If you are going to study gene polymorphism of specific gene in serum and liver of rats after rat experiment and this gene may be affected but you do not have primer sequence to amplify that gene and all papers focused on human samples how can I get the primer sequence? and which better to isolate RNA or DNA and which type of PCR wil be better and more expressive for results
Thanks in advance
Hi If you know the sequence of your gene you can design your own primers using PRIMER BLAST. Its better to do endpoint PCR from isolated DNA from desired tissues.Later perform sequencing, and check for polymorphism using online softwares. You could also use genetic analyzer to analyze your PCR products and study gene polymorphism . Still I could not fully understand you question very well and very confusing.
Hi Thoria,
the answer to your question depends in a series of factors, for example if the SNP you are interested in have been already described or if you are trying to find new SNPs. If they have been already described then an easy way would be to design a pair of primers to amplify the region around the polymorphism and the sequence the PCR produce using one of the primers. Remember to desig the primer separated enough from the SNP so that you can actually se it in the sequencing reaction. Another method that can sometimes be used is a restriction cut. Some SNP create a new restriction site (or destroy one); in this way you could run the PCR and then digest with the specific enzyme, you can determine the presence of a specific SNP based on the digestion pattern. In all this cases I would start with DNA as your template. The problem of using mRNA is that RNA editing could be happening in your sample and affect your results. If you are interested in new SNP you could use the heteroduplex technique. Simply is a technique based in the different mobility of DNA fragments in an acrylamide gel, you can find the details and protocol on line
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