In our work, using ALV subgroup A primer with template DNA extracted from liver lymphoma from dead chickens, we are facing this problem. Using same set of conditions, viz., initial denaturation at 95°C for 3 min followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at 57°C for 30 sec, extension at 72°C for 1 min, and final extension at 72°C for 10 min. Sequences of the oligonucleotide primers (229 bp) for ALV A include :
PA1-Forward 5'-CTACAGCTGTTAGGTTCCCAGT-3', and
PA2-Reverse 5'-GCCTATCCGCTGTCACCACTG-3'
Do you have a positive and a negative control? That your PCR itself is working but that you have no contamination of your reagents?
Thank you all very much for your kind replies. Well, sure positive and negative controls are being included always. The issue is using similar conditions, on the same machine, by the same worker, why the very same samples yield results one day, but do not yield next day or next time same day.
We doubted the initial pick up of the DNA template, which used to be either 1000 ng to 3500 ng / 0.5 to 1 ul. Hence, in one experiment, we diluted the DNA template to a working aliquot of 1000 ng/10 ul, thus initial pick up ranged from 100 ng to 1000 ng / 1 to 10 ul, respectively. Mastermix with 7.5 pM followed by making up of the final volume to 25 ul using DNAs free DW. Positive result was shown only in well with 200 ng DNA template and rest other wells remained negative. Whereas, earlier results had shown up with even 500 ng DNA template onwards, even in 3500 ng DNA template wells.
Well, the genomic DNA has been extracted from lymphoid leukosis suspected dead chicken livers showing clear cut greyish-white tumor foci. Histopathological results are awaited for confirmation. So, this kind of problem is being faced for PCR reactions, when we are trying to confirm our results for avian leukosis virus (ALV) subgroup A infection.
PCR for Marek's disease, few samples were positive and few negative.
Would like to seek advice / guidance in case anyone out there has experienced such kind of issues/ problems.
Thank you once again one and all !!
Ok, next question - why do you want to use such a high amount of template? Your fragment should be 229 bp and for such a short fragment there is normally no need to use too much template, 100-200 ng of template should be sufficient.
And how about your primer specifity? Did you check it via BLASTing? Quality of DNA is still ok? Did you check this with 260/280 ratio?
Well, in our attempt to stabilize repeatability of the PCR, when the reactions did not stabilize, we thought of increasing DNA concentrations to observe reactions at higher concentrations, as well. Surprisingly, the behavior of reactions was same. Another reason was, we picked up just 1 μl directly from the Eppendorf containing DNA without diluting it. Since DNA concentration in original aliquot was high, picking up 1 μl directly yielded 1000 ng to 3500 ng / 1 μl. Avian leukosis field sample is a pure lymphoid nodule [which may or may not be infected with Marek’s disease virus (MDV)], hence isolated DNA concentration is good , 260/280 is 1.7 to 1.9.
Primer is a published primer with specificity of 229 bp for viral gp85 fragment, encoded by viral env gene, specific for ALV subgroup A virus, which specifically binds to TVA receptors on chicken B lymphocytes for virus adsorption and entry.
Thank you in anticipation.
Dear Artur,
Why you say that 35 cycles are a lot with high risk of false positives.
Our problem right now is that samples, which are clearly liver lymphoma ie highly positive for this virus, are yielding positive reactions as well, but on repetitions, very same sample also yields negative result. We have tried many experiments/ combinations since last 2 months, and eventually in our failure came up here for some international suggestions- that we thought some Scientists might also have faced in their system, and hence we could gain by their experiences.
Thank you for your suggestions, anyway.
Hi Alka,
Many researchers have given very nice suggestions for your problem. My suggestions are very simple as this can happen only when forgate or miss to add some reagents by mistake in the sample if your template DNA is in appropriate amount ie. 200 ng/ul not degraded. Make a list of reagents. Make master mixture. Tick one by one after adding them into the tube. Use same master mixture for negative and positive control. Don't get disturbed during experiments. Handle with care i.e. take care when pipetting the reagents. As per you there is no contamination even though you try to avoid any shot of contamination for DNase. Use autoclaved eppendorf tubes, pippet tips ie. DNase free on along with DNase free water. I could not there is any scientific reason rather than human handling errors. Take care for consistancy and reproducibility in the results by keeping your reagents, template and primers separate from your other research colleagues.
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