don't primers melt in elongation of PCR?

[Nathan McCorkle]

So this says anneal at 55 C then elongate at 72 C... won't the primer
melt off if its anneal temp is less than 72C ??? Say the Tm of the
primers is 59 C... what's going on here?

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Jordan Miller]

you anneal at 55, during that time the primers attach, and the polymerase actually also hops on and starts replicating at this lower temp. by this point you can now safely raise the temp to peak polymerase efficiency (72) because you have replicated and elongated the annealed primers enough that the melting temp of those strands is now far above the original primer Tm.

jordan

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[Jordan Miller]

oh... and at that higher elongation temp you also have a very low chance of having new primers incorrectly attaching, so that helps all around.

jordan

[Jordan Miller]

oh sorry, and 72 is not peak efficiency, I don't think. this is taq polymerase after all, which functions at 95C... but I think the prevention of incomplete and incorrect binding events is the reason, in addition to making elongation happen faster.

jordan

[Nathan McCorkle]

On Sun, Apr 17, 2011 at 7:22 AM, Jordan Miller <jrd...@gmail.com> wrote:
> you anneal at 55, during that time the primers attach, and the polymerase actually also hops on and starts replicating at this lower temp. by this point you can now safely raise the temp to peak polymerase efficiency (72) because you have replicated and elongated the annealed primers enough that the melting temp of those strands is now far above the original primer Tm.
>

so if I have 2 primers, one's Tm is 73 and the other's is 77, should I
just anneal at 70? If its OK for the polymerase, and below the Tm of
both primers, it should be good right? Is there some magic number of
degrees less than the lowest Tm that annealment should happen at?

[Pap Dániel]

Tm  55 C is for primer annealing (this temperature is depends on the primer sequence), and 72 C is for activate the enzyme. Melt off: you mean denaturate the two straind is over 94 C

Look:
http://www.btci.org/k12/bft/pcr_geneticscreening.html

Daniel

2011/4/17 Nathan McCorkle <nmz...@gmail.com>

[Jordan Miller]

got this detailed response from our DNA guru... enjoy!!

jordan

"No matter the annealing temp. of the oligos, you always try the standard protocol of anneal at 55C for 50-60 sec, followed by elongation at 72C.  At 55C (or thereabouts), you form a ternary complex of template, primer and polymerase.  The polymerase does not efficiently elongate at this temperature, but the stability of the ternary complex is much higher than just the template-oligo complex, and therefore can survive transiently at 72C.  Since the Taq polymerase's DNA synthesis rate is quite rapid, it will elongate the nascent DNA strand quickly enough that the oligo-template complex doesn't have time to dissociate (and once elongated, the Tm of the complex goes well above 72C).

It is not recommended to do annealing at 70C (even if the oligos are quite long), because the Taq polymerase can start to synthesize new DNA before a stable equilibrium has been reached.  In this non-equilibrium state, primers that have misannealed (i.e. bound in the wrong place), have not had time to dissociate.  So you risk getting a bunch of off-target PCR products.

The 95/55/72 cycling parameter work about 95% of the time without any sequence-specific considerations.  You only consider changing these conditions if you are doing mutagenesis, have a GC-rich template, or have an impure DNA template."

[Mac Cowell]

Wow, so 95% the preset temps of 55 / 72 / 95 are all that are needed?

Even if primer meltings calculators predict a Tm of 65?

231.313.9062 // @100ideas // iPhoned

[Jordan Miller]

yep that's what he said :)

an advantage to optimizing PCR protocols is that you can always take a small aliquot of your reaction and run it on a gel to see if it worked. if, after 35 cycles, it failed to copy your DNA, well the reagents in the reaction vial are still good since they are all thermostable, so you can just try the reaction again with different temp conditions and incubation times. that is how real-time PCR (a.k.a. QPCR) was developed, quantifying DNA yield after successive numbers of cycles.

let us know how it goes!!!

jordan

[Nathan McCorkle]

On Mon, Apr 18, 2011 at 7:08 PM, Jordan Miller <jrd...@gmail.com> wrote:
> got this detailed response from our DNA guru... enjoy!!
>

So I will be adding linkers, which could be considered a form of
mutagenesis from the original strand, right? Could you see what the
Guru says when you are adding linkers to the end of each PCR primer?
I'll be amplifying genes from cDNA and plasmids for directional
cloning, rather than to check for the existence of said gene.

