Genomic PCR - (Dec/09/2003 )
I want to get a promoter sequence from the genomic DNA of human using the PCR technique.
I have done the experment for about 3 month,but the result is not very good. Can anyone give me some adviceĢif this kind of PCR is special ? Is there any thing I should pay attention to?
Thanks a lot!
First of all, you should check the human genome database from NCBI, UCSC or Ensembl to see if the 5'-flanking sequence of your gene is there. Mostly likely you will find it because the genome project is completed. Once you find the sequence you can design primer to amplify the desired portion and clone it.
In the days when the genomic sequences were not available people used the genome walking technique to get the upstream sequence of a gene and there are kits commercially available such as the one from Clontech. The kit enssentially privides 5 genomic libraries which contain genomic DNA cut with different enzymes and linked with adaptors. You use your gene specific primer and the adaptor primer to amplify the unknown promoter sequence.
For any promoter sequence PCR, bear in mind that your promoter sequence may be GC rich (containing CpG islands). In this case, PCR conditions should be optimized.
Thank you very much! hula