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sequencing getting wrong 1 nucleotide :(((

sequencing

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#1 Huongnguyen.miss

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Posted 14 April 2014 - 06:46 AM

My bacteria gene, after i got sequence from company, then i compared with original gene from gene bank. The result is: my gene different just only one nucleotide, so that changed 1 amino acid ( from Leucine to Proline). 
Is my gene okie? can i use that for expression protein?
Hope I have said the problem clearly
thanks in advance


#2 Trof

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Posted 14 April 2014 - 06:55 AM

That's hard to say.

It can be a common polymorphism or a mutation or an amplification error.

Reference sequences can actually differ from "real life" genes,  or multiple variants may exist. You need to search the SNP databases to find out. 


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#3 Huongnguyen.miss

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Posted 15 April 2014 - 12:57 AM

thanks Trof...

but i don't know how to do that...:((

i just did blast in NCBI  



#4 Trof

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Posted 15 April 2014 - 02:07 AM

I've just looked, that for prokayrotes there are generaly no annotations in dbSNP. Unlike in vertebrates, for example.

 

And if you have some rare kind of bacteria, there won't be any info at all.

 

Probably the best way is to search for this specific protein change, if somebody commented on it (is common). If no one did, there is hardly any way to find out if it has a negative effect before you try to express it. You can always try to mutate the vector to have a reference sequence of the gene. If you wanted that specific gene.

 

On the other hand, if this was cloned from real bacteria, and you want to see expression if this particular protein, you may just check, if there is the same substitution in the bacteria. So that it was no amplification mistake.

 

It's really difficult to decide..


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#5 Huongnguyen.miss

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Posted 21 April 2014 - 04:17 AM

thank you so much, i am afraid my protein will lost the function if i continue use that sequence, so now im doing again to make sure my sequence is best to use to express Protein.







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