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| Content Provider | Royal Society of Chemistry (RSC) |
|---|---|
| Author | Trau, Matt Lawrie, Gwen Sova, Pavel Corrie, Simon Battersby, Bronwyn Kiviat, Nancy |
| Copyright Year | 2009 |
| 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. |
| Starting Page | 262 |
| Ending Page | 268 |
| Page Count | 7 |
| File Format | HTM / HTML PDF |
| ISSN | 1742206X |
| Volume Number | 5 |
| Issue Number | 3 |
| Journal | Molecular BioSystems |
| DOI | 10.1039/b813077a |
| Language | English |
| Publisher | Royal Society of Chemistry |
| Access Restriction | Open |
| Subject Keyword | Multiplexing Polymerase chain reaction Flow cytometry Chain reaction SPARC CpG site DNA polymerase Genotyping DNA Bead Cervical cancer Cancer |
| Content Type | Text |
| Resource Type | Article |
| Subject | Molecular Biology Biotechnology |
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