RT-PCR primer designing - (Apr/09/2007 )
Hi there, I am working on a big gene in Drosophila and I have to find out its presence in a specific tissue of the fly from the tissue specific mRNA.
I have questions regarding designing the primers for the gene:
1. I am planning to use the coding region of the gene which is approximately 6kb size for the primer designing. Is this fine or should I select the mRNA sequence of the gene to design the primers?
2. When I am designing the primer for this gene sequence should I feed the whole sequence (mRNA/coding sequence of the gene) in the primer designing program Or should I select a part of the sequence. If so, what are the criteria in selecting the sequence region to design the primer, to get promising results?
3. Can the primers be designed at the 3' or 5' UTR of the mRNA if they have low GC content?
Thanks in Advance for the great help.
Cheers,
Laksh
For primer designing you should take mRNA / coding sequence , because as you are saying that the gene length is 6 kb it is impossible to amplify using Taq DNA poymerase.
You can feed entire coding sequence to the primer making program
it is not advisable to make primer from 3 untranslated region
good luck
Hi ,
I would like to ask coffee...Why do you say "it is not advisable to design primer from 3UTR"? This implies that 5 UTR is more conserved. Is that right?
Thanks
cDNA and mRNA sequences are the same with regard to primer design.
You don't necessarily need to give the whole cDNA sequence to the primer design program.
Since your gene is 6 kb in size, I would design the RT-PCR primers on exons close to the 3 UTR region. The consideration is that RT reaction may not reach the far 5' end of the mRNA.
The most critical things are: your PCR amplicon should span one or more introns, the primers don't bind to other sequences in the genome.
You don't necessarily need to give the whole cDNA sequence to the primer design program.
Since your gene is 6 kb in size, I would design the RT-PCR primers on exons close to the 3 UTR region. The consideration is that RT reaction may not reach the far 5' end of the mRNA.
The most critical things are: your PCR amplicon should span one or more introns, the primers don't bind to other sequences in the genome.
Thanks PCR man, I dont want to sequence the whole length of the gene. I just wanted to sequence a part of the gene in my tissue. For this I wanted to know if deisgining the primers at 3'UTR of the mRNA is a good idea?
This may be a dumb question, but I'm newish at molecular in general and RT-PCR in particular.
A colleague advised that I BLAST my primer sequences. I'm not very familiar with NCBI BLAST. How do I do this?
Just go to this page.
Choose your species. Paste your primer sequence. Click on Format! Wait until the results page builds. Than scroll through the results and check, if the gene your primers are designed for is listed.
K.