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Designing PCR Primers for cloning

primer design cloning

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#1 taiju

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Posted 12 April 2013 - 04:48 AM

Hello,
Urgently need some help on a question for an assignment that has had me stumped for 2 days.

I'm supposed to clone and express a gene that encodes a protein/enzyme known as CobT produced by the bacterium Mycobacterium avium paratuberculosis. Now I need to design a primer to help me amplify, clone and express the protein. Problem is I'm totally confused on how to go about this.

How do you design a primer? I know this is a super basic question but the multitude of tutorials I've seen have me all confused.
I tried using the ncbi primer blast tool but it seemed all the primers it gave me were inside my target sequence which makes no sense, how do you use the ncbi tool to pick primers?

Is there a difference between a primer for cloning and one for PCR?

Any help would be appreciated.

The CobT nucleotide sequence is attached below, the target sequence is highlighted in blue.

Attached Files


Edited by taiju, 12 April 2013 - 04:59 AM.


#2 bob1

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Posted 12 April 2013 - 04:08 PM

OK as you want the full coding sequence of the gene - you have no option about where you put the primers, which is why primer BLAST wasn't working for you. You need to use the first 20-25 bp of the gene and the reverse complement of the last 20-25 bp of the gene, including the start and stop codons respectively. Try and match the GC content and length to give similar annealing temperatures for the primers, but it isn't essential.

If you need to add restriction sites or anything else to the primers, these always go on the 5' end of the primer. If you are doing this you also need to add a few (usually 6) bp to the 5' end before the restriction site so that the RE has somewhere to bind.

Essentially there is no difference between primers for cloning and primers for ordinary PCR, other than that cloning primers frequently have tails added that contain restriction sites or tags (for the protein, e.g. flag tag) added. These aren't essential if you are doing TA cloning or blunt end cloning.

#3 taiju

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Posted 12 April 2013 - 05:58 PM

OK as you want the full coding sequence of the gene - you have no option about where you put the primers, which is why primer BLAST wasn't working for you. You need to use the first 20-25 bp of the gene and the reverse complement of the last 20-25 bp of the gene, including the start and stop codons respectively. Try and match the GC content and length to give similar annealing temperatures for the primers, but it isn't essential.

If you need to add restriction sites or anything else to the primers, these always go on the 5' end of the primer. If you are doing this you also need to add a few (usually 6) bp to the 5' end before the restriction site so that the RE has somewhere to bind.

Essentially there is no difference between primers for cloning and primers for ordinary PCR, other than that cloning primers frequently have tails added that contain restriction sites or tags (for the protein, e.g. flag tag) added. These aren't essential if you are doing TA cloning or blunt end cloning.



Thank you for the great answer. I have a few more questions just so I'm sure I understood what you wrote. Correct me if I'm wrong.

Say this was a DNA sequence and I wanted to amplify the region underlined & in bold:
GAT CAT AAA ACA TGC TTG TAT AAA GGA TGC TGC CAT GTT CCG TGA ACT GGA AGC GAA CAA TCT TGC GGT
ATA TCA GAA AAA GCC AAA GCT GAT TGC AGT GCT TCT TCA GCG TAA TGC TCA
GTT AAA AGC GAA GGT TGT


1. My forward primer would be: 5' (GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC CAT GTT 3'
where the blue bases are the extra bases to allow the restriction enzyme to bind, the red are the restriction site and the orange is the start codon.

2. Reverse primer would be: 5' (CTAAGT CCATGG CTA) TGA GCA TAA CGC TGA AGA AGC ACT 3'
where the yellow are the extra bases, the green is the restriction site and the purple bases are the stop codon.




-When then would you use primer blast or any of those primer design tools?

-During a tutorial we were given a large sequence and told to pick primers to amplify a given region within the sequence. Now we were told we could pick primers from anywhere in the large sequence as long as it flanked our region of interest. Is this another way to pick primers? Wouldn't that lead to you amplifying more bases than are required such that when you cloned the PCR product into a vector and tried to express it you might get another protein product mixing in with the protein you were targeting or maybe even a different protein product?
I've also seen a tutorial where the primers are the bases just before the region of interest i.e blue is sequence of interets, red is the primer.
3' GAT CAT AAA ACA TGC (TTG TAT AAA GGA TGC TGC CAT GTT CCG TGA ACT GGA AGC GAA CAA TCT) 5'
5' CTA GTA TTT TGT ACG

-Is there a reason for putting primers at different locations even though it seems as though its counterproductive?




