I am trying to amplify a gene from streptomyces genome. This is GC rich with 36 start codons (ATG) at different positions of whole gene (1491 bp total gene length). I am not getting the right length of this gene after trying many conditions of PCR including enhancers, stimulants, temperature gradient, template and primers concentrations etc. My questions:
1. Why there are so many ATGs in this gene?
2. Presence of multiple ATGs interferes primer-template binding and amplification?
If I am successful to amplify this gene and later on clone it in expression vector, it will be expressive or not?
Thanks in advance.
Hello,
1) I am a streptomyces researcher and I think it is more likely there is only true start codon. Have you looked for a Shine-Dalgarno ribosome binding sequence? This is a short sequence (~6 nt) of purines (A or G) typically 6-8 nucleotides upstream of the ATG (or GTG, which is also possible in streptomyces) start codon. You could also BLAST the protein sequence of this gene to compare it to homologues, in doing so getting a sense of how large members of the protein family typically are.
2) Not specifically, but high GC streptomycete genomes can be difficult to amplify from. I would recommend trying DMSO and betaine additives to the PCR, if you have not already. I have also used gel-extraction when amplification was really messy.
3) Hopefully, but unfortunately you will just need to try. I have been able to express many streptomycete proteins from E. coli though.
Hope that helps!
Rory Little thank you so much for your detailed answer. I am using enhancers in my PCR mix and betaine is somehow working well. After your suggestion, I checked Shine-Dalgarno ribosome binding sequence in my genes of interest, but I couldn't find that.
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