Polymerization retardation isothermal amplification strategy enables the sensitive and facile investigation of the flanking sequence preference of ten-eleven translocation 2 protein.

Polymerization retardation isothermal amplification strategy enables the sensitive and facile investigation of the flanking sequence preference of ten-eleven translocation 2 protein. Anal Chim Acta. 2020 May 01;1109:140-147 Authors: Chen D, Wang Y, Chen J, Zhang Y, Dai Z, Zou X Abstract Active DNA demethylation process critically relies on the intrinsic properties of ten-eleven translocation proteins (Tets), particularly the flanking sequence preference. Challenged by the fact that the proximate bases to the 5-methylcytosine (5mC) are multitudinous and their influence on the Tets/DNA interplay is minute, the current methodologies are very limited in terms of cost, sensitivity and efficiency. Herein, we propose a polymerization retardation isothermal amplification (PRIA) strategy that enables sensitive and fast study of the flanking sequence preference of Tet. By arranging DNA polymerase to repetitively pass DNA strands through an isothermal replication-scission amplification reaction, the tiny difference in the Tet/DNA interplay can be consecutively accumulated and amplified. Low amount sample (80 ng) even multiple samples can be simultaneously analyzed within 10 h on an easily accessible laboratory real-time quantitative PCR instrument. For a proof-of-concept study, the binding preference (PB) of Tet2 for XmCGX, (X = C, G, T, A) was analyzed by PRIA and computational analysis, showing an order of AmCGT > TmCGA ≈ GmCGC ...
Source: Analytica Chimica Acta - Category: Chemistry Authors: Tags: Anal Chim Acta Source Type: research