Control plasmids carrying a specific target sequence are very useful in PCR analyses, e.g. as positive control or as calibrant. Nowadays, customised plasmids can be easily purchased from service companies by order.
Colleagues using control plasmids often recommend and say, that linearisation is required for reliable amplification in PCR. What is the reason for this? Does anyone know a scientific paper describing this issue?
I don't ever linearize my plasmids, and I use them as positive controls for qPCR. I wonder if your colleagues are worried about secondary structure? Linearization won't fix that, though.
This sounds like scientific superstition to me- I have my own weird biases for various techniques as well. ;)
It is not a matter of control plasmids but a general issue when plasmids are used for quantitative PCR.
Intact plasmid DNA is supercoiled but PCR amplification works optimal with relaxed DNA. After linearization the DNA is relaxed. The difference in conformation between supercoiled and relaxed DNA can make a difference in the results of real-time PCR quantification. Therefore, to be on the safe side, it is better to linearize the plasmids routinely. However, based on the results in my own lab, I can say that the differences between linearized and supercoiled DNA are only minor.
Here is a paper on this topic:
Chen J, Kadlubar FF, Chen JZ (2007) DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair. Nucleic Acids Res. 2007; 35(4):1377-88.
In our work we have not seen any differences between the results with linearised or non-linearized forms , however, it may be plasmid size and type dependent, also different method of preparation generate different ratios of plasmid forms (supercoiled, open circle etc.).
It should be verified case by case, if one need to save time for cleavage and running of subsequent controls of digest.
best regards
Jarka
Thanks for your attention to my question.
Maybe I was not precise enough: I am using plasmid DNA dilution series for validation of the limit of detection (LOD)and my question should read: Is it better to linearise the plasmid for this validation experiment?
I realised now, that a shift of Ct (and amplification) occurs if circular palsmids are used as calibrators. But the question is, whether the absolute LOD (in copy number) is worse if cicular plasmid DNA are used instead of the linearised form.
Apart from the Chen et al. (2007) publication, I found 2 other papers providing evidence for the fact that the conformation of plasmid DNA causes bias in real-time PCR based quantification. But the papers say nothing about worsening the absolute LOD. See:
Lin C-H et al. (2011) Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay. PLoS ONE 6(12): e29101. doi:10.1371/journal.pone.0029101
Hou Y et al (2010) Serious Overestimation in Quantitative PCR by Circular (Supercoiled) Plasmid Standard: Microalgal pcna as the
Model Gene. PLoS ONE 5(3): e9545. doi:10.1371/journal.pone.0009545
What's your assumption? Any idea or scientific paper you have concerning LOD?
Kind regards
Lutz
I would assume that the described difference between relaxed and supercoiled DNA affects not only quantification but also the limit of detection (LOD) and also the limit of quantification (LOQ). However, I would only expect minor effects. I guess, you want to compare (linear) genomic DNA with (circular/linearized) plasmid DNA.
Below is a paper by Milbury et al. in which the authors suggest linearization of the plasmid for determination of LOD (but they don’t discuss this fact). The paper deals with digital PCR (dPCR), not with standard qPCR, but the effects should be the same, even if dPCR is more precise than qPCR and, therefore, also more prone to error.
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Milbury et al. (2014) Determining lower limits of detection of digital PCR assays for cancer-related gene mutations. Biomolecular Detection and Quantification, 1 (1) 8–22.
Both relaxed and supercoiled forms of DNA are efficent in PCR assay but relaxed form gives better results.
Hi Lutz Grohmann,
Very pertinent question raised by you. We can get answer of this question in Journal of General Virology (2004), 85, 3383–3388 DOI 10.1099/vir.0.80039-0 page 3387 paragraph two with proper explanation. Thanks.
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