Hello everyone,
I'm currently working on genotyping the TOMM40 gene, specifically in the context of Alzheimer’s disease (AD) research. However, I'm encountering non-specific amplification during PCR, despite optimizing conditions using a gradient PCR.
Here are a few details about my setup:
- Template DNA: Genomic DNA extracted from PBMC's. - 34ng/µl
- Primers: (F) 5-gtc tcc aac tgc tga cct c-3 (R) 5-ctg cct ttt caa gcc tca g-3
- Mastermix: ( Emerald Amp, Takara, Cat: RR310A)
- Annealing temperature optimized: [67°C]
- Additives used: DMSO
- Controls: No-template control is clean
PCR Conditions:
Initial Denaturation - 95°C - 3 minutes
Denaturation - 95°C - 30 seconds
Annealing - 67°C - 30 seconds
Extension - 72°C - 30 seconds
Final extension - 72°C - 10 minutes
Well map:
•Well 1 - Marker - 50bp DNA Ladder RTU (Simply Biologics)
•Well 2 - N - No template control
•Well 3 - W/o - Without DMSO
•Well 4 - 3 - with 3% DMSO
•Well 5 - 4 - with 4% DMSO
•Well 6 - 5 - with 5% DMSO
Despite adjusting the annealing temperature and DMSO concentration, I still get multiple non-specific bands.
Has anyone faced similar issues while amplifying TOMM40 or working with homopolymer-rich regions? I'd really appreciate suggestions on general troubleshooting tips for non-specificity.
Thank you in advance for your insights!
Some ideas are...
1touchdown pcr,
2 use less primer
3 use a hotstart pcr
4 Re amplify the dirty product with internally nested pcr,
try dmso at up to 10% dmso
redesign the primers checking that you GOI does not have pseudogenes existing in the genome
Hi Yukta,
in addition to what Paul Rutland recommended I wanted to add:
1: The protocol of the EmeraldAmp® GT PCR Master Mix is recommending a denaturation temperature of 98°. As you have a gene with a high GC content (if I see it correctly more than 60% for the human gene) I would follow this advice. Even if only a 10 sec denaturation time is recommended (you can try this) I would think you might benefit from longer denaturation times (e.g. 5 min initial and 1-2 min during cycling). Please check out the recommendations at Thermofisher - I think they are quite good:
https://www.thermofisher.com/de/de/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-cycling-considerations.html#Denatura
They give also some recommendations regarding additives (other than DMSO - however I also would try 10% as recommended by Paul Rudland).
I would also think a hot start PCR is a good idea - as you don't have a hot start polymerase you could simply keep your PCR reaction on ice while starting the PCR machine (lid not closed) and put your samples in in the machine once it reaches 98°C.
KR Dörthe
Other things to try:
1. dilute out your DNA. You say it's 34ng/microliter, but don't say the volumes you are using. At that concentration, 1 microliter would be plenty. "less is more" if there are any PCR-inhibiting molecules in the DNA. Try diluting 1:10
2. increased overall reaction volume. It's easier to measure accurately with larger volumes.
3. make your primers longer. Yours are only 19 bases long, add more to the 5' end of each. That increases specificity too.
4. how many cycles are you using? You can typically increase to 35X
Good luck!
If you've already optimized the annealing and extension temperature gradients to enhance your band and considered the feedback from our colleagues, it might be advisable to purify the band of interest directly from the gel. Running it again afterward could yield better results, as it would eliminate non-specific material.
If feasible, I would also recommend sequencing the purified fragment to confirm that it indeed corresponds to the target sequence you're aiming for.
Wishing you great success in your experiments.
B. R.
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