I am looking to clone a few proteins of interest (using RT PCR of mRNA and then cDNA amplification using gene specific primers). I was wondering how to decide on the correct open reading frame to clone, I have heard anecdotally that in general the longest transcript is taken as being the most "biologically relevant" but wanted to check this.
Any advice would be much appreciated.
Patrick
Dear Patrick,
the "biological relevance" is mainly dependent on your study aim, and the assumption that the longest ORF within a mRNA or the longest transcript are the biological relevant ones came from old decisional parameters that , in my opinion, are no longer valid. What I mean is:
1) think at the Tryptophan operon: is it most biologically relevant the upstream ORF that regulates the expression of the downstream ORF or the downstream ORF that is catalytically active?
2) in higher eukaryotes alternative splicing is the rule and not the exception, and, regardless of the mRNA or ORF length, two or more proteins may be produced by a single gene. These products may have different biological relevance because of the effect you are looking at, as an example think at the TP53 gene, 3 different N-terminal domains and 3 different C-terminal domains are linked by a common central domain. The different combinations of N and C domains affect different cellular processes, so the "most biologically relevant" depends on the effect you are looking at.
Hope this helps in your decision.
Hi patrick
I am agree with alberto. You should first specify your goal in cloning these proteins. Are you analyzing a specific function in those proteins? or studying a particular domain within those proteins?
If you for instance are searching for a domain you can search the domains name in both protein and nucleotide databases. If you do not have any informations about the domains of those proteins then you should read about the proteins structure and functionality and find domains that have the functions of your interest.
good luck
Start with a western blot to determine the number of variants and their molecular weights. Try to find antibodies targeting different epitopes to improve your likelihood of success.
Alternative translation may confound your project.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning