2003
DOI: 10.1002/cbic.200200498
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Platinum(II)‐Based Coordination Compounds as Nucleic Acid Labeling Reagents: Synthesis, Reactivity, and Applications in Hybridization Assays

Abstract: The synthesis, characterization, and molecular interactions of platinum(II) coordination compounds, which contain a distal nonradioactive reporter molecule, with mono- and polynucleotides are described. A [Pt(II)(en)(NH(2)(CH(2))(6)NH-tBoc)Cl](NO(3)) (en=ethylenediamine) entity has been coupled, after removal of the tBoc group, to a number of hapten and fluorophore molecules through succinimide derivatives. The influence of the various tethered reporter groups within these complexes on the reactivity towards g… Show more

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Cited by 33 publications
(25 citation statements)
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“…After 14 d, genomic DNA was isolated with DNAzol (Invitrogen) and shRNA inserts were amplified from genomic DNA by PCR using the primers pRS-T7-fw, 5′-GGCCAGTGAATTGTAATACGAC-TCACTATAGGGAGGCGGCCCTTGAACCTCCTCGTTCGACC-3′ , containing a T7 RNA polymerase promoter sequence, and pRS8-rev, 5′-TAAAGCGCATGCTCCAGACT-3′. After purification (QIAquick PCR purification kit, Qiagen), PCR products were used for linear RNA amplification using the Megascript T7 kit (Ambion), and purified RNA probes (RNeasy, Qiagen) were labeled with cyanine-3 (Cy3) or cyanine-5 (Cy5) fluorescent groups using ULS (Kreatech) and purified over a KreaPure (Kreatech) spin column as described 29 . Labeled RNA probes from untreated and nutlin-3-treated cells were combined and hybridized to oligonucleotide arrays in 40 µl of hybridization mixture (25% formamide, 5× SCC, 0.01% SDS, 25% Kreablock (Kreatech)).…”
Section: Methodsmentioning
confidence: 99%
“…After 14 d, genomic DNA was isolated with DNAzol (Invitrogen) and shRNA inserts were amplified from genomic DNA by PCR using the primers pRS-T7-fw, 5′-GGCCAGTGAATTGTAATACGAC-TCACTATAGGGAGGCGGCCCTTGAACCTCCTCGTTCGACC-3′ , containing a T7 RNA polymerase promoter sequence, and pRS8-rev, 5′-TAAAGCGCATGCTCCAGACT-3′. After purification (QIAquick PCR purification kit, Qiagen), PCR products were used for linear RNA amplification using the Megascript T7 kit (Ambion), and purified RNA probes (RNeasy, Qiagen) were labeled with cyanine-3 (Cy3) or cyanine-5 (Cy5) fluorescent groups using ULS (Kreatech) and purified over a KreaPure (Kreatech) spin column as described 29 . Labeled RNA probes from untreated and nutlin-3-treated cells were combined and hybridized to oligonucleotide arrays in 40 µl of hybridization mixture (25% formamide, 5× SCC, 0.01% SDS, 25% Kreablock (Kreatech)).…”
Section: Methodsmentioning
confidence: 99%
“…PCR products were labelled with cyanine-3 or cyanine-5 fluorescent groups using the Universal Linkage System (ULS; Kreatech Biotechnology) and purified over a KreaPure (Kreatech Biotechnology) spin column as described previously 28 . PCR products from two samples were combined and hybridized to oligonucleotide arrays in 40 ml of 25% formamide, 5£SCC, 0.01% SDS (containing poly d(A), yeast transfer RNA and COT-1 DNA).…”
Section: Sirna Bar-code Screensmentioning
confidence: 99%
“…These reactions, however, are inevitably biased toward regions close to the 3Ј end of the ORF due to the processivity nature of RT. To circumvent this potential problem and preserve the signals from longer transcripts, we adapted a direct, nonenzymatic labeling approach (Heetebrij et al 2003). This method involved the use of a platinum-based compound to directly couple a fluorescent dye to RNA molecules.…”
Section: Genome-wide Identification Of Genes That Rely On the Set2-rpmentioning
confidence: 99%
“…Briefly, in Round A amplification, a degenerate primer with a universal oligo tag at the 5Ј end was used in a two-round primer extension reaction catalyzed by T7 sequenase version 2.0 (Upstate Biotechnology); onesixth of Round A products was then subjected to Round B amplification with ExTaq (Takara) using the universal primer. DNA was subsequently purified using Qiagen PCR purification kits and was chemically labeled using a platinum-based coordination compound as described previously (Heetebrij et al 2003).…”
Section: Chip-chip and Transcription Profilingmentioning
confidence: 99%