Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries

Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermed...

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Veröffentlicht in:BioTechniques. - 1993. - 58(2015), 6 vom: 13. Juni, Seite 301-7
1. Verfasser: Murgha, Yusuf (VerfasserIn)
Weitere Verfasser: Beliveau, Brian, Semrau, Kassandra, Schwartz, Donald, Wu, Chao-Ting, Gulari, Erdogan, Rouillard, Jean-Marie
Format: Online-Aufsatz
Sprache:English
Veröffentlicht: 2015
Zugriff auf das übergeordnete Werk:BioTechniques
Schlagworte:Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Oligopaint FISH probes fluorescent in situ hybridization (FISH) in vitro transcription (IVT) microarrays oligonucleotide library pools reverse transcription (RT) single-stranded DNA (ssDNA) libraries mehr... DNA Primers DNA, Single-Stranded Fluorescent Dyes Oligonucleotide Probes RNA 63231-63-0
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245 1 0 |a Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries 
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500 |a Date Revised 13.11.2018 
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520 |a Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization 
650 4 |a Journal Article 
650 4 |a Research Support, N.I.H., Extramural 
650 4 |a Research Support, Non-U.S. Gov't 
650 4 |a Oligopaint FISH probes 
650 4 |a fluorescent in situ hybridization (FISH) 
650 4 |a in vitro transcription (IVT) 
650 4 |a microarrays 
650 4 |a oligonucleotide library pools 
650 4 |a reverse transcription (RT) 
650 4 |a single-stranded DNA (ssDNA) libraries 
650 7 |a DNA Primers  |2 NLM 
650 7 |a DNA, Single-Stranded  |2 NLM 
650 7 |a Fluorescent Dyes  |2 NLM 
650 7 |a Oligonucleotide Probes  |2 NLM 
650 7 |a RNA  |2 NLM 
650 7 |a 63231-63-0  |2 NLM 
700 1 |a Beliveau, Brian  |e verfasserin  |4 aut 
700 1 |a Semrau, Kassandra  |e verfasserin  |4 aut 
700 1 |a Schwartz, Donald  |e verfasserin  |4 aut 
700 1 |a Wu, Chao-Ting  |e verfasserin  |4 aut 
700 1 |a Gulari, Erdogan  |e verfasserin  |4 aut 
700 1 |a Rouillard, Jean-Marie  |e verfasserin  |4 aut 
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773 1 8 |g volume:58  |g year:2015  |g number:6  |g day:13  |g month:06  |g pages:301-7 
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