Polymerase synthesis of four-base DNA from two stable dimeric nucleotides.

摘要: We document the preparation and properties of dimerized pentaphosphate-bridged deoxynucleotides (dicaptides) that contain reactive components two different nucleotides simultaneously. Importantly, dicaptides are found to be considerably more stable hydrolysis than standard dNTPs. Steady-state kinetics studies show dimers exhibit reasonably good efficiency with Klenow fragment DNA polymerase I, we identify thermostable enzymes process them efficiently at high temperature. Experiments dAp5dT dimer successfully acts as a combination dATP dTTP in primer extension reactions, dGp5dC dGTP dCTP. The promote successful 4-base extension. final byproduct reaction, triphosphate, is shown less inhibitory pyrophosphate, canonical byproduct. Finally, PCR amplification dimeric nucleotides, can under extended conditions when normal dNTPs fails. These represent novel simple approach for increasing stability avoiding inhibition from pyrophosphate.