has part has part realizes is_specified_output_of concretizes continuant nucleotide sequencing assay evidence rapid amplification of cDNA ends polymerase chain reaction evidence 2015-11-04T09:48:38Z 5' RACE analysis was employed to localize the espR promoter using RNA extracted from Mtb H37Rv grown to mid-log phase. The espR transcript starts with a poly-G (7) sequence 144 bp upstream of the translational start codon. 5’ RACE-PCR 5' and 3' RACE-PCR utilize different protocols to amplify an unknown end using the known sequence of the center of the transcript. For cDNA synthesis, 5' RACE-PCR uses an anti-sense oligonucleotide primer (gene specific primer (GSP)) that recognizes a known sequence in the middle of the gene of interest and the reverse transcriptase to add base pairs to the 3' end of the primer. Next, terminal deoxynucleotidyl transferase (TdT) is used to add a string of identical nucleotides to the 3' end of the cDNA. A PCR reaction is then carried out, using a second anti-sense gene specific primer (GSP2) that binds to the known sequence, and a sense (forward) universal primer (UP) that binds the homopolymeric tail added to the 3' ends of the cDNAs to amplify a cDNA product from the 5' end. In contrast, 3' RACE-PCR utilizes the naturally occurring 3' polyA tail of the transcript and uses an Oligo-dT-adaptor primer (a primer with a short sequence of deoxy-thymine nucleotides) that complements the polyA tail and adds a special adaptor sequence to the 5' end of each cDNA. PCR is then used to amplify 3' cDNA from a known region using a sense GSP, and an anti-sense primer complementary to the adaptor sequence. Using 5' rapid amplification of cDNA ends (RACE), we mapped the transcriptional start sites of rpoQ and VF_A1016 (Fig. 4A). Neither start site was detected in the DeltarpoQ mutant, supporting the idea that transcription of rpoQ and VF_A1016 requires RpoQ (data not shown). ECO:0001820 CollecTF RACE PCR is frequently used to verify transcription start sites relevant to the function of transcription factor binding sites such as repression. RACE PCR eco swolfish A type of nucleotide sequencing assay evidence in which RT-PCR (cDNA synthesis) is first used to produce a cDNA copy of a region of the RNA transcript being investigated followed by PCR to capture either the unknown 5' or 3' end of the transcript for sequencing, depending on whether 5' RACE-PCR or 3' RACE-PCR are being undertaken. The 5'-end of the espA mRNA was located 66 bp upstream of the translation start codon using 5' RACE (Fig. S3). 3’ RACE-PCR assay sequence feature identification objective in vitro design rapid amplification of cDNA ends study design