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Molecular Human Reproduction, Vol. 9, No. 12, pp. 815-820, 2003
© European Society of Human Reproduction and Embryology 2003; all rights reserved

Detection of cystic fibrosis alleles from single cells using molecular beacons and a novel method of asymmetric real-time PCR

Kenneth E. Pierce1, John E. Rice, J.Aquiles Sanchez and Lawrence J. Wangh

Department of Biology, MS-008, Brandeis University, Waltham, MA 02454-9110, USA

1 To whom correspondence should be addressed. e-mail: pierce{at}brandeis.edu

We present a method for rapid and accurate identification of the normal and {Delta}F508 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 {Delta}F508 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.

Key words: cystic fibrosis {Delta}F508/fluorescent oligonucleotide probes/linear DNA amplification/preimplantation genetic diagnosis/quantitative PCR


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Home page
 Nucleic Acids ResHome page
J. J. Li, Y. Chu, B. Y.-H. Lee, and X. S. Xie
Enzymatic signal amplification of molecular beacons for sensitive DNA detection
Nucleic Acids Res., April 1, 2008; 36(6): e36 - e36.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
K. E. Pierce, J. A. Sanchez, J. E. Rice, and L. J. Wangh
Linear-After-The-Exponential (LATE)-PCR: Primer design criteria for high yields of specific single-stranded DNA and improved real-time detection
PNAS, June 14, 2005; 102(24): 8609 - 8614.
[Abstract] [Full Text] [PDF]


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