Asymmetric polymerase chain reaction provides alternatives for preparation of (GT)₅-tailed duplex DNA promoter for promoter trapping.
Synthesis of (GT)₅-tailed duplex DNA promoter is an important first step for purifying transcription complexes by promoter trapping purification. In our previous publication, we showed that the purification of the c-jun promoter using lambda exonuclease digestion of polymerase chain reaction (PCR) produced DNA with single-stranded tails. Asymmetric PCR can also produce tailed single strands that can be annealed to yield the desired promoter. An effective method uses 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.
Department of Chemistry, University of Texas at San Antonio, San Antonio, TX 78249, USA.
Yanwen
Y
Zhou
Yinshan
Y
Jia
Harry W
HW
Jarrett
D026501:Blotting, Southwestern; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000032:analysis / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism; D016758:Genes, jun / Q000235:genetics; D006801:Humans; D008979:Moloney murine leukemia virus / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D004277:DNA, Single-Stranded; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D005092:Exonucleases; D004262:DNA Restriction Enzymes