of diagnostic errors was due to allele drop-out among heterozygous lymphoblasts, 4/119 (3.4%), and contamination among homozygous DeltaF508 lymphoblasts, 1/57 (1.8%). LATE-PCR offers a new strategy for preimplantation genetic diagnosis and other fields in which accurate quantitative detection of single copy genes is important.
Pierce
Kenneth E
KE
Department of Biology, MS-008, Brandeis University, Waltham, MA 02454-9110, USA. pierce@brandeis.edu
Rice
John E
JE
Sanchez
J Aquiles
JA
Wangh
Lawrence J
LJ
D000483:Alleles; D001483:Base Sequence; D045744:Cell Line, Tumor; D003550:Cystic Fibrosis / Q000175:diagnosis*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D005456:Fluorescent Dyes; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods
C505032:CFTR protein, human; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator