Real time PCR for gene family - (Jun/08/2006 )
HELLO!
We are interesting in gene expression of gene family in Arabidopsis.
This family contains 9 genes that share high homology, so there are no regions with out some homology.
We want to design primers that will be specific for each one of the gene in this family, we hop we will be able to see the expression of each one of the genes separately from the others.
We are planning to check the specificity by using plasmid contains these genes.
We would like to know:
What should be the differences between the primers (how many different nucleotides) so they will be specific?
Since it quit difficult to do it by eyes, maybe you know if there is a computer program that will do it for us?
Thank you for your help
Shilo
do sequence alignment of the genes and select regions which are unique to each gene. Its a bit lengthy but you will find some regions. Sequence alignment tools are on NCBI web site or at the this site http://sciencegateway.org/tools/index.html. Try to see that your primers 3' ends are unique and match completly to your gene of interest.
Thank you
Actually, I look for a program that able to take the alignment file from
clustalW and to find specific primers for each one of the genes taking in account the specificty of the primers.
Shilo
Actually, I look for a program that able to take the alignment file from
clustalW and to find specific primers for each one of the genes taking in account the specificty of the primers.
Shilo
Definitely possible, colleagues in my old lab developed primer primer probe sets for all 39 human HOX genes which show high degree of homology. They used Primer Express but they did need to do a fair bit of playing around with the criteria Primer Express uses to select primer probe sites.