Hello everybody,
maybe my auestion can appear stupid but i must have an answer as soon as possible. I have already the sequence for real time qPCR primers for the human GAPDH. How can i see if they are suitable also for the same gene in other species (particularly MUS MUSCULUS and RATTUS)?
Thanks in advance for your help
Hi, you could use primer blast.
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
And use the "Primer Pair Specificity Checking Parameters" where you can select taxonomic unit which you want to check (e.g. Chordata).
Alternatively, you can download the cDNA sequence of all available GAPDH, align them and see if your primers are universal, then check the specificity in Primer Blast.
Hope it helps.
Primer blast is a good idea.
If you want to do it by the old slow manual way, the standard molecular biology web tools are:
Use NCBI gene http://www.ncbi.nlm.nih.gov/gene/ to find the human, mouse, and rat GAPDH genes. For each, go down to the mRNA sequences (NM...).
Click on the NM... link and then find the FASTA sequences.
human: http://www.ncbi.nlm.nih.gov/nuccore/576583518?report=fasta
rat: http://www.ncbi.nlm.nih.gov/nuccore/402691727?report=fasta
mouse: http://www.ncbi.nlm.nih.gov/nuccore/576080554?report=fasta
copy-paste these sequences one after the other including the ">gi..." line into the window for a clustal program:
http://www.ebi.ac.uk/Tools/msa/clustalo/
then add the sequence of your forward primer after a line like: >forward_primer
run the clustal program and you will see the alignment of the four sequences and be able to see if the primer has reasonable homology.
Then calculate the reverse complement of your reverse primer:
http://www.bioinformatics.org/sms/rev_comp.html
and repeat the clustal alignment replacing >forward_primer with >reverse_primer
- Badges
- Science method