title: Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.
abstract: 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.
authors.affiliation: MYcroarray, Ann Arbor, MI.
authors.forename: Yusuf
authors.initials: Y
authors.lastname: Murgha
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA.
authors.forename: Brian
authors.initials: B
authors.lastname: Beliveau
authors.affiliation: MYcroarray, Ann Arbor, MI.
authors.forename: Kassandra
authors.initials: K
authors.lastname: Semrau
authors.affiliation: MYcroarray, Ann Arbor, MI.
authors.forename: Donald
authors.initials: D
authors.lastname: Schwartz
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA.
authors.forename: Chao-Ting
authors.initials: CT
authors.lastname: Wu
authors.affiliation: MYcroarray, Ann Arbor, MI.|Department of Chemical Engineering, University of Michigan, Ann Arbor, MI.
authors.forename: Erdogan
authors.initials: E
authors.lastname: Gulari
authors.affiliation: MYcroarray, Ann Arbor, MI.
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Rouillard
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics*; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D048348:Reverse Transcription; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012313:RNA
keywords: Oligopaint FISH probes; fluorescent in situ hybridization (FISH); in vitro transcription (IVT); microarrays; oligonucleotide library pools; reverse transcription (RT); single-stranded DNA (ssDNA) libraries