primer Tm reasoning?

69 views
Skip to first unread message

Jonathan Nesser

unread,
Jan 27, 2012, 11:43:46 AM1/27/12
to diy...@googlegroups.com
Sorry to start yet another new thread for such a small question, but I don't want to pollute other threads with off topic posts... I've been trying to understand why primer melting temperatures have to be in the 55-65C range when PCR cycles call for 94C for DNA melting... I've tried to find an explanation for this but haven't been able to... Wouldn't that result in the primers splitting off from the template DNA before you reach the extension cycle of 75C (or around there)? I'm sure I'm wrong because obviously these PCR programs work, but I don't know WHY I'm wrong, and would like to understand. Thanks again for explaining something that is probably so common knowledge that nobody feels the need to address it in writing :)

Jonathan Nesser
Message has been deleted

PCR Assays

unread,
Jan 27, 2012, 12:21:19 PM1/27/12
to diy...@googlegroups.com

Jonathan;

You are not wrong..if you can design a primer that doesn’t produce a bunch of cross reactivities that has a high Tm..then so be it.

 

Signal Diagnostics has a patented technology termed Dynamic Flux Amplification, where we use annealing at temperatures much elevated from the conventional 50-65C range.  It works beautifully..

Brian

--
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To view this discussion on the web visit https://groups.google.com/d/msg/diybio/-/3XwKiPRnTtIJ.
To post to this group, send email to diy...@googlegroups.com.
To unsubscribe from this group, send email to diybio+un...@googlegroups.com.
For more options, visit this group at http://groups.google.com/group/diybio?hl=en.

Cathal Garvey

unread,
Jan 27, 2012, 1:36:52 PM1/27/12
to diy...@googlegroups.com
Technically you're right, but..
When the primers bind, polymerases tend to bind pretty quickly to the
loose 3' end of the primer, and immediately start extending. Sure,
they're not very fast at that temperature, but they have long enough to
make that short primer long enough that the melting temperature is much
higher by the time the next step rolls around.

Secondly, the melting temperature isn't the absolute temperature beyond
which no DNA is bound; it's calculated as something of a midway point
between "No dissociation" and "All primers dissociated", so there's a
lot of wiggle-room. I also imagine that once a primer has bound, it has
a certain intrinsic stability that helps keep it there a while longer.

Of course, longer primers usually still work fine and have a higher Tm.


--
www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey

veera

unread,
Jan 27, 2012, 11:57:36 AM1/27/12
to diy...@googlegroups.com
generally annealing temperatures will be just 3-4 C below Tm of primers. if the melting point of primers is very high like you said in the range of 90 , at that hight tempertaure all double stranded dna will split off and so annealing will not take place., because annealing happens only in the range of 50 - 65 C. hope i'm correct.. if i'm wrong anyone can correct me...

Jonathan Nesser wrote:

Nathan McCorkle

unread,
Jan 31, 2012, 10:36:50 AM1/31/12
to diy...@googlegroups.com
I think its wrong to say annealing only happens between 50-65 C, but
that's the general range for most primers 15-25 bp in length

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

Sigma70

unread,
Jan 31, 2012, 12:54:23 PM1/31/12
to DIYbio
Quick note here

Annealing temp needs to be just low enough for primers to re-associate
with complementary strands. This is based on affinity, so, as a
previous poster pointed out, primer association % is in a state of
equilibrium.

Dissociation / Denaturing / Melting (whatever you want to call it)
tempurature is dependent on the bond strength between the template
strands, and the energy needed to dislodge Taq. The high (94 degree)
temp ensures that G-C (triple bond) rich regions are thoroughly
split. double-stranded DNA is super stable, so once the double helix
is formed (post elongation), you really need to blast the system with
heat energy before those bonds start to break.


On Jan 31, 10:36 am, Nathan McCorkle <nmz...@gmail.com> wrote:
> I think its wrong to say annealing only happens between 50-65 C, but
> that's the general range for most primers 15-25 bp in length
>
>
>
>
>
> On Fri, Jan 27, 2012 at 11:57 AM, veera <drveeramanikan...@gmail.com> wrote:
> > generally annealing temperatures will be just 3-4 C below Tm of primers. if
> > the melting point of primers is very high like you said in the range of 90 ,
> > at that hight tempertaure all double stranded dna will split off and so
> > annealing will not take place., because annealing happens only in the range
> > of 50 - 65 C. hope i'm correct.. if i'm wrong anyone can correct me...
>
> > Jonathan Nesser wrote:
>
> > Sorry to start yet another new thread for such a small question, but I
> > don't want to pollute other threads with off topic posts... I've been
> > trying to understand why primer melting temperatures have to be in the
> > 55-65C range when PCR cycles call for 94C for DNA melting... I've
> > tried to find an explanation for this but haven't been able to...
> > Wouldn't that result in the primers splitting off from the template
> > DNA before you reach the extension cycle of 75C (or around there)? I'm
> > sure I'm wrong because obviously these PCR programs work, but I don't
> > know WHY I'm wrong, and would like to understand. Thanks again for
> > explaining something that is probably so common knowledge that nobody
> > feels the need to address it in writing :)
>
> > Jonathan Nesser
> > jonathan.nes...@gmail.com <mailto:jonathan.nes...@gmail.com>
> > diybioandneurosci.blogspot.com <http://diybioandneurosci.blogspot.com>
>
> > --
> > You received this message because you are subscribed to the Google
> > Groups "DIYbio" group.
> > To view this discussion on the web visit
> >https://groups.google.com/d/msg/diybio/-/3XwKiPRnTtIJ.
> > To post to this group, send email to diy...@googlegroups.com.
> > To unsubscribe from this group, send email to
> > diybio+un...@googlegroups.com.
> > For more options, visit this group at
> >http://groups.google.com/group/diybio?hl=en.
>
> > --
> > You received this message because you are subscribed to the Google Groups
> > "DIYbio" group.
> > To post to this group, send email to diy...@googlegroups.com.
> > To unsubscribe from this group, send email to
> > diybio+un...@googlegroups.com.
> > For more options, visit this group at
> >http://groups.google.com/group/diybio?hl=en.
>
> --
> Nathan McCorkle
> Rochester Institute of Technology
> College of Science, Biotechnology/Bioinformatics- Hide quoted text -
>
> - Show quoted text -

Russell Durrett

unread,
Jan 31, 2012, 10:34:49 PM1/31/12
to DIYbio
In response to Jonathan's initial question, you're not wrong - the
primers will denature before 94, but the full-length PCR product
you're trying to amplify is a lot longer and requires a higher melting
temp to dissociate.

also, if you're designing primers you can find some convenient code at
https://github.com/russelldurrett/Python_Primers



On Jan 27, 11:43 am, Jonathan Nesser <jonathan.nes...@gmail.com>
wrote:
> jonathan.nes...@gmail.com
> diybioandneurosci.blogspot.com
Reply all
Reply to author
Forward
0 new messages