Fluorescent labelling of cRNA for microarray applications.
Authors't Hoen PA, de Kort F, van Ommen GJ, den Dunnen JT
JournalNucleic Acids Res
PubMed ID12595569
'Microarrays of oligonucleotide expression libraries can be hybridised with either cDNA, generated from mRNA during reverse transcription, or cRNA, generated in an Eberwine mRNA amplification procedure. While methods for fluorescent labelling of cDNA have been thoroughly investigated, methods for cRNA labelling have not. To this purpose, we developed an aminoallyl-UTP ... More
Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling.
AuthorsGraham MR, Smoot LM, Migliaccio CA, Virtaneva K, Sturdevant DE, Porcella SF, Federle MJ, Adams GJ, Scott JR, Musser JM
JournalProc Natl Acad Sci U S A
PubMed ID12370433
'Two-component gene regulatory systems composed of a membrane-bound sensor and cytoplasmic response regulator are important mechanisms used by bacteria to sense and respond to environmental stimuli. Group A Streptococcus, the causative agent of mild infections and life-threatening invasive diseases, produces many virulence factors that promote survival in humans. A two-component ... More
Non-radioactive labeling and detection of nucleic acids. IV. Synthesis and properties of digoxigenin-modified 2'-deoxyuridine-5'-triphosphates and a photoactivatable analog of digoxigenin (photodigoxigenin).
AuthorsMühlegger K, Huber E, von der Eltz H, Rüger R, Kessler C
JournalBiol Chem Hoppe Seyler
PubMed ID2076201
'The chemical syntheses of novel digoxigenin-derivatized compounds are described which are modified substrates for enzymatically or photochemically non-radioactive digoxigenin labeling of nucleic acids. Various activated digoxigenin-haptens are coupled to 5-aminoallyl-substituted 2''-deoxyuridine-5''-triphosphate. This results in digoxigenin-modified nucleoside triphosphates of variable spacer lengths (Dig-[4]-dUTP/Dig-[11]-dUTP/Dig-[16]-dUTP) which can be used as substrates for enzymatic ... More
Preparing fluorescent probes for microarray studies.
AuthorsXiang CC, Brownstein MJ
JournalMethods Mol Biol
PubMed ID12710665
Sequence-specific and hydrolytic scission of DNA and RNA by lanthanide complex-oligoDNA hybrids.
AuthorsKomiyama M
JournalJ Biochem (Tokyo)
PubMed ID8576074
'Totally synthetic and sequence-specific nucleases and ribonucleases, which hydrolyze DNAs and RNAs selectively at target sites, have been prepared. Lanthanide ions, which efficiently hydrolyze the phosphodiester linkages in nucleic acids, are attached to the 5''-end of synthetic DNA oligomers (sequence-recognizing sites) by use of an iminodiacetate ligand. Under physiological conditions, ... More
Comparison of probe preparation methods for DNA microarrays.
AuthorsFarbrother P, Müller S, Noegel AA, Eichinger L
JournalBiotechniques
PubMed ID12398197
Multiplex detection of RNA expression in Drosophila embryos.
AuthorsKosman D, Mizutani CM, Lemons D, Cox WG, McGinnis W, Bier E
JournalScience
PubMed ID15297669
We present a fluorescence-based, multiplex in situ hybridization method that permits the simultaneous detection of five differently labeled antisense RNA probes and up to seven differ-ent transcripts in a single Drosophila embryo. We also show that it should be possible to increase the number of detected transcripts substantially with nascent ... More
Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group-labeled 2'-deoxyribonucleoside-5'-triphosphates.
AuthorsGiller G, Tasara T, Angerer B, Mühlegger K, Amacker M, Winter H
JournalNucleic Acids Res
PubMed ID12736313
Fluorescent-labeled DNA is generated through enzymatic incorporation of fluorophore-linked 2'-deoxyribonucleoside-5'-triphosphates (dNTPs) by DNA polymerases. We describe the synthesis of a variety of dye-labeled dNTPs. Amino-linker-modified 5'-triphosphates of all four naturally occurring nucleobases were used as precursors. Commercially available dyes were coupled to the amino function of the side chain. In ... More
Fluorescent DNA hybridization probe preparation using amine modification and reactive dye coupling.
AuthorsCox WG, Singer VL,
JournalBiotechniques
PubMed ID14740493
Fluorescent nucleic acid hybridization probes traditionally have been generated by enzymatic incorporation of dye-labeled nucleotides, even though incorporation efficiency is low and variable from dye to dye. Alternatively, 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP) is enzymatically incorporated to generate amine-modified DNA, which is then chemically labeled with an amine-reactive fluorescent dye. We optimized ... More
Simple method for preparation of fluor/hapten-labeled dUTP.
AuthorsNimmakayalu M, Henegariu O, Ward DC, Bray-Ward P
JournalBiotechniques
PubMed ID10723566
Many projects, such as multiplex-fluorescence in situ hybridization (M-FISH) karyotyping, require the use of relatively large amounts of multiple fluor- or hapten-labeled nucleotides for the preparation of DNA probes. Such a requirement makes these experimental approaches prohibitively expensive for many researchers. The cost of such nucleotides can be reduced approximately ... More
Amine-modified random primers to label probes for DNA microarrays.
AuthorsXiang CC, Kozhich OA, Chen M, Inman JM, Phan QN, Chen Y, Brownstein MJ
JournalNat Biotechnol
PubMed ID12089562
DNA microarrays have been used to study the expression of thousands of genes at the same time in a variety of cells and tissues. The methods most commonly used to label probes for microarray studies require a minimum of 20 microg of total RNA or 2 microg of poly(A) RNA. ... More
Use of RNA and genomic DNA references for inferred comparisons in DNA microarray analyses.
In most microarray assays, labeled cDNA molecules derived from reference and query RNA samples are co-hybridized to probes arrayed on a glass surface. Gene expression profiles are then calculated for each gene based on the relative hybridization intensities measured between the two samples. The most commonly used reference samples are ... More
Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI.
AuthorsBodnar JW, Zempsky W, Warder D, Bergson C, Ward DC
JournalJ Biol Chem
PubMed ID6317689
The cleavage of specific DNA sequences by the restriction endonucleases AluI, DdeI, HinfI, RsaI, and TaqI has been studied by monitoring the effect of various nucleotide modifications on the rate of DNA digestion. Bacteriophage fd DNA was completely substituted in one strand with a single nucleotide analog, using an in ... More
Non-isotopic RNA probes. Comparison between different labels and detection systems.
AuthorsGiaid A, Hamid Q, Adams C, Springall DR, Terenghi G, Polak JM
JournalHistochemistry
PubMed ID2613556
Several studies have shown the use of non-radioactive labelled DNA probes for in situ hybridisation, mainly to identify cellular DNA. In this study mRNA in situ hybridisation was performed on rat pituitary with biotinylated complementary (c) RNA probes for rat prolactin and growth hormone (GH), and compared with radioactive 35S-radiolabelled ... More