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When designing a PCR forward primer for LIC why do I need to remove the...
...signal peptide sequence? I have a target gene sequence I want to amplify using PCR (LIC) but I've been told when designing my primer that I should remove the signal peptide (cleavage site at the 26th amino acid) which corresponds to removing the first 78 or so nucleotides in my sequence.
Why is this necessary? Is it just a matter of not wasting resources on replicating a sequence we don't need in vitro when all we want is the target gene/protein?
I'm using my designed primer in association with pET vector. I know my primer must be preceded by a particular string in order to fit the vector so this orimer (~25 bp) will be extremely specific. SO i don't understand how we can have the (idealised) forward primer form:
<overhang code to fit pET vector> <ATG><remove signal peptide><the rest of my primer>
It's probably because the signal peptide is common to many different proteins, so that primer will bind to many different genes. Your amplification will be much cleaner if you choose a gene-specific sequence.
|designing , forward , lic , pcr , primer , remove|
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