I am working for a snake genus and I amplified multiple samples of the same species with a set of ND4 primers used in other 90% of the studies on snakes. However, when I cross checked for pseudogene amplification, I found five samples with a nonsense codon standing right in the middle of my 650bp (194-195-196 position) sequence length. The chromatograms were amplified and sequenced very well. I repeated the amplification and sequencing one more time to validate the condition. The result is the same as earlier. However, I have no such record in other individuals of same species collected from different location. Now, I wonder whether pseudogenes get amplified every time? Any idea whether any pseudogenes are functional (I know they are not! but still I wonder)? Then why it is happening?
It is not true that pseudogenes are for sure non-functional. You should read e.g. this paper and search for others by PP Pandolfi.
A coding-independent function of gene and pseudogene mRNAs regulates tumour biology.
Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ, Pandolfi PP.
Nature. 2010 Jun 24;465(7301):1033-8.
The most thorough way to test this would be to clone the PCR product into a plasmid vector; Then you pick individual colonies and prepare plasmids separately, each representing one original molecule from the PCR mixture (a "clone library"). Now you can sequence the plasmids separately, and get a count of all the different genes and pseudogenes in approximately their original ratios.
I found it to be numt gene, what I am picking. Any idea how to amplify the mt gene with same primer without picking numt?
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