04 - Put the M into STEM: Quantitative Techniques for Biotechnology
many cycles before the fluorescence can be detected (high Cq). Conversely, if the target DNA is abundant in the starting material, the fluorescence will increase to measurable levels relatively quickly (low Cq). The exact number of target DNA molecules in a sample can be determined by comparing its Cq value to those from samples of known concentration using a standard curve. To create a standard curve, a DNA template is diluted over several orders of magnitude (for example, from microgram to picogram quantities), and the Cq is determined for each sample (Figure 4). Plotting Cq on the y-axis and the log 10 of the known DNA concentration on the x-axis results in a straight line. The equation of this line is used to determine the starting concentration of our unknown sample by substituting the measured Cq value into the equation.
APPLICATIONS OF QPCR TECHNOLOGY Real-time PCR is a commonly used technique in the both the research and the diagnostic laboratory because it is fast, sensitive, and requires less material and technical skill than traditional techniques like Northern or South- ern blotting. For example, microbiologists commonly use qPCR to both identify and quantify microorganisms in food and water samples. Physicians may use qPCR to establish the exact level, or titer, of a particular bacteria or virus present in a specific patient sample. Because qPCR can differentiate between specific strains of a particular pathogen (like influenza A and B), it is a powerful diagnostic and informational tool for health professionals. qPCR can also be used to determine the extent that a specific gene is “turned on,” i.e., how much RNA is
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being transcribed from that particular gene. First, the RNA must be converted into DNA before it can be quantified. This process, known as reverse transcription, creates a complementary DNA (cDNA) se - quence from an RNA template. Once the cDNA is produced, qPCR can be used to quantify the amount of cDNA — and, by extension, the amount of original RNA — present in the sample. This is very useful when biotechnology companies need to determine the effects of experimental medications on specific biological pathways. For these reasons, qPCR has become an essential technique for today’s scientists. The experiment we are using in this workshop is adapted from EDVO-kit #103, Principles of PCR (https:// www.edvotek.com/103). This experiment explores the principles of DNA amplification using samples with increasing quantities of DNA. Using agarose gel electrophoresis, we will observe the relationship between cycle number and amount of DNA present in a sample at the beginning of qPCR. Students will perform data analysis to support this observation.
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Standards Unknowns
Correlation Coefficient: 0.999 Slope: -3.488 Intercept: 39.204 Y=-3.488 X=39.204
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