Clinical Chemistry AACC Online Job Center
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 52: 1855-1863, 2006. First published August 17, 2006; 10.1373/clinchem.2006.068817
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow 068817.Supplemental Data
Right arrow All Versions of this Article:
clinchem.2006.068817v1
52/10/1855    most recent
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Amicarelli, G.
Right arrow Articles by Makrigiorgos, G. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Amicarelli, G.
Right arrow Articles by Makrigiorgos, G. M.
Related Collections
Right arrow Molecular Diagnostics and Genetics
(Clinical Chemistry. 2006;52:1855-1863.)
© 2006 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Genotype-Specific Signal Generation Based on Digestion of 3-Way DNA Junctions: Application to KRAS Variation Detection

Giulia Amicarelli1, Daniel Adlerstein2,a, Erlet Shehi2, Fengfei Wang3 and G. Mike Makrigiorgos3

1 Department of Biotechnology and Biosciences, University of Milano Bicocca, Milano, Italy.
2 DiaSorin SpA, Saluggia (VC), Italy.
3 Dana Farber-Brigham and Women’s Cancer Center, Harvard Medical School, Boston, MA.

aAddress correspondence to this author at: Diasorin SpA, Piazza della Scienza 4, 20126 Milano, Italy. Fax 0039-02-6448-3565; e-mail daniel.adlerstein{at}diasorin.it.

Background: Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens.

Materials and Methods: We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction.

Results: The system detected and genotyped KRAS sequence variants down to ~0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%.

Conclusion: OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.




The following articles in journals at HighWire Press have cited this article:


Home page
 Nucleic Acids ResHome page
G. Amicarelli, E. Shehi, G. M. Makrigiorgos, and D. Adlerstein
FLAG assay as a novel method for real-time signal generation during PCR: application to detection and genotyping of KRAS codon 12 mutations
Nucleic Acids Res., October 11, 2007; (2007) gkm809v1.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2006 by the American Association for Clinical Chemistry.