Dear all,
One of my friends has to amplify some species-specific genes from cooking oil, which contained little and mostly degraded DNA. I think paleontologists often work on degraded ancient DNA. Could someone suggest me a protocol? My friend has isolated the DNA using a membrane-based method. Now it's all about the primer design and PCR. Thanks in advance
That seems a rather good choice.
There is nothing you can do regarding PCR buffers etc. Your only option is to select small size products and hope at least one fragment of your fragmented DNA contains the whole sequence.
Thus, you could also help by increasing DNA concentration (in the hope that such molecule containing the whole sequence gets into the reaction). However this only works to a certain limit.
You could use the Quick Multiplex Consensus PCR protocol, which is basically a 2 stage nested PCR. It is designed for the amplification of poor quality DNA. The first stage involves multiplexing a number of targets using outer primers. In the second stage, the 1:100 dilution of the 1st stage pcr product is used as a template in singleplex PCR in which nested primers amplify targets. See this article: J Clin Pathol 2010;63:134-140 doi:10.1136/jcp.2009.070508
You could also use the simple 2 stage pcr which involves amplifying the targets in two stages using the same primers and cycling conditions. See: clinmedjournals.org › ijccr › ijccr-2-018.
I hope these help.
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