isPCR Not Working?

[Aanandi Munshi]

Hi, 

We have been trying to use the isPCR tool on the genome browser for primers that we found in the literature and we are not sure if what we are doing anything wrong but we wanted to see if there was any way to get on a Zoom call with a technician to see if what we are doing is wrong. The problem that we have been having is that we have combed through a lot of articles looking for qPCR primers for PUMA, RRM2B, and BAX genes. Unfortunately, every time we plug those forward and reverse primers into the isPCR, it says no matches. We are not sure why this is happening and if we are doing something we are not meant to. We have other primers in our lab that show up as a match, so we are confused about these two genes. Sometimes we have found that the primers might have a match in the mouse genome but mostly it does not have a match at all. The other part of the problem is that sometimes when we try to look up the forward or reverse sequences individually, one of them might not have a match in the human genome at all. However, we have specifically designed these in the NCBI's primer designing tool using the human version of these genes so our hope was that it should show a match in the isPCR tool using the human genome as the reference. 

Also, we are wondering about the size that isPCR tells us when it shows a PCR product. Is this at the transcript level and only including the exons or does it contain the genomic size and also have the introns?

We contacted Kent Informatics about this issue and they have told us to contact this email for further assistance. Please let me know if you have any recommendations for us in solving our problem. Thank you!

Best, 

Aanandi Munshi

Research Technician III | Linardic Lab

Department of Pediatrics | Hematology-Oncology 

Duke University School of Medicine 

Levine Science Research Center | Room B361 | 308 Research Dr., Durham, NC, 27708

https://sites.duke.edu/corinnelinardiclab/ 

[Gerardo Perez]

Hello, Aanandi.

Thank you for your interest in the UCSC Genome Browser and for your question about isPCR.

Which human assembly version are you using with isPCR? The sequences change between assemblies and primers that match one assembly version may not match a different assembly version. Did you try the "GENCODE Genes" as the Target option to find matches? If your primer straddles an exon-intron-exon boundary, then you must use the "GENCODE Genes" target option. Here is an image of where to find this option for the hg38 assembly:

I hope this is helpful. Please reply to gen...@soe.ucsc.edu. All messages sent to that address are archived on a publicly-accessible Google Groups forum. If your question includes sensitive data, you may send it instead to genom...@soe.ucsc.edu.

Gerardo Perez
UCSC Genomics Institute

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