Hello friends,
I ligated my gene of interest in pET vector and transformed into BL21 cells and confirmed my insert through colony PCR. Further, I picked those PCR confirmed colonies for sequencing, but my sequencing result shows only 99% sequence identity to the reference mRNA sequence in NCBI or ensemble database, which include 7 nucleotide mismatches and 4 nucleotide deletions in my query mRNA sequence compared to reference sequence (Repeated sequencing shows same result). As a result I noticed a frameshift in amino acid sequences giving something else protein but not my protein of interest. So my questions are;
1. Do I need to have 100% sequence identity at amino acid or nucleotide level to the reference mRNA or protein?
2. I used Taq polymarase to amplify my cDNA, should I choose high fidelity polymarase?
3. What other methods I can approach to avoid these mutations in my protein sequence and be able to produce recombinant protein ?
Hello Bidur,
Beside what others have already indicated/suggested, I'd like to make a few questions and comments.
What is the origin of your coding sequence ?
A high mutation rate is not necessarily stemming from reverse transcription and PCR with unfaithful DNA pol ... even if it is often the case ... it can stem from multiple gene natural polymorphism (some coding sequence are more akin to polymorphism than others) and it can also comes from alternative splicing events.
What we do in my lab when we want to be 100% sure of the inserted coding sequence, we make it synthesized! That's definittelythe best way ... and not expensive since now a 1500-bp long coding seuqnce costs as low as 400 euros. Moreover, you can optimize the codon bias (though it is not that important).
Best regards,
Prof Philippe Urban
Hi Bidur
I recommend Pfu polymerase based PCR enzyme (high fidelity PCR) in stead of Taq polymerase.
http://en.wikipedia.org/wiki/Pfu_DNA_polymerase
This is a best way to make a expression vector.
Thanks Hisashi, I just started my experiment using pfu turbo polymerase..Let's see how it goes.
Hello Bidur
Taq polymerase is fast, but when it comes to cloning a gene, speed is not your main concern. Remember that most of the Taq add an A at the beginning of your sequence, resulting in a frameshift mutation. Use a high-fidelity polymerase. As Hisashi, I suggest Pfu, but it's not the only one available, so pick one and start again ;-)
Hi Bidur!
You should definitely not introduce mutations, as it will introduce uncertainty in interpretation of downstream experiments. And, as said by others, you should avoid taq polymerase. That said, the number of mutations sounds high. Error rates of taq varies, but I have rarely seen reported error rates above 1 in 3000 give or take. If switching enzymes don't help, the mutations are probably present in the template, whether it's genomic or cDNA.
Best of luck! Vegard
Hi Bidur,
there are now better enzymes than Pfu for HiFi PCR. I strongly recommend Takara PrimeSTAR GXL for high fidelity and long amplification (up to 30kb). Or PrimeSTAR Max a premix for highest fidelity and fast PCR.
Please contact clontech (www.clontech.com; +1.800.662.2566) to find out about samples availability.
Anyway prior to protein expression I recommend sequencing the PCR insert to confirm accurac=y of the sequence.
Regards and good luck, F-X
Hello Bidur,
Beside what others have already indicated/suggested, I'd like to make a few questions and comments.
What is the origin of your coding sequence ?
A high mutation rate is not necessarily stemming from reverse transcription and PCR with unfaithful DNA pol ... even if it is often the case ... it can stem from multiple gene natural polymorphism (some coding sequence are more akin to polymorphism than others) and it can also comes from alternative splicing events.
What we do in my lab when we want to be 100% sure of the inserted coding sequence, we make it synthesized! That's definittelythe best way ... and not expensive since now a 1500-bp long coding seuqnce costs as low as 400 euros. Moreover, you can optimize the codon bias (though it is not that important).
Best regards,
Prof Philippe Urban
Thank you all for the great ideas. I already started my cloning . I amplified ( my primers include RE sites) my cDNA using two sets of High-Fidelity DNA Polymerases, pfu turbo and Phusion ( available in my lab). Will let you all know if I get it to work this time.
Before you go to expression, just clone your gene and sequence without expression of protein
Hi Bidur,
You didn't say what your template was-cDNA , plasmid etc. if it is a 'clonal' source like a plasmid then sequence this first before you start as many clones available from libraries contain errors.
Definitely avoid Taq unless it is absolutely the only enzyme that will amplify your gene , which I doubt, and switch to any high fidelity polymerases already mentioned (I personally like KOD for general cloning use). Also bear in mind that there are many enzymes sold or labelled as high -fidelity but they vary enormously in their fidelity and processivity.
If any of the mutants appear in your primer sequence just re-order/re-synthesise.
Silent mutations are generally not a problem. Don't worry about them unless they either introduce unwanted cloning sites that would complicate your cloning or give rise to strings of very low usage codons if your expressing in E.coli.
Good Luck!
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