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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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IMPACT FACTOR 2016: 3.432

CiteScore 2016: 2.21

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1437-4331
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In This Section
Volume 45, Issue 7 (Jul 2007)

Issues

A novel application of melting curves: utility of peak area calculation for relative methylation quantification

Ugur Deligezer
  • 1Department of Basic Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey
/ Ebru Esin Akisik
  • 2Department of Basic Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey
/ Nejat Dalay
  • 3Department of Basic Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey
Published Online: 2007-07-08 | DOI: https://doi.org/10.1515/CCLM.2007.146

Abstract

Background: DNA methylation markers appear to be useful in cancer detection, in evaluating the prognosis associated with disease progression, and in detecting the metastatic potential of tumors.

Methods: We present an approach for relative quantification of methylated gene sequences. The technique combines conventional PCR and LightCycler fluorescence PCR. In the conventional PCR step, bisulfite-modified molecules are selectively enriched, irrespective of their methylation status, and used as template in the second step, the LightCycler fluorescence PCR, which includes methylation-specific primers and fluorescently labeled probes. After amplification, a stepwise increase in temperature leads to the generation of melting curves reflecting fluorescence emission induced by disassociation of the probe-target complexes. Quantification is based on calculation of the peak areas for melting curves using a Gaussian fit curve. The area under this curve indicates the relative amounts of methylated sequences in each sample.

Results: By normalizing the peak areas by template concentrations, a methylation index was calculated for each sample. This approach was then used to carry out a relative quantification of p16INK4A gene methylation in bone marrow and blood mononuclear cells from patients with positive translocation status.

Conclusions: If validated in further studies, this approach represents a highly specific technique that can be used in analyzing paired samples of cancer patients.

Clin Chem Lab Med 2007;45:867–73.

Keywords: leukemia; melting curve; methylation; methylation index; peak area calculation

About the article

Corresponding author: Prof. Dr. Nejat Dalay, Department of Basic Oncology, Oncology Institute, 34390 Capa, Istanbul, Turkey Fax: +90-212-5348078


Received: 2006-12-04

Accepted: 2007-03-05

Published Online: 2007-07-08

Published in Print: 2007-07-01



Citation Information: Clinical Chemical Laboratory Medicine, ISSN (Online) 14374331, ISSN (Print) 14346621, DOI: https://doi.org/10.1515/CCLM.2007.146. Export Citation

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[1]
Ugur Deligezer, Yesim Eralp, Ebru E. Akisik, Elif Z. Akisik, Pinar Saip, Erkan Topuz, and Nejat Dalay
Clinical Chemical Laboratory Medicine, 2008, Volume 46, Number 3
[2]
Pietro Spitali, Hans Heemskerk, Rolf HAM Vossen, Alessandra Ferlini, Johan T den Dunnen, Peter AC 't Hoen, and Annemieke Aartsma-Rus
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Laboratory Investigation, 2010
[4]
Ugur Deligezer, Yesim Eralp, Elif Z. Akisik, Ebru E. Akisik, Pinar Saip, Erkan Topuz, and Nejat Dalay
Annals of the New York Academy of Sciences, 2008, Volume 1137, Number 1, Page 175
[5]
Despina K. Alexiadou, Andrea K. Ioannou, Sofia A. Kouidou-Andreou, Anastasios N. Voulgaropoulos, and Stella Th. Girousi
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