I'm trying to amplify up a very GC rich (87%) region for pyrosequencing PCR. I'm getting lots of non-specific amplification - I've tried varying concentrations of DMSO (10%, 5%, 2.5%) , betaine (1.5M) etc without much success. Another complication is that the DNA is bisulphite treated.
I've used the Qiagen Multiplex PCR kit to amplify up, as I've found this reliable for difficult PCRs previously and I get some product with 10% DMSO but it is terribly unreliable.
Does anyone have any hints or tips from their experience?
We have also had good success with decreasing the annealing time, this could be a "quick fix" and wouldn't require any special reagents.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727727/
Would that make any difference? The whole region is GC rich (at least 2kb in each direction)
Try to use a GC enhancer provided with the kit AmpliTaq Gold 360 DNA Polymerase!
Good Luck
Hi Andrew.
Try formamide, works great!
You can use both DMSO and formamide without problem. I don't have my lab-book here and I don't remember the exact concentations. Anyways, following papers can help:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC332902/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55707/
Another option is trying the AmpliTaq's Stoffel fragment, it's a little bit more expensive than normal Taqs, but works fine.
Good luck!
Grigorios: I've done an annealing temp gradient, my primer set is only any good at 56C
Eberhard: My DNA is highly fragmented, both as a factor of the bisulphite conversion process and because it is from FFPE tissues. I won't have any fragment sizes beyond 500 bases
Sebastián: I have some formamide in the fridge, that sounds like a great idea - thanks!
For a different approach read the my paper Palmirotta R, et al. Hum Mutat. 2012 May;33(5):895-8. doi: 10.1002/humu.22043. An AT-rich region in the APC gene may cause misinterpretation of familial adenomatous polyposis molecular screening.
Hi Andrew
I found this, try it and maybe your problems with PCR will disappear
http://www.ncbi.nlm.nih.gov/pubmed/18714299
Good Luck
We use Failsafe buffer J (or you can try the other buffers, I think there is a kit). They are made by Epicentre.
phusion highfidelity polymerase from finzymes is good. use the GCbuffer provided.u may also try DMSO gradient 3-10% along with this.
Are you getting any amplification? most of the time the concentration of dna after bisulfite conversion will be very less resulting in no amplification
Hi, use the mix in this paper, is a mixture of betaine, DMSO and DTT: A part of improve amplification of high GC content primer avoid aspecific products
High GC content can pose a problem in PCR.By using DMSO or other chemicals and higher annealing temperature one may be able to get aroung the problem. Also use of different DNA polymerases does help.
Moussaoui Wardi has set a very good article link.
GC-rich sequences are refractory to amplification. flanking primers allows amplification of short length amplicon, and large pathogenetic GC rich cannot be amplified using flanking primers. there are a lot of methods for detecthin of triplet repeat, such as:
Conventional PCR, Southern blot analysis, Triplet repeat primed PCR (TP PCR), repeat-primed fluorescent PCR, STR-primed PCR, CCG-chimeric primer, Cloning, Capillary electrophoresis, Repeat Expansion Detection Method (RED), direct identification of repeat expansion and cloning technique (DIRECT), RAPID cloning, hybridization signal intensity, fluorescence polymerase chain reaction (PCR) and GeneScan®, Single-Surface DNA Hybridization Assay with Enzyme-Linked Electrochemical Detection, Bisulfite-Converted DNA, XL PCR, mass spectrometry (MS).
also, there are important reagent for reducing effect of GC rich region, such as 7-deaza-dGTP , Betaine, dimethyl sulfoxide (DMSO), GC reagents.
I recommend Triplet repeat primed PCR (TP PCR) as method and substitution of 7-deaza-2-deoxyGTP for dGTP GC-melting reagents and DMSO.
fortunately i have prepared a power pointabout TP-pcr but its not completed. it might be helpful for you.
We have also had good success with decreasing the annealing time, this could be a "quick fix" and wouldn't require any special reagents.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727727/
Yes, it's right, annealing time is dependent to type of region and primer. You can play with it
I did PCR for a bisulfit treated Gc rich sequence. You can see my paper entitled:
Analysis of Promyelocytic Leukemia in Human Embryonic Carcinoma StemCells During Retinoic Acid-Induced Neural Differentiation.
Iran J Biotech. 2016 September;14(3): e1358
try this one
Article PCR-amplification of GC-rich regions: 'slowdown PCR'
For problematic and GC rich template amplifications I have used this reagent from Invitrogen, it worked well for me; PCRx Enhancer System, Invitrogen Cat. No. 11495-017; along with Platinum Taq DNA Polymerase, Invitrogen Cat. No. 10966-018. It will work even for 70% GC rich DNA, however needs little optimization by titration with the Enhancer concentration.
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