I have obtained the source sequence of p5cs from NCBI. The title of the genes varies with partial cd, complete cd, mRNA and sometimes only gene. What does this signify? In case of expression study, do we have to design primer based on cd seq or is it fine if we use the whole gene sequence? If I have more than one sequences coding for the same enzyme, then HOW should I align all the mRNAs and design specific primers?
You can check your PCR primers on the UCSC Genome Browser website to investigate whether or not these primers are specific for your gene only, use this link:
http://genome.ucsc.edu/cgi-bin/hgPcr?command=start
you should use complete cd sequences for alignment. Align the sequences and then design the primer for most common sequences.... remember starting and stop codons in your sequences for expression studies during primer designing.
you can see the stat and stop codons in your sequences on Expacy Translate tool.
you will get there 6 reading frames out of them select largest one as expressible. based on translated sequences you can search their positions in the cd sequences you have downloaded.
all the best....
Do you want to expres this protein, or do you want to measure the expression by qPCR? Every gene has more more then one splice variant , but normally the longest variant (protein) with all exons present, produces the functional protein, sometimes intron retention occurs, but this will cause premature termination of the protein.
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