Posted 10 October 2011 - 05:54 AM
I am reading a paper, which has been published in 2000 year, and I came across something that can not understand........
In brief, authors explore the usefulness of a particular fixative for molecular studies. As a house-keeping gene they have used GAPDH with size of amplicon 682 bp.
During the time of experiments they say that the genomic DNA sequence information for this particular gene was not available and they did not know if the amplicon spans any intron sequences.
To control if the amplified fragment originates from mRNA, or contaminationg gDNA they have performed mock RT-PCR and they found that even with low amounts of total RNA template (150 ug) there was amplification. This result is not shown and they do not say if this product has the same length as the expected one (i.e. 682 bp).
Well, now the gDNA sequence for GAPDH is available and I found that the authors` amplicon spans 4 (!?!) intron sequences. Then, how it is possible?
They explain their result with the assumption that ''housekeeping genes like GAPDH usually share homologous sequences with genomic pseudogenes and such sequences in contaminating genomic DNA in total RNA preparations would be amplified preferentially by RT-PCR''.
I just read that ''Pseudogenes are genomic DNA sequences similar to normal genes but non-functional; they are regarded as defunct relatives of functional genes. '' but still don`t understand anything............
Any help will be highly appreciated.
Posted 11 October 2011 - 05:04 AM
Moreover, there are also processed pseudogenes, i.e. there is an mRNA of this pseudogene in the cell.
I hope this was helpful.
Posted 11 October 2011 - 06:54 AM
What was more amazing, is that SCAND2 has completely different protein sequence and function, because the sequence is read in a different frame. It is like reading portion of book backwards and geting another complete book. Fascinating!
I never trust anything that can't be doubted.
Posted 17 October 2011 - 07:17 AM
Posted 17 October 2011 - 11:15 PM
Posted 18 October 2011 - 12:33 AM
I see. Thank you!