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
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Last Updated 11-01-2004 |
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