JMD etoc alert
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Originally published online as doi:10.2353/jmoldx.2008.070076 on December 28, 2007

Published online before print December 28, 2007
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jmoldx.2008.070076v1
10/1/33    most recent
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
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 Google Scholar
Google Scholar
Right arrow Articles by Gustafson, K. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gustafson, K. S.
Journal of Molecular Diagnostics 2008, Vol. 10, No. 1
Copyright © 2008 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2008.070076

Technical Advance

Locked Nucleic Acids Can Enhance the Analytical Performance of Quantitative Methylation-Specific Polymerase Chain Reaction

Karen S. Gustafson

From the Department of Pathology, The Johns Hopkins University, Baltimore, Maryland

Abstract

Aberrant DNA methylation of tumor suppressor genes is a frequent epigenetic event that occurs early in tumor progression. Real-time quantitative methylation-specific polymerase chain reaction (QMSP) assays can provide accurate detection and quantitation of methylated alleles that may be potentially useful in diagnosis and risk assessment for cancer. Development of QMSP requires optimization to maximize analytical specificity and sensitivity for the detection of methylated alleles. However, in some cases challenges encountered in primer and probe design can make optimization difficult and limit assay performance. Locked nucleic acids (LNAs) demonstrate increased affinity and specificity for their cognate DNA sequences. In this proof-of-principle study, LNA residues were incorporated into primer and probe design to determine whether LNA-modified oligonucleotides could enhance the analytical performance of QMSP for IGSF4 promoter methylation in human cancer cell lines using either SYBR Green or fluorogenic probe detection methods. Use of LNA primers in QMSP with SYBR Green improved analytical specificity for methylated alleles and eliminated the formation of nonspecific products because of mispriming from unmethylated alleles. QMSP using LNA probe and primers showed an increased amplification efficiency and maximum fluorescent signal. QMSP with LNA oligonucleotides and either detection method could reliably detect five genome equivalents of methylated DNA in 1000- to 10,000-fold excess unmethylated DNA. Thus, LNA oligonucleotides can be used in QMSP optimization to enhance analytical performance.






HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2008 by the American Society for Investigative Pathology and the Association for Molecular Pathology.