Edited by taiju, 12 April 2013 - 06:06 PM.


#4 bob1

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Posted 13 April 2013 - 05:07 PM

The primers look good to me. SOme people advocate putting a 3-6 bp between the restriction site and the start/stop codon, but it shouldn't be necessary.

-When then would you use primer blast or any of those primer design tools?

-During a tutorial we were given a large sequence and told to pick primers to amplify a given region within the sequence. Now we were told we could pick primers from anywhere in the large sequence as long as it flanked our region of interest. Is this another way to pick primers? Wouldn't that lead to you amplifying more bases than are required such that when you cloned the PCR product into a vector and tried to express it you might get another protein product mixing in with the protein you were targeting or maybe even a different protein product?
I've also seen a tutorial where the primers are the bases just before the region of interest i.e blue is sequence of interets, red is the primer.
3' GAT CAT AAA ACA TGC (TTG TAT AAA GGA TGC TGC CAT GTT CCG TGA ACT GGA AGC GAA CAA TCT) 5'
5' CTA GTA TTT TGT ACG

-Is there a reason for putting primers at different locations even though it seems as though its counterproductive?

You could use Primer BLAST or other tools in the situation that you just described, though they won't ensure that the primers will result in in-frame sequence. The advantage of using the tools is that they check for dimerisation and hair-pin formation, ensure that the GC content and Tm are similar, and give you primers that don't have any other binding sites in the sequence of interest (or genome if you used Primer BLAST), otherwise you have to do all that manually which is time consuming and very difficult for the other binding sites bit. Expression is not necessarily the reason you might want to clone a sequence, it could be that it harbours a siRNA or is the binding site for a DNA binding protein, etc.

#5 mdfenko

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Posted 15 April 2013 - 05:24 AM

minor comments about your proposed primers:

the third triplet from the added bases of the reverse primer: you wrote "taa", i think you meant "tta"

you end both primers with a "t", it's good practice to end with a "g" or a "c" to clamp the primer.
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#6 taiju

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Posted 15 April 2013 - 06:52 AM

minor comments about your proposed primers:

the third triplet from the added bases of the reverse primer: you wrote "taa", i think you meant "tta"

you end both primers with a "t", it's good practice to end with a "g" or a "c" to clamp the primer.


Thanks, I hadn't spotted that.

About ending the primers with a "G" or "C"...the primers I made are basically just complements of my templates, to make them end with "G" or "C" is the idea to just shorten my primer until I get a "C" end? i.e for the forward primer:

FROM:
5' (GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC CAT GTT 3'

TO:
5' (GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC 3'

The recommended primer length of 18-30bp should include all these other bases right? So your (actual primer sequence+additional binding bases+restriction site and start and stop codons) should all add up to a maximum of 30?

If your sequence starts in ATG then its not really necessary to add a start codon right?

How would you use a primer design tool in such a way that it put your primers where you needed them, like in my case I need primers at the beginning of my coding sequence for the CobT protein as I want to be able to express CobT.


_______________________________________________________________________________________________________________________

Okay, so I just used this information to make primers for my actual project.
The right primer is okay.
The left is giving me issues with regards to GC content, its at 68-70%. Is there a way to lower this other than trying to change up the primers I use, my ending sequence is pretty much full of G and C bases. These are the last 36bp of my protein sequence:
CAG GCC GGT GTG TCC GAC CCG TCC GCT CAC CCG TGA

Edited by taiju, 15 April 2013 - 07:49 AM.


#7 memari

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Posted 15 April 2013 - 07:00 AM

Also concider this page

Cleavage Close to the End of DNA Fragments
https://www.neb.com/...f-dna-fragments
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#8 mdfenko

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Posted 15 April 2013 - 08:27 AM

About ending the primers with a "G" or "C"...the primers I made are basically just complements of my templates, to make them end with "G" or "C" is the idea to just shorten my primer until I get a "C" end? i.e for the forward primer:

FROM:
5' (GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC CAT GTT 3'

TO:
5' (GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC 3'

The recommended primer length of 18-30bp should include all these other bases right? So your (actual primer sequence+additional binding bases+restriction site and start and stop codons) should all add up to a maximum of 30?

