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Primer non specific binding sites

primer non specific binding software

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7 replies to this topic

#1 Mariam

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Posted 28 January 2015 - 01:12 AM

Hello,

I have designed a pair of primers for amplifying an truncated gene. Since I wanted to clone the PCR product in pET28a vector, I have added the restriction sites for NheI and XhoI to the forward and reverse primer sequences, respectively. The product size should be around 600bp. But after ampilfication, I just detected an single band of around 500bp. I was wondering if the primers have nonspecific binding sites in the template. 

Would you please introduce me an online software which can show all possible binding sites of a designed primer in the template sequences?

 

Thanks



#2 Trof

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Posted 28 January 2015 - 04:10 AM

BLAST

Specifically if you are looking for potential binding site within ysome custom sequence, use Align sequences (it's not exactly align, but BLAST of one sequence aginst other). Nucleotide BLAST can be used to search against existing database of human or other genome, mRNAs etc.

 

If you added restriction site at the ends, you need to BLAST the specific part only, of course.

 

But if you don't know BLAST, I wonder how did you design and check primers in the first place?

 

In my oppinion, getting a clear single band with different size than expected is not a result of non-specific binding, in that case you would have also your expected band present.
So its either a mistake in design (product is actually supposed to be 500 bp in the right sequence), incorrect reading of the fragment length, or in some rare cases there may be actually different sequence (deletion) in the template, than in databases used for design, that could not be predicted.


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


#3 Mariam

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Posted 29 January 2015 - 03:42 AM

BLAST

Specifically if you are looking for potential binding site within ysome custom sequence, use Align sequences (it's not exactly align, but BLAST of one sequence aginst other). Nucleotide BLAST can be used to search against existing database of human or other genome, mRNAs etc.

 

If you added restriction site at the ends, you need to BLAST the specific part only, of course.

 

But if you don't know BLAST, I wonder how did you design and check primers in the first place?

 

In my oppinion, getting a clear single band with different size than expected is not a result of non-specific binding, in that case you would have also your expected band present.
So its either a mistake in design (product is actually supposed to be 500 bp in the right sequence), incorrect reading of the fragment length, or in some rare cases there may be actually different sequence (deletion) in the template, than in databases used for design, that could not be predicted.

Thanks for your tips.

I always use nucleotide BLAST (NCBI) to check the designed primers. If the problem is one of the cases you mentioned in your last paragraph, what is the solution? 



#4 Mariam

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Posted 29 January 2015 - 03:48 AM

 

BLAST

Specifically if you are looking for potential binding site within ysome custom sequence, use Align sequences (it's not exactly align, but BLAST of one sequence aginst other). Nucleotide BLAST can be used to search against existing database of human or other genome, mRNAs etc.

 

If you added restriction site at the ends, you need to BLAST the specific part only, of course.

 

But if you don't know BLAST, I wonder how did you design and check primers in the first place?

 

In my oppinion, getting a clear single band with different size than expected is not a result of non-specific binding, in that case you would have also your expected band present.
So its either a mistake in design (product is actually supposed to be 500 bp in the right sequence), incorrect reading of the fragment length, or in some rare cases there may be actually different sequence (deletion) in the template, than in databases used for design, that could not be predicted.

Thanks for your tips.

I always use nucleotide BLAST (NCBI) to check the designed primers. If the problem is one of the cases you mentioned in your last paragraph, what is the solution? 

 

I am sure about the fragment nucleotide size and I have checked it many times since I got the wrong PCR product.Could the problem be solved if different pairs of primers are designed? 



#5 Trof

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Posted 29 January 2015 - 10:15 AM

First, you can sequence your 500bp product to see, what it is, i.e. what part is missing.

 

Hmm, another idea.. is your template cDNA? Genes can by spliced into different mRNA transcripts, you may have expected one, that has one ~100bp exon in addition and that may be a minority transcript. You would see that by sequencing the product as well.

 

Depends what is the purpose, if the source material is the one you want to check in vitro, then you probably have the right product, but with different size then you expected. Since you talk about truncated gene, I have no idea what is it you want to do and why is it truncated. Also, if it's a mutation, the sequence does not of course correspond with the wild-type gene in the database, so you somehow must desided what the truncated sequence is and how long it should be. I don't know nothing about it, but if there was a mistake in that, your product is right, but your expectations were wrong.


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


#6 Mariam

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Posted 30 January 2015 - 08:53 AM

First, you can sequence your 500bp product to see, what it is, i.e. what part is missing.

 

Hmm, another idea.. is your template cDNA? Genes can by spliced into different mRNA transcripts, you may have expected one, that has one ~100bp exon in addition and that may be a minority transcript. You would see that by sequencing the product as well.

 

Depends what is the purpose, if the source material is the one you want to check in vitro, then you probably have the right product, but with different size then you expected. Since you talk about truncated gene, I have no idea what is it you want to do and why is it truncated. Also, if it's a mutation, the sequence does not of course correspond with the wild-type gene in the database, so you somehow must desided what the truncated sequence is and how long it should be. I don't know nothing about it, but if there was a mistake in that, your product is right, but your expectations were wrong.

It is not a cDNA.

It is a bacterial outer membrane gene that has been truncated based on the conserved areas and the location of good MHC binder epitopes. Since our final goal is to design fusion from three truncated genes, we are supposed to check and compare the in vitro expression and in vivo experiments of singles and the fusion. The complete gene is 1044bp and the truncated form is 604bp. I have designed the truncated form in silico based on the gene sequence of the specific bacterial strain submitted in Genbank and then the same standard strain of the bacteria was used for in vitro production of the truncated gene. I have checked the size of both many times, but I did not make a mistake. Moreover, previously, PCR , cloning and expression of the complete gene was successfully done by us! The sequencing result was satisfactory. I used the complete gene cloned in pET28a as a template for amplification of the truncated form. I am completely confused about what is going on there in the PCR tube!!



#7 Trof

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Posted 30 January 2015 - 11:38 AM

I see, so you PCRed a gene from a plasmid? So to check what do you really have in plasmid, take some plasmid primers and amplify/sequence it. If you got correct length (i.e. your 604 bp gene part + some plasmid sequence) then you should sequence it to find out why the gene primers amplify different size than you expect.  If the length will be again 100bp short, then you actually now have something different in the plasmid, than you think.
In theory it may happen, plasmids may recombine, but you first need to find out if something is wrong with your plasmid you use as a template of with your primers or what.


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


#8 Mariam

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Posted 31 January 2015 - 08:32 AM

I see, so you PCRed a gene from a plasmid? So to check what do you really have in plasmid, take some plasmid primers and amplify/sequence it. If you got correct length (i.e. your 604 bp gene part + some plasmid sequence) then you should sequence it to find out why the gene primers amplify different size than you expect.  If the length will be again 100bp short, then you actually now have something different in the plasmid, than you think.
In theory it may happen, plasmids may recombine, but you first need to find out if something is wrong with your plasmid you use as a template of with your primers or what.

Thank you very much for your valuable tips and your time. I will check the plasmid by sequencing to check if something is wrong or not. 







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