influence of MgCl2 in PCR - (Jan/18/2005 )
How does the MgCl2 concentration influence the amplification of PCR?
Is it possible that there is still amplification with 10mM MgCl2?
And what exactly is the MgCl2 titration?
afaik Mg2+ complexes the single nucelotides in your pcr reaction, and only Mg/nucleotide-complexes are the substrate for the dna-polymerase. so, no Mg2+, no amplification.
amplification is possible at 10mM Mg2+, in my oppinion. titration means you should try various concentrations of Mg2+ (e.g. 1, 3, 5, 7, 9 mM) for the same pcr reaction, since the concentration of Mg2+ influences the productivity and fidelity of polymerases. no pcr-product at 1mM Mg2+ and only smear at 10mM Mg2+ doesn't mean this reaction isn't going to work at all.
Thanks a lot for the quick response.
It really helped.
I have another question when it comes to the Polymerase.
I have also been varying the concentration of Taq Polymerase and I noticed that the Ct values are not really effected by an increase of concentration.( 1.25Units-31.25 Units)
Why is that the case?
I'm not 100% sure but I would think, that once you reached the optimal concentration of taq, more enzyme will have no beneficial effect, as possible "faster" amplification anymore. so, naively speaking, when every primer that annealyed to a template has a polymerase molecule attached to it, more taq molecules will idle around and do nothing...
The role of Mg++ in PCR is dual: promoting DNA/DNA interactions and forming complexes with dNTPs that are the actual substrates for Taq Polymerase. Typically, the dependence of the PCR yield on Mg++ is a bell curve with a broad maximum. When Mg++ is too low, primers fail to anneal to the target DNA. When Mg++ is too high, the base pairing becomes too strong and the amplicon fails to denature completely when you heat to 94°C. (J. F. Williams 1989 BioTechniques 7: 762. and D. L. Elsworth et al. 1993 BioTechniques 14: 214.)
Part 2: varying the polymerse concentration will not affect the Ct value. A typical PCR reaction starts with 1 e 4 copies of template/100 uL reacation. 1.25 Units of AmpliTaq equals 3.20 e 10 molecules of enzyme. 31.25 Units of AmpliTaq equals 8.01 e 11 molecules of enzyme.
At Ct, there is more enzyme than template regardless of the number of Units you are adding. Amplification is exponential until the number of molecules of enzyme euals that of template.
After 25 cycles of amplification, ABI expects 1.8 e 12 molecules of product.
The thermodynamic driving force for normal PCR is the molar excess of reagents with respect to template. Consumption of reagents other than the primers is not an issue. Limiting amounts of Taq DNA polymerse generates more artifacts than limiting primer.
More than 4 Units/100 µL reaction is not recommended as it can result in increased non-specific amplification and reduced yield of the desired product due to the intrinsic 5’ > 3’ exonuclease activity of Taq.
(M. J. Longley et al. 1990 NAR 18:7317, A. D. Sardelli 1993 Amplifications 9:1 and D. A. Bell and D. M. DeMarini 1991 NAR 19:5079.)
I thought Taq doesn't have an exonuclease-activitity - or is it only the 3' -> 5' exonuclease activity thats missing?
What are "Ct values"?
The intrinsic 5’ > 3’ exonuclease activity of Taq is what makes the TaqMan Assay possible. See http://www.vetscite.org/issue1/tools/leute_2_0800.htm and other websites for an explanation.
Ct values are obtained when performng QPCR on a real-time thermal cycler. "The point on the curve in which the amount of fluorescence begins to increase rapidly, usually a few standard deviations above the baseline, is termed the threshold cycle (Ct value). The plot of Ct versus template is linear, thus a comparison of Ct values between multiple reactions enables one to calculate the concentration of the target nucleic acid. The slope of this line provides a measure of PCR efficiency." (http://www.realtimeprimers.org/)