Making Deletions constructs:
Once we know the TSS, the next thing is to clone the region 5' to the TSS in a reporter vector. Progressive deletions from 5' and 3' region can be made to narrow down the region which has the core promoter.
There are several approaches to make deletion constructs but the method of choice is PCR methodology. This method can be used when the sequence is known.
PCR Methodology:  Primers are designed for the region of interest in the following way. Both the  forward and the reverse primers should have restriction enzyme (RE) site in their 5'region for two different enzymes. For eg: one can incorporate Kpn I in the forward primer and Hind III in the reverse primer. The other important thing is that the RE sites, for the enzymes used in primer, should not be present anywhere in the region to be amplified.
PCR is performed with these primers. If you get a single band then the PCR product can be directly cleaned using PCR purification columns. If you get non-specific bands in the PCR, then cut the band of your interest and elute the DNA  from the gel. and the product obtained can be purified and digestion with the respective enzymes or they can be cloned in a T/A vector. The latter approach is easier i.e., cloning in T/A vector, since some PCR products will not get digested easily when the Restriction site is near their ends. So, cloning will help in the

 

Last Updated 

11-01-2004

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