6 replies to this topic

[Paja]

Hallo everybody,
I am trying to amplify (and eventually sequence) promoter region of my gene. The problem is high content of GC (73%). I tried almost everything possible and now I am without any new idea.    Does somebody have some advice what else can I try before going into some special kits enabling PCR amplification of difficult templates? I found several such a helper kits but want to be sure I did all possible before buying some of them.

The whole amplicon is supposed to be 507 bp long. My primers were designed using Primer Express Software, forward primer has length 19 nucleotides, %GC 63, Tm 60°C, reverse primer is 20 nucleotides long, %GC 60, Tm 60°C.
The whole amplicon has Tm 89°C, Ta 65°C, and as I already mentioned % GC 73.

I am using AmpliTaq Gold polymerase (1u into 20 ul reaction), 3.5 mM Mg2+, 1000 nM final primer concentration,
First I ran my “regular” PCR cycling conditions: 96°C for 13’, than 35 x 96°C for 20’’, Ta for 15’’, 72°C for 30’’, final extension 72°C for 1’. Ta: 58°C, 60°C and 62°C. All what I got was signal at annealing temperature 58°C: 1 weak band around 300 bp, another one around 1000 bp, and two very weak bands 200 bp and 400 bp…  

Then I changed cycling conditions the following way: 95°C 13’, than 35 x 96°C for 40’’ to allow complete denaturation, Ta for 40’’, 68°C for 2’ and final extension at 68°C for 10’. Ta: 54°C, 56°C and 58°C. Simultaneously I had serial dilution of primers: original 1000 nM, 600 nM and 200 nM – I have read that high primer concentration may lead to formation duplex (of course) that traps all the polymerase. And because I tried also 3 concentrations of DMSO (0%, 5% and 10%) that inhibits Taq polymerase I assumed the trick with lowering primer concentration may help…

And I got the following:

200 nM primers: 0
600 nM primers: terribly weak band around 110 bp when using 5% DMSO, but also in NTC so I assume this is just primer/dimmer/whatever mix
1000 nM primers: weak band around 110 bp everywhere (also in NTC) regardless Ta or  % of DMSO used.

So, please, any suggestions?
Thank you so much in advance!
Paja

Edited by Paja, 15 September 2004 - 01:14 PM.

[Paja]

Hmmm, it seems I scared you with detailed description of my troubles...  

[rassen]

Hi Paja,

Have you tried BD's Advantage GC PCR kit?  You can find info about the kit here http://www.bdbioscie...antage-GC.shtml

It boasts of amplifying template with 90% GC content. I myself have not used this kit, but have used the GC-melt, a component of it.

I forgot to mention that Sigma's JumpStart RedTaq makes a big difference.

Rassen

Edited by rassen, 15 September 2004 - 11:13 PM.

[kant0008]

Hi,
from personal experience AmpliTag is not a very good enzyme. At least in my hands it wasn't.
If I have problems with PCR using standard Taq (I use the one from Promega), then I go to Qiagen's HotStar. It works on most difficult templates. Also they provide something called Qsolution (some buffer I guess), which also can make a difference.
If you want to try with your Taq, add a little  formamide or glycerol (work similarly to DMSO) they help with amplifying GCrich templates.
Good luck

[iamm]

Hi,
I am the one suffering with this problem.. :(
however, when i tried with the resolution solut

[smesme]

Hi Paja,

try using the 7-deaza-G analogue which decreases G:G binding affinity.

Also, in some cases it helps just to increase denaturation temperature (96-97 c) and maybe also denaturation time. You may need to add a little extra TaqPol as it degrades faster at this temperature. This increases denaturation of the difficult templates.

Søren

Søren M. Echwald, MSc., Ph.D.
-----------------------------
Exiqon A/S
Bygstubben 9
DK-2950 Vedbaek
Denmark

www.ProbeLibrary.com
One Real-time PCR kit, which covers 38.565 genes

[dadada]

Hi,

I had almost exaclty the same problem: a promoter of 466 bp, 73% GC content. It worked beautifully with Pfx using de 10xPCR enhancer solution.