Development of a multiplexed bead-based assay for detection of DNA methylation in cancer-related genes

Corrie, Simon, Sova, Pavel, Lawrie, Gwen, Battersby, Bronwyn, Kiviat, Nancy and Trau, Matt (2009) Development of a multiplexed bead-based assay for detection of DNA methylation in cancer-related genes. Molecular Biosystems, 5 3: 262-268. doi:10.1039/b813077a


Author Corrie, Simon
Sova, Pavel
Lawrie, Gwen
Battersby, Bronwyn
Kiviat, Nancy
Trau, Matt
Title Development of a multiplexed bead-based assay for detection of DNA methylation in cancer-related genes
Journal name Molecular Biosystems   Check publisher's open access policy
ISSN 1742-206X
1742-2051
Publication date 2009-12-24
Year available 2008
Sub-type Article (original research)
DOI 10.1039/b813077a
Open Access Status Not Open Access
Volume 5
Issue 3
Start page 262
End page 268
Total pages 7
Place of publication Cambridge, United Kingdom
Publisher Royal Society of Chemistry
Collection year 2009
Language eng
Abstract Herein we report a method for the detection of methylated CpG dinucleotides located within CpG islands in genomic DNA using multiplexed bead-based assays and standard flow cytometry instrumentation. Four CpG "clusters" were identified in the TFPI2 and SPARC CpG islands whose methylation status was highly correlated with the incidence of invasive cervical cancer in our previous studies. Eight probes in total were designed for both the methylated and unmethylated forms of each cluster and attached to different fluorescently-encoded organosilica bead sets. Probe design was investigated by changing either the length of probes whilst keeping the melting temperature constant, or changing the melting temperature and keeping the probe length constant. Asymmetric polymerase chain reaction (PCR) methods designed without methylation-specific primers were used to prepare fluorescently-labelled targets based on bisulfite-converted genomic DNA. After investigating the specificity of the probes in a model system using fluorescently-labelled synthetic oligonucleotides, cancer cell-line DNA was analysed and the constant length probe design facilitated the correct genotyping of all clusters with respect to negative controls.
Keyword organosilica microspheres
flow-cytometry
lung-cancer
cpg islands
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Pre-publication title "Multiplexed Biomarker Detection Assay for DNA Methylation Genotyping" Received 05 Aug 2008, Accepted 01 Dec 2008 First published on the web 24 Dec 2008

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Chemistry and Molecular Biosciences
 
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Created: Thu, 03 Sep 2009, 18:40:05 EST by Mr Andrew Martlew on behalf of Faculty of Science