TY  - JOUR
TI  - Visualization of RNA 3’ ends in Escherichia coli Using 3’ RACE  Combined with Primer Extension
AU  - Wang, Xun 
AU  - Jeon, Heung Jin
AU  - Abishek N, Monford Paul
AU  - He, Jin 
AU  - Lim, Heon M.
VL  - 8
IS  - 5
PY  - 2018
DA  - 2018/03/05
SP  - e2752
C1  - Bio-protocol 2018;8:e2752
DO  - 10.21769/BioProtoc.2752
UR  - https://doi.org/10.21769/BioProtoc.2752
AB  - In this assay, 3’ RACE (Rapid Amplification of cDNA 3’ Ends) followed by PE (primer extension), abbreviated as 3’ RACE-PE is used to identify the mRNA 3’ ends. The following protocol describes the amplification of the mRNA 3’ ends at the galactose operon in E. coli and the corresponding visualization of the PCR products through PE. In PE, the definite primer is 5’ end-labeled using [γ-(32) P] ATP and T4 polynucleotide kinase, which anneals to the specific DNA molecules within the PCR product of the 3’ RACE. The conventional PE can only be used to locate the 5’ end of an mRNA transcript since reverse transcriptase (RTase) polymerizes only in the 5’ → 3’ direction. Thus, Taq polymerase is used instead of RTase, PCR is performed. Therefore, we are able to locate the 3’ end of the mRNA using this assay. The relative amount of the 3’ end can be directly visualized and quantified by way of separating DNA products in a denaturing 8% urea-PAGE (Polyacrylamide Gel Electrophoresis) gel. The exact position of the 3’ ends can be sequenced by comparison of these final DNA products with the corresponding DNA sequencing ladder.
KW  - 3’ RACE
KW  - Primer extension
KW  - RNA 3’ ends
KW  - E. coli
KW  - Galactose operon
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False