INNOVATIVE METHOD FOR CELLULAR GENOME TAGGING USING CELLS OWN REPLICAT PROTEINS TO INCORPORATE MODIFIED DEOXYNUCLEOTIDES
Awadelkareem Masaad, Sara Abdelatif, , Mazin Ahmed , , Amna Adil, Moawia.M.Moawia.
Introduction : DNA sequencing lead to revolution in biology and breakthrough in diagnostic medicine and drug development via understanding of different genetic effect on disease manifestation and drug response. This breakthrough was facilitated via discovery of heat stable polymerases that used in thermal cyclers.
Objectives: Innovation of new method for cellular DNA tagging depending on cells own replication protein’s to incorporate florescent dNTPS in their genome in various cells.
Material and method: Rhodamine-12-dCTP and Aminoallyl-dUTP-xx-AF488 fluroscent dNTPS along with natural dATP and dGTP. Isolated peripheral lymphocytes, , THP1 cell line , Leishmania major promastigotes, and fungi were incubated in RPMI1640 complete media supplemented with mm of tagged dNTPs for 48 hours upto 7 days. The uptake and incorporation of the fluorescent tagged DNTPs was documented using ZIES fluorescent microscope
Results: almost 90% of THPI cells , 95% of Leishmania promastigotes ,nearly 100% of fugus and near to 60% of peripheral lymphocyte(60%) nuclei were stained by the florescent green.
Conclusion : living eukaryotic cells used in the experiment were able uptake and incorporate the tagged dNTPS.
Recommendation : further testing of this new method in diagnostic medicine and biology will advances these fields via:
– use of this technique in cell detection methods (culture and rapid microscopy detection).
– To explore the possibility of using the tagging technique to develop a new DNA sequencing method (sequencing by cell synthesis).
Conflict of interest: No conflict of interest
Key words: DNA polymerases , Mass genome tagging, sequencers, florescent dNTPS.