qPCR is an acronym for quantitative PCR and is a DNA amplification technique used to measure the concentration of specific target DNA molecules. It is used in applications such as medical diagnostics, pharmacogenomics, biotechnology, and forensics. The quantitative PCR machine allows users to control various parameters during analysis. quantitative PCR uses two different primers that are labeled with fluorescent dyes after hybridization. If the target sequence is present, both primers hybridize and fluorescence is detected by the probe at excitation wavelengths of 480 nm (green) or 535 nm (red). If the target sequence is not present, one primer hybridizes while the other does not bind. In this case fluorescence from only one dye will be detected at either green or red wavelength. This creates a positive or negative readout based on the absence or presence of target locus of interest.
DNA amplification
DNA amplification is the first step of qPCR machines. The goal is to produce enough copies of the target DNA sequence so that it can be measured accurately. The polymerase chain reaction (PCR) is the most common method for achieving this. PCR uses two short strands of DNA as primers that hybridize to the ends of the target DNA. Next, DNA polymerase initiates the synthesis of a new DNA strand by adding nucleotides to the 3' end of each primer. The polymerase adds nucleotides one at a time to each DNA strand. Once the growing DNA strand has reached the other end of the target DNA, the strands are separated, and new primers are added to the reaction to start the process over again. The process is repeated many times to increase the number of copies of the DNA sequence.
Cycle of qPCR machine
A qPCR machine consists of a thermal cycler that manages the cyclic process of heating, cooling, and sample addition. The first step is to denature the DNA. This is done by heating the sample to 94 degrees Celsius to separate the two strands of DNA. When the temperature is lowered to 60 degrees Celsius, the primers are added and hybridize with the target DNA. The DNA polymerase is added next. After a short heating and cooling cycle, the DNA polymerase will start adding nucleotides to the 3' end of the primers. qPCR machines have an internal detection system that monitors the amplification by measuring the fluorescence of the sample. This is done at specific cycles in the amplification process. The cycle time is the time between sample additions. Cycle times are usually between 10 and 30 seconds.
Quantification with data analysis
The number of DNA molecules in a sample is determined by the amount of fluorescent signal detected during the amplification cycle. The data is analyzed by plotting the amount of fluorescence against the cycle number. The graph obtained is used to determine the number of recombinant molecules present in the sample. The qPCR machine will have a control program to determine the number of amplification cycles required to get a standard curve. The standard curve is used to calculate the number of molecules in the sample.
Limitations of the qPCR machine
qPCR is used to quantitate DNA molecules in a sample. The number of molecules in the sample is determined by the number of DNA molecules amplified. The number of DNA molecules in a sample depends on the number of initial molecules in the sample and the rate of loss of those molecules through degradation, mis-amplification, or inactivation of the DNA polymerase. The reliability of qPCR results is dependent on the accuracy of the initial sample preparation, the specificity of the primers, the efficiency of the DNA polymerase, and the accuracy of the data analysis. One of the limitations of the qPCR machine is that it is affected by contamination. Contamination refers to the presence of foreign DNA in the sample. The presence of foreign DNA will cause inhibition or mis-amplification of the sample nucleic acid.
The results of qPCR are affected by all the aspects of the sample preparation and PCR, as well as data analysis. The results can be verified by the melting temperature of the PCR product. The melting temperature is the temperature at which two strands of DNA are released from each other. This can be compared to the expected melting temperature of the known DNA sequence. The qPCR machine is a powerful tool that can be used for DNA quantitation and analysis.
