Hello everyone. I would be really grateful if you can help me solve my problem.
I have purchased Real Time PCR primers for ureC gene (glmM) of Helicobacter Pylori which the sequences were in an article. I have checked the specificity in NCBI primer BLAST and the results show that they cannot bind to the genome of E. coli.
But in practical effort when I use these primers in classic PCR (not Real Time) they can amplify E. coli DNA too and the size of the product band on gel is the same as the size of product in Helicobacter pylori.
P.S. I check E. coli because there is a high possibility that it interferes with my samples in Real Time PCR.
I have checked all my primers and master mix. There is no contamination.
Primers do not have to be a 100% perfect match to amplify DNA, they just have to be "close enough". That's probably why the BLAST information & your data are not in agreement.
Try changing the annealing temperature using a grading of different temperatures. Include DNA from E. coli as a control at each temperature.
Good luck!
Your primers may amplify genes from other organisms due to several reasons related to primer design and the complexity of the DNA samples being analyzed: 1. **Cross-Reactivity of Primers**: Primers designed to target specific genes can sometimes bind to similar sequences in other organisms. This cross-reactivity can occur if the primer sequences are not perfectly optimized to match the target sequence only. For example, 16S rRNA gene targeting primers have been reported to cross-react with eukaryotic DNA present in complex environmental DNA samples. 2. **Complexity of DNA Samples**: In complex samples such as environmental DNA (eDNA) or clinical samples, there is a high diversity of DNA from different organisms. Primers designed for a specific organism might inadvertently bind to similar sequences from other organisms present in the sample. 3. **Primer Design Flaws**: Inadequate primer design, such as mismatches in the primer sequence, can lead to non-specific amplification. This can occur if the primer sequences are not sufficiently specific to the target gene or if they are too similar to other genes in the genome of the organism or other organisms. 4. **Metabarcoding and Metagenomics**: In applications like metabarcoding or metagenomics, where the goal is to amplify a specific region of the genome from many different organisms, the primers must be carefully designed to minimize cross-reactivity. However, even with careful design, some off-target amplification can occur due to the inherent complexity of the samples and the primer sequences. To mitigate these issues, it is crucial to carefully design primers with specificity in mind, possibly using tools that predict cross-reactivity and ensuring that the primer sequences are as unique as possible within the context of the target genome and the expected background DNA. Additionally, using controls and validation experiments can help confirm the specificity of the primers in real-world samples.
That is why it's better to opt for a TaqMan real-time PCR. In addition to your couple of primers, an added probe allows us to be more specific when targeting a short gene sequence as in qPCR.
- Badges
- Science topic
- Similar topics
- Engineering
- Petroleum Engineering
- Oil