Primer dimer discussion

An actively discussed topic on primer dimer in bionet newsgroup and biowww.net user comments.


So, my degenerate PCR worked. But I had more Primer dimers than product
using Pwo. (nothing at all with Taq)

I wanted to have a DIG Probe (with the deg Primers), which didn't work
when I took genomic DNA as template. I cut out the PCR band from the
first rxn and used it as a template with the Expand Pol from my DIG
labeling kit. I got a product AND the primer dimers were gone (mostly)!

Of course I'm happy about the outcome but am wondering why suddenly the
primer dimers are gone¡Â- Polymerase? Buffer?

Link

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In genomic DNA you have many places where degenerate primer could prime
(if you¡�r not working with the absolute optimum and even then).
Furhtermore you need more cycles to get the same amount of product because
in e.g. 5 ng of genomic template the wanted sequence is present only
rarely. And the more you amplify the larger the fraction of nonspecific
product will be. If you use a purified PCR-template every bit of DNA is
template - this makes it dificult for the primers to misprime and do their
primer dimer stuff.

That¡�s why for a amplification from a excised band you only need to tip
your pipete tip once in the template and once in your reaction (instead of
pipeting 0,1 ul or less) and 25 cycles in order to get your product. With
genomic DNA you need 5 ng or so and 30 cycles.

Link

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>> If you use a purified PCR-template every bit of DNA is
>> template - this makes it dificult for the primers to misprime and do their
>> primer dimer stuff.
>
>Thanks, but I know all that. (It's kind of a theoretical question, since I
>got what I wanted)
>
>I thought the formation of primer dimers involves _just _ the primers -
>that's why I couldn't come up with an explanation .

Just what I was going to say! However can you be sure yourself that what
you were getting was primer/primer and not some very short product? The
only real way to tell would be to clone the small product and sequence.
In theory a real Hot Start would cut out the dimers as they should only
form at low Tm's i.e. the time it takes to prepare the PCR to
denaturation.

Link

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I did sequence that low molecular stuff once and got overlapping sequences
- seems that it is not *one* but a mixture of products (every reaction is
different in this respect, I suppose)

Link

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I've been seeing a lot of this lately, and have a provisional explanation. If
your primers are really bad, they will make primer dimers all the time. If they
are really good, they will never make primer dimers, even if there is no
template present. However, if you have to work with less than perfect primers,
the formation of the primer dimer may be a very low probability event, only
occurring late in the cycle if there is no template present. If there is
template present, the desired PCR reaction outcompetes the primer dimer
reaction. Thus, if you got primer dimer and desired band when you were using
whole genomic template, which has very few target sites, and you then used cut
out band material, which will have a huge number of specific sites for the
primer, the reamplification will deplete the reagents before the primer dimer
reaction can get going.
Although I haven't rigorously tested this scenario, I have used serial
dilutions of gel purified PCR product, and as the amount of template dropped off
the primer dimer product increased in intensity.

Link

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Hi all
I want to make a Dig probe. My pcr product is 1.4kb. The retio of dTTP:DIG-dUTP that I use is 1:6. my pcr condition is 95 C 5min, 5 cycles 94 C 1min 50 C 1min 72 C 1min, 5 cycles 94 C 1min 55 C 1min 72 C 1min, 30 cycles 94 c 1min 62 C 1min 72 C 1min and finally 72 C 5min. The genomic concentration that I use is 475ng. When I use unlabelled dNTP my pcr band is sharp but when I use labelled dNTP I see don¡¯t see any band. When I use pcr product as a template for labelling I take sharp band. What can I do to take sharp band with genomic DNA.


2001-02-07 03:41:28
shamsa_m (shamsa_m_2000@yahoo.com)
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Clone the 1.4Kb product into a TA-vector like pGEM'T'Easy, determine the direction its in, cut it, and make a riboprobe using run off transcription. You only need a single digested miniprep for many run-offs, and they're better than double stranded probes, and dirty PCR probes. A lot more work though!

2001-11-21 11:34:01
Jonathan (jon@molbiol.net)
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Primer-dimers:
I have attempted amplification of two rDNA sites through the design of specific primers. It is very likely that inserts should be present, and some of these at least should be between 3 and 5 kb. However, most of the bands I obtain are 200-250 bp or less. The primer sizes are up to 21 bp, and the sequence between the last base in the primer and the beginning of the insertion site is not long enough to explain these fragments sizes. Could I be getting primer-dimers?

2002-04-02 03:19:23
Natasha (natashazt@hotmail.com)

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Last update 10-Nov-2005, Rating n/a of 10 votes.