Document 0209
 DOCN  M9580209
 TI    Rapid screening of open reading frames by protein synthesis with an in
       vitro transcription and translation assay.
 DT    9506
 AU    Switzer WM; Heneine W; Retrovirus Diseases Branch, Centers for Disease
       Control and; Prevention, Atlanta, GA 30333, USA.
 SO    Biotechniques. 1995 Feb;18(2):244-8. Unique Identifier : AIDSLINE
       MED/95244141
 AB    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.
 DE    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 ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).