-Nathan

[Jordan Miller]

what do you mean linkers? you mean synthetic additions on the 5' end of the primers that will not anneal? is it to help you ligate to something else after amplification and then digestion?

maybe you can tell us *exactly* what you're doing...? then we will have a better chance of giving you proper info. or feel free to ping me at jsmi...@seas.upenn.edu

jordan

[Cory Tobin]

> what do you mean linkers? you mean synthetic additions on the 5' end of the primers that will not anneal? is it to help you ligate to something else after amplification and then digestion?

I think he's adding restriction sites to the end of the primers so he
can cut with restriction enzymes specific for that sequence and then
ligate into some plasmid. The primer would look like:

3'-primer_sequence-GAATTCGCG-5'

Where 'GAATTC' is the sequence targeted by EcoRI and 'GCG' is the GC
clamp on the end.

-cory

[Jordan Miller]

ya so I'm wondering if that's why his reported melting temp is so high, because it's calculated for the full length even if it's not all binding initially. so hopefully we get the full story of what he's trying to do... there may be other gotchas we can help him avoid

jordan

[Nathan McCorkle]

>
>
> On Apr 19, 2011, at 12:10 AM, Cory Tobin <cory....@gmail.com> wrote:
>
>>> what do you mean linkers? you mean synthetic additions on the 5' end of the primers that will not anneal? is it to help you ligate to something else after amplification and then digestion?
>>

yes

>> I think he's adding restriction sites to the end of the primers so he
>> can cut with restriction enzymes specific for that sequence and then
>> ligate into some plasmid.  The primer would look like:
>>
>> 3'-primer_sequence-GAATTCGCG-5'
>>
>> Where 'GAATTC' is the sequence targeted by EcoRI and 'GCG' is the GC
>> clamp on the end.
>>

yep, except I hadn't thought of the GC clamp, good idea.

>>
>> -cory
>>

On Tue, Apr 19, 2011 at 12:31 AM, Jordan Miller <jrd...@gmail.com> wrote:
> ya so I'm wondering if that's why his reported melting temp is so high, because it's calculated for the full length even if it's not all binding initially. so hopefully we get the full story of what he's trying to do... there may be other gotchas we can help him avoid
>

yeah I used invitrogen's primer design tool, pasted in my sequences
and added the linker sites to both from the drop-down list, then chose
the forward and reverse by selecting the two with closest pairs of Tm
(forward, reverse), (forward with linker, reverse with linker).

I am cloning some genes from cDNA, chromosomal DNA, and plasmid DNA
using specific primers, with linker (REase sites) added to the 5' end
of the forward primers and to the 3' end of the reverse primers. I'll
then amplify, gel purify, cut, ligate, repeat for all remaining
DNAs...

> jordan

[Cory Tobin]

> yep, except I hadn't thought of the GC clamp, good idea.

Yeah, when you add a restriction site to the end of the primers its
usually a good idea to add the GC clamp as well. 2 reasons: 1) help
with annealing 2) the extra bases help with the digestion since some
enzymes won't cut near the end of the fragment. See here:
http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_linearized_vector.asp

> yeah I used invitrogen's primer design tool, pasted in my sequences
> and added the linker sites to both from the drop-down list, then chose
> the forward and reverse by selecting the two with closest pairs of Tm
> (forward, reverse), (forward with linker, reverse with linker).

Hmm, I don't know what algorithm Invitrogen is using to calculate the
annealing temp, but I imagine if they give you the option to add to
the 5' end it's *probably* taking that into account.

