Semiautomated clone verification by real-time PCR using molecular beacons

R. C.A.A. Van Schie, S. A.E. Marras, J. M. Conroy, N. J. Nowak, J. J. Catanese, P. J. De Jong

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) detection system that swiftly and accurately confirms marker content in template containing common repeat elements. A simple, one-tube, real-time PCR assay system was developed to specifically detect regions containing CA and GATA repeats. Ninety-six samples can be confirmed for marker content in a closed-tube format in 3 h, eliminating product confirmation on agarose gels and avoiding crossover contamination. Multiple STSs can be detected simultaneously in the same reaction tube by utilizing molecular beacons labeled with multicolor fluorophores. Template DNA from 260 RPCI-11 bacterial artificial chromosome (BAC) clones was examined for the presence of CA and/or GATA repeats using molecular beacon PCR and compared with conventional PCR results of the same clones. Of the 205 clones containing CA and GATA repeats, we were able to identify 129 clones (CA, n = 99; GATA, n = 30) by using molecular beacons and only 121 clones (CA, n = 92; GATA n = 29) by conventional PCR amplification. As anticipated, 55 clones that contained sequences other than CA or GATA failed molecular beacon detection. Molecular beacon PCR, employing beacons specific for tandem repeat elements, provides a fast, accurate, and sensitive multiplex detection assay that will expedite verification of marker content in a multitude of template containing these repeats.

Original languageEnglish (US)
Pages (from-to)1296-1308
Number of pages13
JournalBioTechniques
Volume29
Issue number6
StatePublished - Dec 1 2000

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Amplification
Real-Time Polymerase Chain Reaction
Assays
Clone Cells
Gels
Throughput
Sequence Tagged Sites
Polymerase Chain Reaction
Tandem Repeat Sequences
Fluorophores
DNA
Chromosomes
Electrophoresis
Sepharose
Contamination
Bacterial Artificial Chromosomes
Staining and Labeling

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Van Schie, R. C. A. A., Marras, S. A. E., Conroy, J. M., Nowak, N. J., Catanese, J. J., & De Jong, P. J. (2000). Semiautomated clone verification by real-time PCR using molecular beacons. BioTechniques, 29(6), 1296-1308.
Van Schie, R. C.A.A. ; Marras, S. A.E. ; Conroy, J. M. ; Nowak, N. J. ; Catanese, J. J. ; De Jong, P. J. / Semiautomated clone verification by real-time PCR using molecular beacons. In: BioTechniques. 2000 ; Vol. 29, No. 6. pp. 1296-1308.
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Van Schie, RCAA, Marras, SAE, Conroy, JM, Nowak, NJ, Catanese, JJ & De Jong, PJ 2000, 'Semiautomated clone verification by real-time PCR using molecular beacons', BioTechniques, vol. 29, no. 6, pp. 1296-1308.

Semiautomated clone verification by real-time PCR using molecular beacons. / Van Schie, R. C.A.A.; Marras, S. A.E.; Conroy, J. M.; Nowak, N. J.; Catanese, J. J.; De Jong, P. J.

In: BioTechniques, Vol. 29, No. 6, 01.12.2000, p. 1296-1308.

Research output: Contribution to journalArticle

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AU - Catanese, J. J.

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Van Schie RCAA, Marras SAE, Conroy JM, Nowak NJ, Catanese JJ, De Jong PJ. Semiautomated clone verification by real-time PCR using molecular beacons. BioTechniques. 2000 Dec 1;29(6):1296-1308.