I've been trying to design primers for qPCR to detect NCF1 cDNA using Primer3Plus and NCBI Primer-BLAST, but I couldn't find primers that are specific to the gene. Any help ?
Thanks !
Using alternative splicing information to design primers can indeed help in distinguishing between the gene of interest and its pseudogenes. Here’s a detailed approach to designing primers for NCF1 using alternative splicing:
Steps to Design Specific Primers Using Alternative Splicing
Example Workflow
1. Identify Splice Variants
- Retrieve the exon and transcript information for NCF1, NCF1B, and NCF1C from Ensembl or UCSC Genome Browser.
2. Compare Exons
- Align the sequences of NCF1, NCF1B, and NCF1C to identify unique exons in NCF1.
3. Design Primers
- Use primer design tools to create primers that span the unique exon-exon junctions.
4. Validate Primers
- Use NCBI Primer-BLAST or similar tools to check the specificity of the primers against the entire genome.
Tools and Software
- Ensembl or UCSC Genome Browser: For retrieving exon and transcript information.
- Clustal Omega or MUSCLE: For multiple sequence alignment to compare exons.
- Primer3Plus: For designing primers.
- NCBI Primer-BLAST: For validating primer specificity.
i'd just that that NCBI primer-blast is good for checking the targets of primers but I hate it for primer design and it doesn't vary primer location much. i could recommend IDT's design tool and you can put in the exon-exon junctions area as a target for either primer. then blast those primers in NCBI blastn to check for off-target binding. as long as the 3'end cannot bind then you should be good.
good luck!
I would suggest you
1- to download the gene and pseudogend sequences and then do a blast two sequence.
if there are differences you can use them to insert them into the primers better in the 3' region
you could also use the point differences (better C or G) and make them coincide with the 3' of the primer.
2- are the 5' and 3' UTRs also identical?
Amal Zammeli
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