Method of the Month: SYBR Green qPCR
February 2024
Background
What happens when you think you get sick and need to be tested for a specific disease? Doctors can start by taking a swabbing for a sample and determining the type of foreign DNA in this sample. There are various ways to test DNA, one of them being real-time quantitative PCR, aka qPCR. More specifically, this could be a method called SYBR Green qPCR.
SYBR Green qPCR measures DNA amplification as a PCR reaction progresses. By adding a fluorescent dye to the sample, the qPCR machine, a specialized thermocycler with fluorescence detection modules, can measure the colored light emitted and graph it for data interpretation.
Method
The method begins with a basic PCR technique using a DNA or cDNA sample. cDNA is DNA that has been reverse-transcribed from an RNA sample. Obtaining this cDNA is part of the initial assembly step, in which scientists mix an RNA sample from a patient with DNA polymerase, reverse transcriptase, and a buffer.
During the assembly step, several other components are also added to the cDNA: primers, Taq polymerase, Base Pairs, a template strand of DNA, and SYBR Green. The primers, Taq polymerase, and base pairs are specifically selected for the target DNA and work together to amplify all the DNA in the sample. SYBR green is a dye produced largely by ThermoFisher that binds only to the minor grooves of the double-stranded target DNA.
Once the complete sample is assembled, the qPCR process can proceed in three steps: Denaturation, Annealing, and Extension. All of these steps occur in the thermocycler.
Denaturation is the process of separating double-stranded DNA. In the thermocycler, this occurs at around 95 degrees Celsius. Two rounds of this occur, one round lasting five minutes and the second lasting thirty seconds.
Annealing is the process of the primers and polymerases attaching to the single strands of DNA. This happens at around 56 degrees Celsius for thirty seconds.
Extension is the process of copying the target DNA by adding nucleotides onto the ends of the primers. This occurs at around 72 degrees Celsius for five minutes. At the end of this step, the SYBR green dye binds to the double-stranded DNA.
Throughout this process, the thermocycler measures the amount of fluorescent dye emitted. The intensity of light measured quantifies how much target DNA is in the sample. All of this is displayed on a graph to interpret later.
The main downside to SYBR Green qPCR is the possibility of amplifying and monitoring DNA that was not target DNA. Since the SYBR green dye is non-specific, attaching to any double-stranded segment of DNA, it is possible that foreign DNA could interfere with testing. This problem can be mitigated by repeating the test multiple times to ensure accurate melting curves (graphs of fluorescence). An incorrect melting curve means that the target DNA is contaminated and the results from that test are faulty.
Despite this downside, SYBR Green is still one of the most commonly used laboratory tests for infected samples. The results of this method are an easy, affordable, and fairly accurate way to determine the progression and identity of diseases such as COVID-19 or HPV.
For more information on SYBR Green qPCR, check out the following link!
