2 replies to this topic

[BGS]

Hi!

I would really appreciate if anyone has any comment on my sequencing results.  

I am studying parasitic factors in plant parasitic nematodes.
In a study of the sequence variability of a particular parasitic gene, I have determined over a hundred sequences of this gene from populations of species A. I have also determined the mRNA/cDNA sequence from the same species, where as expected, the sequence is missing all the introns.

When I used the same primers and genomic DNA of a closely related species, I got amplification in size identical to cDNA sequence from species A, and in fact the sequencing revealed very similar sequence to cDNA of species A, where all the introns are missing.

I am not so familiar with introns, but this is the first case I see a lack of introns in one species and presence in a relative species.

Have anyone a similar experience?

Is there any way that a mRNA template could amplify by a regular Taq polymerase? RNAse was used in gnomic DNA extraction though…

I have double checked my experiments, and I am sure the results are correct and that there could be no mix up of samples. However, I am still puzzled by the lack of introns in one species, especially as I can not get repeated amplification using genomic DNA of species B as a template.    

There is no sequence of this particular gene in species B in the public databases yet.

Any suggestions?

Thanks!

[smu]

BGS, on May 12 2009, 05:33 AM, said:

Hi!

I would really appreciate if anyone has any comment on my sequencing results.  

I am studying parasitic factors in plant parasitic nematodes.
In a study of the sequence variability of a particular parasitic gene, I have determined over a hundred sequences of this gene from populations of species A. I have also determined the mRNA/cDNA sequence from the same species, where as expected, the sequence is missing all the introns.

When I used the same primers and genomic DNA of a closely related species, I got amplification in size identical to cDNA sequence from species A, and in fact the sequencing revealed very similar sequence to cDNA of species A, where all the introns are missing.

I am not so familiar with introns, but this is the first case I see a lack of introns in one species and presence in a relative species.

Have anyone a similar experience?

Is there any way that a mRNA template could amplify by a regular Taq polymerase? RNAse was used in gnomic DNA extraction though…

I have double checked my experiments, and I am sure the results are correct and that there could be no mix up of samples. However, I am still puzzled by the lack of introns in one species, especially as I can not get repeated amplification using genomic DNA of species B as a template.    

There is no sequence of this particular gene in species B in the public databases yet.

Any suggestions?

Thanks!

so, just to get your method straight, you made genomic DNA from species B, did PCR on it using your primers from species A and then sent it off for sequencing? Is it possible that your PCR product is just contamination (i.e. you have cDNA floating around the lab and you amplified this instead)? And when you say very similar, is it that it is exactly like sequence A but missing the intron? If so, I would again suspect contamination. If there are few differences in the exon sequences then I would be more likely to believe that your sequence B just happens to lack an intron.

[bob1]

contamination is my first thought too.  It is possible to get genes without introns though, so it could happen.