5 replies to this topic

[Blackeyed]

Hello,

I'm very new to the primer design story and for my first experiment I already got a difficult one to crack, hopefully someone can help.

I'm designing primers for a RT-PCR to measure mRNA levels of a number of genes in Sheep ( Ovis) lung tissue.
The problem is that the sequences are not yet known in sheep. I based my primer design on Cow (Bos) but again there are only 'predictions' of the sequences.
I don't know how valid these are and if I should design my primers based on these sequences??

I can also use Mouse sequences ( are known) but if I blast my primers then should a good primer also have similarity with the prediction of the Cow sequence??


Can anyone help me?
Thanks

[Prep!]

http://www.csiro.au/...eep-Genome.html

according to this, both the aniomals have more than 90% maps similar so best luk and acc to me you can go ahead using bovine as your reference and tryin it out for sheep!!!

[Blackeyed]

even if the sequence for the Bos Taurus is just a prediction?

[phage434]

There are highly conserved sequences in many genes, which can be used for designing primers. Do an alignment of the gene amino acid sequences from as many species that are available. You may find that there are regions which are essentially unchanged. You can then design nucleotide sequences that will prime those sequences. Single amino acid codons (such as met, trp) will be useful in reducing uncertainty. Try to make the 3' end of primers as specific and correct as possible.

[Blackeyed]

Okey, If I found such a conserved region in my sequence, which program can I then use to design primers specific for that sequence?
I'm now using PerlPrimer. That program does not have an option to select where you want your primer designed.
Thanks

[Trof]

You can use Primer3 to design primers in certain areas (target areas), using the square brackets.