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Titolo:
Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase
Autore:
Allawi, HT; Dong, F; Ip, HS; Neri, BP; Lyamichev, VI;
Indirizzi:
Third Wave Technol Inc, Madison, WI 53719 USA Third Wave Technol Inc Madison WI USA 53719 ol Inc, Madison, WI 53719 USA
Titolo Testata:
RNA-A PUBLICATION OF THE RNA SOCIETY
fascicolo: 2, volume: 7, anno: 2001,
pagine: 314 - 327
SICI:
1355-8382(200102)7:2<314:MORASB>2.0.ZU;2-T
Fonte:
ISI
Lingua:
ENG
Soggetto:
INTERCELLULAR-ADHESION MOLECULE-1; ANTISENSE OLIGONUCLEOTIDES; COMPLEMENTARY OLIGONUCLEOTIDES; MESSENGER-RNA; SELECTIVE-INHIBITION; STRUCTURED RNA; DNA-POLYMERASE; HYBRIDIZATION; EXPRESSION; KINETICS;
Keywords:
antisense; extendible sites; hybridization; ICAM-1; invasive cleavage; RNA detection;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
57
Recensione:
Indirizzi per estratti:
Indirizzo: Lyamichev, VI Third Wave Technol Inc, 502 S Rosa Rd, Madison, WI 53719 USAThird Wave Technol Inc 502 S Rosa Rd Madison WI USA 53719 SA
Citazione:
H.T. Allawi et al., "Mapping of RNA accessible sites by extension of random oligonucleotide libraries with reverse transcriptase", RNA, 7(2), 2001, pp. 314-327

Abstract

A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR witha primer corresponding to the tag sequence and a second primer specific tothe target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotidelibraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated res (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta -globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 29/03/20 alle ore 12:16:57