> I am cloning some genes from cDNA, chromosomal DNA, and plasmid DNA
> using specific primers, with linker (REase sites) added to the 5' end
> of the forward primers and to the 3' end of the reverse primers. I'll
> then amplify, gel purify, cut, ligate, repeat for all remaining
> DNAs...

I don't think adding to the 3' end of the reverse primer will work.
It's gotta be the 5' end on both primers.

-cory

[Cathal Garvey]

The GC clamp is a 3' addition; it's suggested so that the 3' end will bind strongly to the target sequence, as Taq or whatever pol you're using does all the magic at the 3' end.

The 2-3bp addition to the 5' end for enzyme cleaving sites can be any trio of nucleotides, so adapt at will for whatever your needs are or just go with ATA.

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[Jordan Miller]

so then you don't want to include the initially non-binding bases in the Tm calculation. if you had tried 70 for annealing it definitely wouldn't have worked!

jordan

[Nathan McCorkle]

sorry it is on the 5' end of the reverse... but its on the 3' end of the gene

> I don't think adding to the 3' end of the reverse primer will work.
> It's gotta be the 5' end on both primers.
>
>
> -cory
>

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[Nathan McCorkle]

On Tue, Apr 19, 2011 at 8:39 AM, Jordan Miller <jrd...@gmail.com> wrote:
> so then you don't want to include the initially non-binding bases in the Tm calculation. if you had tried 70 for annealing it definitely wouldn't have worked!

As I mentioned the Invitrogen calculator gave both melting temps, one
including the linkers and one without, without are all lower temps...
so I was thinking that I could the first few PCR cycles a few degrees
lower than the lowest melting temp (of the two melt-temps given by the
primer design tool), then after 3-6 cycles increase it to a few
degrees less than the higher of the two melting temps. My thinking is
that the startup cycles would allow the primers to bind to sites they
aren't totally complementary to, but might also allow for non-specific
annealing, so after 3-6 cycles there should be enough of the "first
strands" with linkers successfully added, so the higher annealing
temperature would then be tolerable and good to amplify only the gene
plus linker sequences.

[Jordan Miller]

apparently i missed that part.

wait, why do you have the linker on the 3' end of the reverse primer? you always want the linker on the 5' end. if you put it on the 3' end of the primer it won't work because the polymerase would proceed from bound->unbound primer. polymerase only works in the 5'->3' direction.

Did you do a search of the other contaminating DNA sequences for potentially annealing with the linker portions of your primer? In general this will not matter unless you have opposite matching sequences that are within the extension time of your elongation. If they are not, contaminating sequences can only increase linearly while your species of interest will increase exponentially so it won't matter that you are amplifying some single strands of contaminating species.

jordan

[Nathan McCorkle]

On Tue, Apr 19, 2011 at 1:21 PM, Jordan Miller <jrd...@gmail.com> wrote:
> apparently i missed that part.
>
> wait, why do you have the linker on the 3' end of the reverse primer? you always want the linker on the 5' end. if you put it on the 3' end of the primer it won't work because the polymerase would proceed from bound->unbound primer. polymerase only works in the 5'->3' direction.

sorry, I just realized how bad I may have presented this... there are
no linkers on 3' ends of any of the primers I have... they are on the
5' ends in all cases... I meant to say I had linkers upstream of the
sequence start, and downstream of the sequence end.

>
> Did you do a search of the other contaminating DNA sequences for potentially annealing with the linker portions of your primer? In general this will not matter unless you have opposite matching sequences that are within the extension time of your elongation. If they are not, contaminating sequences can only increase linearly while your species of interest will increase exponentially so it won't matter that you are amplifying some single strands of contaminating species.
>

I tend to hate blast because I haven't learned what all the numbers
and coefficients mean... and things that I know are in the NCBI
database don't match for me... so basically I suck at BLASTing.... but
what you say about linear/exponential growth makes sense.

So it seems like I shouldn't worry about changing the annealing temp,
is that right? Why do people run temp gradients then?