one question on reverse transcription-PCR - (Apr/09/2007 )

Hi, all
I made an insertional mutant of one gene of my research interest. I did this by first amplying around 200bp fragment within that gene and put it into a sucide plasmid. After conjugation, the sucide plasmid was integrated into the bacterial genome via one event of recombination. Then I designed primers, extracted RNA and checked if there was still gene expression of this gene via reverse transcription PCR.

It was so weired that the mutant still had the same PCR product as that of WT strain! I then double checked the primer landing sites. It turned out the forward primer landed within that 200bp region while the reverse primer landed outside. So in thoery, there should be no prodcut at all. Right?

can anyone help with this?

thanks a lot!

VFinMadison

Even if only one primer is working (eg. reverse) reaction still goes on, but instead of getting product of defined size, there are many products of variable sizes.

If your 200bp is close to the start of the gene and forward primer is still binding (it does not distinguish between wt and mut [if mutation is included in primer binding site] ) then the same product for mut as for wt is good.

thanks for the note--yes, the 200bp fragment is indeed closed to the start of the gene.

but I still cannot understand why WT and mutant would have the PCR products of same size if the insertion mutantion occurs at the priming site of forward primer? I would assume the insertion of that plasmid would mass up the priming site (or at least partially) so that there would be no PCR product at all for mutant.

actually I checked the priming site again this morning and found the site is 42bp downstream of the 1st nt of 200bp fragment. Since I have no idea at all where the insertion occurred, I do not know the possibility of occurrance of insertion before the priming the site. if the insertion occurred Before the priming site and there were readthrough of the plasmid, that would explain why mutant and wt have the PCR product of the same size (like what I have observed in RT-PCR result). But if insertion occured after or right in the priming site, I would assume no PCR product at all for the mutant..

am I right in reasoning like this? Please clarify! thanks so much

VF

If insertion is big, long enough to ‘break’ primer binding site (eg 10nt) then only reverse primer would bind.

How do you now where primer binds, do you have the sequence?
You could make the primers outside the gene, based on the vector sequence, or use universal primers. Do the sequencing, of both wt and mut, compare them and if the there is mutation you will see what and where it is.

Maybe in the wt primer is binding to the same site, so everything before that site in mut is insertion itself. Also if the insertion is smallish, depending how long the gene is, you probably would not pick the size difference on the gel.

I would do the sequencing, and even before that, stick with DNA of the recombinant thing.

thanks for the explaination. Here I attached the drawing I made trying to explain why there is PCR product in both WT and mutant.

The priming site of the Forward primer is like what presented in Situation 1 and 2. And I am pretty sure there is strong promoter in the antibiotic resistance gene in the plasmid. So if there is readthrough, there would always be the PCR product..

as I noted in the drawing, only if I could design the forward primer in the upstream of the 200bp fragment, there would be no PCR product in the mutant. But unfortunately, that upsteam region is extremely high in AT. so hard to pick a good primer....

please take a look and see if there is anything wrong with the drawing..

thanks a lot,

VF

Drawing changes how I imagined it all (side effect of using plasmid only)
Wrote some thoughts next to it.

great--your comments and extra drawing made more sense to me!

thanks so much for all the effort and inputs!

VF