Asymmetric PCR provides alternatives for preparation of (GT)5 tailed duplex DNA promoter for promoter trapping

摘要

Synthesis of (GT)5 tailed duplex DNA promoter is an important first step for purifying transcription complexes by promoter trapping purification. In our earlier publication we have shown that the purification of the c-jun promoter using lambda exonuclease digestion of PCR produced DNA with single stranded tails. Asymmetric polymerase chain reaction (PCR) can also produce tailed single strands which can be annealed to yield the desired promoter. An effective method utilizes asymmetric PCR and double digestion. After PCR, first a restriction enzyme, in this case SacII, cuts duplex strands remaining after asymmetric PCR, leaving 5’-phosphoryl ends susceptible to a second digestion with lambda exonuclease to effectively degrade any duplex. The resulting single strands are then annealed to produce a duplex DNA with a single stranded (GT)5 tail at the 3’ end of each strand of the duplex. Unlike the previously described method, this novel procedure produces the desired tailed promoter devoid of any untailed duplex.