6 replies to this topic

[Nephrite]

Hello.

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.

Nephrite

[tea-test]

pseudogenes can lack introns in the genome, so you will detect this genomic DNA also with intron spanning primers. If I remeber corrct, they originate from mRNA which was reverse transcribed and inserted back into gDNA.

Moreover, there are also processed pseudogenes, i.e. there is an mRNA of this pseudogene in the cell.

I hope this was helpful.

[Trof]

I was once designing primers for PHD2 prolyl hydroxylase mRNA, but BLAST was always picking sequences from a different gene, on every primer combination. So i looked deeper and compared the sequences and found that PHD2 has a homologue called SCAND2, that was probably created by retroposition of large parts of PHD2 mRNA (so as tea-test said, no introns) into different genomic location.

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!

[Nephrite]

Tea-test and Trof, thank you very much!

[Nephrite]

One more question - when we do RT-PCR with primers spanning introns, how can be sure that the amplified fragment doesn`t come from contaminating pseudogene? Always to run ''mock'' sample?

[tea-test]

You have to do a BLAST search and look if your primer sequence also detects pseudogenes. If so, you have to choose a more specific region of your gene for primer design.

[Nephrite]

tea-test, on 17 October 2011 - 11:15 PM, said:

You have to do a BLAST search and look if your primer sequence also detects pseudogenes. If so, you have to choose a more specific region of your gene for primer design.

I see. Thank you!