Entering edit mode
4.7 years ago
Yoosef
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60
Hello I am trying to design primer pair for Real time PCR. The problem is that my gene has a lot of pseudogenes which interferes with my products. I need to know how can i align the sequence of these pseudogenes and my own gene by using their NM number? or which one of alignment programmes can be used for more than thousands of sequences.
You can use PrimerBLAST, combining Primer3 primer optimization and BLAST to avoid off-targets (if possible).
thanks for ypu response First i need to find the sequence of my mRNA which isn't the same with the pseudogene and then design primers for that sequence. I nedd to align the sequences to find which part is unique in my cDNA sequence. how can i align these sequences using their NM number?
That is not necessary. Just go to PrimerBLAST, enter the NM number of your gene and then run the program. It will design you a primer that is unique to your gene (if that is possible) while considering the entire genome as background including the pseudogenes. Please read the manual of PrimerBLAST. There is no need to to custom alignments. The program does it for you and will return a primer if that is possible. If there is too much similarity with the pseudogenes it will report that as well.
Thanks again I an designing the primers same way you told. But i always get the pseudogenes among my blast results. how can i avoid amplifying those pseudogenes ? I thought that maybe alignning NM of my gene with these pseudogenes may find a seuquence where is unique and i can design primer for that sequence I will be so pleased if you could help me with that. I get a lot of pseudogenes in my blast results and there no way to get rid of them...
Well if PrimerBLAST cannot find any unique primer than there simply aren't any. You cannot fight the biological reality, if the genes are two similar then primer-based qPCR is not the way to go. I do not know what exactly the aim of your study is but maybe some long-read RNA-seq might help here.
I simply want to evaluate its expression level in sperm cell to analyse its effect on sperm motility and morphology using real time PCR. unfortunately all these pseudogenes will interfere with my results.
In that case real time PCR might not be a good experimental strategy. The genome does not allow investigation of every region using short primer-based techniques of short read sequencing due to its redundant nature. If the gene is protein-coding maybe Western blot might help here.
Thanks a lot for your help.