Rapid screening of open reading frames by protein synthesis with an in vitro transcription and translation assay. NLM AIDSLINE Important note: Information in this article was accurate in 1995. The state of the art may have changed since the publication date.

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Rapid screening of open reading frames by protein synthesis with an in vitro transcription and translation assay.

Biotechniques. 1995 Feb;18(2):244-8. Unique Identifier : AIDSLINE MED/95244141
Switzer WM; Heneine W; Retrovirus Diseases Branch, Centers for Disease Control and; Prevention, Atlanta, GA 30333, USA.

Abstract: The analysis of open reading frames (ORFs) to predict full-length or truncated proteins in genes is conventionally achieved by DNA sequencing. This method becomes labor-intensive when a large number of specimens or when large genes are to be examined. To circumvent this problem, we used an in vitro transcription and translation (TT) assay to identify full-length or truncated proteins in PCR-amplified genes. A total of 47 nef genes from the simian immunodeficiency virus (SIV) were cloned from 13 SIV-infected monkeys and were screened for ORFs by using the TT assay. Of these 47 genes, 20 had an intact ORF and 27 had premature stop codons at variable positions in the nef gene. All 20 nef genes with intact ORFs produced full-length proteins, while truncated proteins of different sizes were synthesized from all 27 nef genes with premature stop codons. In addition, we validated a simplified TT protocol that allows the direct screening of ORFs from transformed bacterial colonies, thus eliminating the need for plasmid preparations. By being rapid, simple and cost-effective, this technique should be widely applicable to examine the integrity of the ORF of any gene.
Keywords: Animal Base Sequence Genes, nef Genetic Techniques Haplorhini In Vitro Molecular Sequence Data Open Reading Frames/*GENETICS SIV/GENETICS Transcription, Genetic/*GENETICS Translation, Genetic/*GENETICS Viral Proteins/*BIOSYNTHESIS TECHNICAL REPORT JOURNAL ARTICLEKWDanimalbasesequencegenes,nefgenetictechniqueshaplorhiniinvitromolecularsequencedataopenreadingframes/KWDgeneticssiv/geneticstranscription,genetic/KWDgeneticstranslation,genetic/KWDgeneticsviralproteins/KWDbiosynthesistechnicalreportjournalarticle
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