Sequence error in cloned expressed gene - how to correct it? - (Jul/24/2006 )

My goodness! I felt so stoopid now!

I am currently doing a 8 months long project which requires me to clone out a gene to express. So far, I have spent like 3 months. I have just finished cloning and was sooo happy when I realise that something was wrong!!!!!

Previously when I checked the sequences after sequencing result, I happily check and didnt realise that since I used blast from NCBI, the Reverse reaction starts from the end of the gene.And unfortunaltely, there is a mismatch at the end of the gene reading from the end of the gene, I get CCA when it was supposed to be CCG. After I translated it, the amino acid remains the same. BUt as I hav realised, i was supposed to read it the other way round, meaning now the mismatch is from GCC to ACC which have two totally diferent amino acid!!!!!!!!!!!

I don have much time left so I don know wat to do. Should I jus express it and pray that it works since the mismatch is at the end of the gene, it is at about 1400 when the whole gene is 1500 bp long. DOes anyone know whether the conformation of the protein will change??? Should I just go on and express cos relly not much time for me. But I realise that there is actually two types of antibodies for this gene which is one hav the immunogen at N terminal the other is at C terminal. can I use the one at the C terminal , will it still affect? Wat do I do?

SOmeone pls save me!!!!

please clear me more. r u trying to clone some chDNA???

what do u want to express?? is it like a cassette?? that means a lacZ or something like that and a promoter and a terminator region with a marker gene

if you want to transfrom any gene like that expression cassette tell me, may be i can help you

I think you should do a quickchange mutagenesis. It doesn't require so long and you can change your aa to the original one you wanted to have

Dear T. reesei

I need to clone a a human gene that is to be expressed in a pichia vector. I am not very sure about wat is an expression cassette but THe who;e plasmid that I am expressing will consist of a secretory factor, my gene of interest and then some histidine tag. Is it helpful???


As for dnafactory.

Does that mean I need to buy a kit for the mutagenesis? I am not very familiar with the process,too. THats why I worried i might not hav enough time. Moreover, fund is limited.

[quote name='Fossil' date='Jul 25 2006, 07:29 PM' post='61193']
Dear T. reesei

yea i understand your case, i am also doing the same experiment. so you will transform a DNA fragment in pichia. that is may be homologous intigration. so you can cut your DNA fragment without your erorr portion, and then isolate it from gel and then transformation. i think there is no problem but for successful intigration you need atleast 1-2 kb upstream and downstream region. so be careful about it.

Hi Fossil,

Your aa change may merely represent an allelic variant of your gene. It may even be the most common allele in the population, and the version at NCBI may be a rare variant. Without population information, it's impossible to tell. If your variant is at a key position for the function of the protein, then you may have to pay close attention to it and possibly do mutagenesis as others have suggested. Try to find out if there are any known SNPs in your gene, and also check other clones to see if they have the NCBI sequence - your DNA source may be heterozygous at this locus. Or try getting your cDNA from another individual. Variation is a natural thing, so don't assume that your clone is "wrong".

Well, I am not integrating my gene of interest into the yeast genome, The only worry for me is that one mismatch may affect the 3D structure of the protein to be expressed which may not be able to be detected by the commercial antibody that I am using for my Western blot at later stage.So SHould I just make a bet and try expressing?

I agree with wbla3335 -- if you do have a single amino acid mutation, there's no evidence to support that NCBI's sequence is "correct" and yours is "wrong". How many entries are there for this gene in GenBank? Are the from different labs? Are they all exactly the same? Are you using the exact same genus/species/strain of whatever organism this is (what organism is it, BTW) as was used to generate the sequence in the database?

Why did it take 3 months to clone the gene? I'm not being critical, it's just that if I found myself in the same situation, I'd just re-clone the gene rather than try a mutagenesis procedure or kit, but I'd expect recloning to take more like three days rather than three months.

Also, I do not fully understand what happened here. Can you re-explain this:

in that case mutation may be solve of your problem
may be u can try with 1 sapmle........ur original one (with mismatch)
and mutated one
best of luck