One of the great features of PCR is its excellent sensitivity as we know. And many articles describe real-time qPCR as an added leap forward in that sensitivity – to the point where it has become a standard feature of a new assay description
Indeed, I’m currently developing a new qPCR assay to replace a published nested PCR – and showing the two assays have equivalent sensitivity.
However, do I believe qPCR is an inherently more sensitive technique than conventional PCR (in this description, meaning standard agarose gel electrophoresis)? No, I don’t. The issue for me? The comparison is usually apples vs oranges.
Older gel-based assays in the comparisons are often amplifying larger products and with different enzyme systems (i.e. non hot-start) which may be less sensitive. A design rule of qPCR assays traditionally has been to use small amplicon sizes and of course, it’s a lot easier to complete 100 metres (100bp) in a sprint than 1km (1kb). Even among qPCR chemistries, “but it has a probe in the reaction” is sometimes used as a further claim of increased sensitivity of something like a hydrolysis probe (to be MIQUEy about it) over a dye-based assay such as SYBR Green. Yes, a particular assay may show this increased sensitivity but many SYBR assays are more sensitive than their probe assay version.
This topic of conventional PCR vs qPCR sensitivity was the subject of an excellent ’round table’ discussion1 and was followed by a paper dedicated this issue2
So what did I leave out of the description of the assay development at the beginning of this note? Well, the published nested PCR uses Taq, has a 1200bp first round assay and an almost 900bp second round. I’m comparing that to a 75bp real-time qPCR assay.
So what would happen if I ran the 75bp assay on a conventional gel-based platform using similar enzymes and buffer systems as the qPCR master mix? Well, I did – and in this instance, the 75bp assay showed equivalent sensitivity by bands on a gel as fluorescent curves on a screen. However it is worth repeating, in this instance.
But hey, a 45 minute qPCR beats a nested PCR protocol for me any day! Tell us about your examples of PCR sensitivity comparisons? Do you feel one platform or chemistry is more sensitive?
References 1Experts Roundtable: Real-time PCR and microbiology. In Real-time PCR in microbiology: from diagnosis to characterisation? ed: Ian M. Mackay. Caister Academic Press 2J. Clin. Microbiol. 2008 Jun;46(6):1897-900.
PCR screening is great for identifying potential positive clones after cloning/subcloning. However, you’ll still want to confirm that the DNA sequence is correct for any positives identified. The easiest and quickest way to do this is by restriction digestion. This guide will tell you about the theory and practice of using this technique. The Theory […]
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