yes, sort of...

you can increase as well as decrease. also, you don't have to make the change with triads, you can change one base at a time so you could just remove the "tt" to get to the "g" or you could have added a "c" (or "cc") to the end to set up the clamp.

remember, you are extending from the end of the primer so you don't have to make changes in threes to maintain the coding sequence.

the necessary primer length takes into consideration only the actual primer sequence. the rest doesn't anneal.
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#9 taiju

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Posted 15 April 2013 - 09:01 AM


About ending the primers with a "G" or "C"...the primers I made are basically just complements of my templates, to make them end with "G" or "C" is the idea to just shorten my primer until I get a "C" end? i.e for the forward primer:

FROM:
5' (GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC CAT GTT 3'

TO:
5' (GCATGC GGATCC ATG) TTG TAT AAA GGA TGC TGC 3'

The recommended primer length of 18-30bp should include all these other bases right? So your (actual primer sequence+additional binding bases+restriction site and start and stop codons) should all add up to a maximum of 30?

yes, sort of...

you can increase as well as decrease. also, you don't have to make the change with triads, you can change one base at a time so you could just remove the "tt" to get to the "g" or you could have added a "c" (or "cc") to the end to set up the clamp.

remember, you are extending from the end of the primer so you don't have to make changes in threes to maintain the coding sequence.

the necessary primer length takes into consideration only the actual primer sequence. the rest doesn't anneal.


You just answered a question I was about to ask, thank you as I was wondering about what happens to the rest of the bases you add but if they don't anneal how do you get an amplicon that has the restriction site and the start/stop codons, like if only the actual primer sequence anneals and is replicated and the additional bases are just kind of hanging how do they get into the subsequently produced PCR products.. I hope my question makes sense.

Edited by taiju, 15 April 2013 - 11:30 AM.


#10 bob1

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Posted 15 April 2013 - 12:38 PM

The primer ends get copied by the subsequent cycles of PCR, its just the initial cycles where there are "free" ends.

#11 taiju

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Posted 15 April 2013 - 01:31 PM

The primer ends get copied by the subsequent cycles of PCR, its just the initial cycles where there are "free" ends.


Thanks. It hadn't quite clicked that each primer replicates all the way across until it gets to the end of the gene so it would replicate the free ends.

You have all been so helpful.

#12 taiju

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Posted 16 April 2013 - 12:45 AM

Okay, so I just used this information to make primers for my actual project.
The right primer is okay.
The left is giving me issues with regards to GC content, its at 68-70%. Is there a way to lower this other than trying to change up the primers I use, my ending sequence is pretty much full of G and C bases. These are the last 36bp of my protein sequence:

CAG GCC GGT GTG TCC GAC CCG TCC GCT CAC CCG TGA


Is there anyway around this? The lowest I've managed to get the GC for the right primer is 66% using only 15 bases. Will this be okay or should I change my reverse primer and add a few bases from outside my coding sequence to even out the GC count. Would doing so cause issues later on?

Is there anything to worry about if I have a lot of hypothetical proteins?

Edited by taiju, 16 April 2013 - 12:52 AM.


#13 bob1

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Posted 16 April 2013 - 05:57 PM

If this is the last 20 or so bp of the gene coding sequence then there isn't much you can do about it, unless you want to include some of the 3' UTR and use a primer further out.

The G/C content can matter if it leads the primers to form hairpins, you can usually get around this with DMSO in the PCR.

#14 taiju

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Posted 16 April 2013 - 06:42 PM

If this is the last 20 or so bp of the gene coding sequence then there isn't much you can do about it, unless you want to include some of the 3' UTR and use a primer further out.

The G/C content can matter if it leads the primers to form hairpins, you can usually get around this with DMSO in the PCR.


Thanks for the reply. So there'd be no issue in shifting the primer slightly out to include bases in the 3' UTR?
If so I think I would prefer to do that just to get the GC content down.

This is a theoretical question I'm working on btw, so I won't actually be doing any PCR.

Edited by taiju, 16 April 2013 - 06:43 PM.


#15 bob1

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Posted 17 April 2013 - 12:47 PM

So long as you include the coding sequence, there shouldn't be a problem with using primers that are outside the coding region. However, for the forward you don't want it to be too far from the promoter such that it falls off the sequence before reaching the ATG.





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