Rapid screening of open reading frames by protein synthesis with an in vitro transcription and translation assay.
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.
Switzer
W M
WM
Retrovirus Diseases Branch, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Heneine
W
W
D000818:Animals; D001483:Base Sequence; D015754:Genes, nef; D005821:Genetic Techniques; D000882:Haplorhini; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics*; D015302:Simian Immunodeficiency Virus / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis*
D014764:Viral Proteins