Preparation of Promoter DNA Templates for Transcription Experiments

To study the mechanism of mRNA synthesis in a biochemically-defined system, we use a strong promoter, i.e., a promoter that can support a high level of transcription. Such a promoter will have a near-perfect TATA box and a good initiator element. The core element of the adenovirus major-late (AdML) promoter possesses these very properties. Indeed this promoter is the promoter of choice for in vitro transcription studies by a number of laboratories that study the function of RNA Polymerase II. We have sub-cloned the core AdML promoter region, including approximately 60 base-pairs, into an M13 vector to facilitate future manipulation of specific promoter sequences.

For transcription experiments, a ~440 base-pairs DNA fragment is synthesized by a polymerase chain reaction (PCR), and is subsequently purified. The location of the promoter within the fragment allows for a maximal transcript size of approximately 350 nucleotides.

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