TP-PCR or three primer PCR - TP-PCR or three primer PCR (Mar/01/2011 )

Hello every one.... just have a bit confusion about TP-PCR- involving three primers and two templates.. one of the primer is reverse, other one forward and the third one is internal... Can anyone tell me how cans we design the primers for this PCR and what's the purpose of this PCR?

Bilal Malik on Wed Mar 2 04:17:24 2011 said:

Bilal,

You should be telling us the purpose of the PCR. We can all help, if the PCR does not work or if you want to confirm if primers designed by you look ok....

If you have a specific question you need to tell more like gt_ameya said, but if you simply dont understand what TP-PCR means, then you might want to check the paper I have included and if that doesnt help, ask a a more specific question because its not clear what you want to ask?

I have a slightly more detailed question. I am trying to stick two sequences together (initally from a plasmid and the other is cDNA). I am having problems getting product bands on my gels from my PCRs. Am I right in that I put all three primers in the tube with the linking/internal primer at half the concentration as the others? I think one of my main issues is the relative concentrations, but Ive never done this before so I cant be sure. Sometimes if Im really lucky and increase the exposure time, I can see very feint bands. I would love any advice you can give me



Currently x20 cylces
5ul Green GoTaq buffer
1ul dNTPs (10mM each)
0.5ul Taq
0.5ul Forward primer (10uM all primers)
0.25ul linking primer
0.5ul Reverse primer
0.5ul plasmid DNA (5ng/ul)
2ul MgCl2 (25mM)
total volume = 20ul w/ water

wanderingstranger on Wed Mar 2 19:06:00 2011 said:

Two things come to my mind,

1. Increase number of cycles... make it 30-35...
2. Depending on what you get after say 35 cycles, try Mg concentrations...

pito on Wed Mar 2 12:42:06 2011 said:

1st of all thanks guys for your replies.....

Now coming to my point..

I am actually looking into this exon 19 deletion in EGFR gene (Epidermal growth factor receptor). So I came across this three primer-PCR technique (Attached publication) which employs three primers. In this paper they have used it for BRCA1 del (ex 19-21). So I had a confusion in designing of the internal primer. I was thinking what if I take a few base pairs up-stream and a few down-stream to the deleted exon (19) in order to cover that deleted area..Hope this time I have made my self clear

Bilal,

You can definitely do as you suggest in order to detect the deleted exon. Its the simplest way to do it.

However, with a two primer PCR, if you dont see a band, there is always a chance that the PCR has not worked.

The reason, a 3 primer strategy is used, is that it acts as a control in itself and tells you that the PCR has worked fine and the absence of a particular band is purely because it is deleted.

I think that answer's your first question.

Well I think that we can also make use of this technique to see whether its a homozygous deletion or a heterozygous in a single step..

Bilal Malik on Thu Mar 3 06:13:48 2011 said:

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So this can be done by making use of three primers, forwards (P1) and Reverse (P2)for wild type and the third (P3) which would be reverse and specific for deleted exon 19 in EGFR. So this P3

Should be designed according to the downstream sequence of the deleted exon.. And yeah your right-alongside our PCR's working will also be justified

Bilal Malik on Thu Mar 3 06:28:27 2011 said:

Your primers P1 and P2 must work all the time and you will not get a product with P3, if the exon is deleted. Is that what you are trying to say? Then yes, you are right. But P1 and P2, must be quite away from the deletion prone region. Also, your PCR conditions must be suitable for amplification with primers P1 and P2, in competition to P3.

Probably, I am not getting what you are trying to say, but it is difficult to determine the homozygous/ heterozygous deletion. Cause, whether present in homo or hetero state, the primers will give you a PCR product. Could you elaborate on, what you are thinking?