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Published on: May 29, 2016
[Cloning E. coli mRNAs with poly (A) tails by restriction digest polymerase chain reaction]
Zi-You Hu1, Wen-Li Ma, Yan-Bin Song
1Institute of Molecular Biology, First Military Medical University, Guangzhou 510515, China.
Objective:
To investigate the polyadenylation at the 3' terminal of the mRNAs in E.coli.
Methods:
mRNAs of E.coli was enriched from total RNA with oligo (dT)-cellulose, and reverse transcription was performed using oligo(dT)18 as primer prior to synthesis of double strands cDNA which was digested with Sau 3A I to produce multiple gene fragments that were then ligated with adapters. Restriction digest-polymerase chain reaction (RD-PCR) was employed to divide the fragments into 10 groups using 10 different combinations of the 4 primers, and the products were cloned into T-vectors.
Results:
More than 100 gene fragments were cloned, 30 of which were sequenced.
Conclusion:
Polyadenylation of E.coli mRNA is not a biochemical curiosity, and very likely, it is a general attribute of the mRNAs of bacteria.
