Capping-RACE: a simple, accurate, and sensitive 5' RACE method for use in prokaryotes.

Abstract

Rapid amplification of cDNA ends (RACE) is a prevalent technique used to obtain the 5′ ends of transcripts. Several different 5′ RACE methods have been developed, and one particularly simple and efficient approach called CapFinder relies on the 5′ cap-dependent template-switching that occurs in eukaryotes. However, most prokaryotic transcripts lack a 5′ cap structure. Here, we report a procedure to capture primary transcripts based on capping the 5′ triphosphorylated RNA in prokaryotes. Primary transcripts were first treated with vaccinia capping enzyme to add a 5′ cap structure. First-strand cDNA was then synthesized using Moloney murine leukaemia virus reverse transcriptase. Finally, a template-switching oligonucleotide with a tail containing three ribonucleic acid guanines was hybridized to the cDNA 3′ poly(C) and further used as template for reverse transcriptase. It is oligonucleotide sequence independent and is more sensitive compared to RLM-RACE. This approach specifically identified the transcription start sites of ompA, sodB and shiA in Escherichia coli and of ompA, rne and rppH in Brucella melitensis. Furthermore, we also successfully identified the transcription start sites of small noncoding genes ryhB and micC in E. coli and bsnc135 and bsnc149 in B. melitensis. Our findings suggest that Capping-RACE is a simple, accurate, and sensitive 5′ RACE method for use in prokaryotes.