I'm trying to design qPCR primers and probes to detect DNA from a specific cell line (IMR32 neuroblastoma cell line) and would not be present in normal white blood cell DNA.
I found a qPCR primer and probe sequence in literature against the PHOX2B gene but it was designed to detect cDNA for RT-qPCR. The amplicons spanned an intron of at least 500bp. But I am interested in quantifying genomic DNA. Is it possible to change the primers so that gDNA is detected?
I think it should be possible. You could redesign the primers so that the Forward is within an intron, and the reverse is within an exon. Or two primers within an exon. In addition to providing a nice visual, NCBI's primer blast has a specific section for Exon/Intron selection. This site should be very helpful to you! Good luck!
https://www.ncbi.nlm.nih.gov/tools/primer-blast/
Yes, Samantha has provided the perfect tool, PrimerBLAST. Further, as you have mentioned that the same primers would be having greater than 500bp region if bound to genomic DNA, these would not be suitable for real time PCR. Accuracy of quantification would be great if you could select primers below 200bp amplicon size for this experiment using one of the primers from currently available pair and designing new primer within 200bp span.
- Agree with both answers
- Situate one your primers within an intron and you will only target gDNA
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