04 - Put the M into STEM: Quantitative Techniques for Biotechnology
Introduction You’ve planned the experiment and collected data, now let’s discuss the best way to analyze it. In this workshop, we’ll use PCR and ELISA to bring quantitative data analysis and statistics to the lab. Mastery of these skills is crucial to prepare students for careers in biotechnology and STEM. Step 1: qPCR PRINCIPLES OF QUANTITATIVE PCR
The products of conventional PCR are most often analyzed by agarose gel electrophoresis. If a target DNA sequence is present in the starting material and is amplified by the PCR reaction, a band of DNA will be visible when the gel is stained (Figure 1). Therefore, conventional PCR coupled with electro- phoresis produces a “yes/no” qualitative result. In contrast, quantitative PCR (qPCR, also known as “real-time” PCR) can determine the exact amount of target DNA in the starting material by measuring the accumulation of DNA as the reaction progresses. Electrophoresis is not required because amplifica - tion and quantitation of the DNA occur simultaneously. Similarly to conventional PCR, each cycle of real-time PCR doubles the amount of the DNA in the sample. Mathematically, this doubling can be expressed as an exponential relationship – if we begin with a starting copy number of m, then after n cycles, we will have m x 2 n copies of our DNA target. For example, if we start with one copy of our target, we will have two copies after the first PCR cycle, four after the second PCR cycle, eight after the third PCR
Figure 1 Results of a conventional PCR experiment as analyzed by agarose gel electrophoresis.
cycle, and so on. After many cycles (regardless of the amount of DNA present in the starting material) the amount of DNA produced reaches a maximum where a product curve flattens out, known as the plateau (Figure 2). This leveling off of the curve is due to the depletion of reaction components like primers and nucleotides and the loss of Taq polymerase activity. In contrast to conventional PCR, real-time PCR samples contain special fluorescent dyes that produce light when bound to double-stranded DNA (Figure 3). This allows the user to measure the amount of DNA in a sample as it is being synthesized. The amplification is performed in a thermal cycler that can
excite the fluorescent molecules and detect the signal that they produce. A measured increase in fluorescence directly relates to an increase in the amount of ampli- fied DNA in the sample. In early cycles of PCR, fluorescence is low because there is not a lot of DNA present in the sample. As the number of cycles increases, the PCR product accumulates, and so fluorescence increases. The cycle during which the fluorescence reaches a set threshold is known as the quantification cycle, or Cq (Figure 4). As the concentration of DNA template increases, the number of cycles it takes to reach the Cq decreases. For example, if the DNA template is present in the sample in low levels, it takes
Exponential phase
Non Exponential plateau phase
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Threshold line
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Figure 2 Graph showing the exponential phase and plateau phase of PCR.
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