Quantitative Polymerase Chain Reaction (qPCR) measures DNA amplification in real-time, allowing for the precise quantification of a specific DNA sequence in a sample. SYBR Green dye is a common and economical choice for monitoring this process. This approach is widely used in laboratories for applications like gene expression analysis and pathogen detection. This guide details the steps required to execute a SYBR Green qPCR protocol.
The Mechanism of SYBR Green Dye
The utility of SYBR Green in qPCR comes from its unique interaction with DNA. SYBR Green is a fluorescent molecule that functions as an intercalating dye, inserting itself into the minor groove of double-stranded DNA (dsDNA). This binding allows the reaction to be monitored in real-time.
When the dye is free in the solution, its fluorescence is minimal, close to the background signal. Once the dye binds to newly synthesized dsDNA, its fluorescence increases dramatically. The qPCR instrument detects this significant increase in light emission to track DNA amplification during each cycle.
Because the dye binds to any dsDNA, the fluorescence signal is directly proportional to the total amount of dsDNA present. This non-specific binding makes SYBR Green cost-effective as it does not require a custom-designed probe for every target. However, this lack of specificity means the dye will also bind to unwanted products, such as primer-dimers or non-target sequences, necessitating an additional quality control step after the reaction.
Assembling the Reaction Mixture
Successful qPCR depends on the preparation of the reaction mixture, which contains all components required for amplification. The DNA template is the starting material, which can be complementary DNA (cDNA) derived from RNA, or genomic DNA (gDNA). A low concentration of template is typically required, such as 1–10 nanograms (ng) of cDNA or 10–100 ng of gDNA per reaction, to ensure accurate quantification.
The reaction also requires forward and reverse primers. These synthetic oligonucleotides define the specific region of the template DNA that will be amplified. Primers are typically used at a final concentration between 300 to 800 nanomolar (nM), with 400 nM being a common recommendation. The simplest preparation method uses a commercial SYBR Green Master Mix, which combines the remaining components in a single, concentrated solution.
The Master Mix contains:
- Thermostable DNA polymerase (often a hot-start version to prevent non-specific amplification before cycling begins).
- Deoxyribonucleotide triphosphates (dNTPs) for building new DNA strands.
- A reaction buffer to maintain optimal pH.
- The SYBR Green dye.
The final reaction volume is often 20 microliters ($\mu$L), though volumes as low as 10 $\mu$L are feasible. It is standard practice to include a 10% overage when preparing a bulk master mix for multiple reactions to account for minor volume errors.
Setting the Thermal Cycling Program
Once the reaction mixtures are loaded into the instrument, the thermal cycler executes a program consisting of several stages. The first step is Initial Denaturation, a single heating step typically set at 95°C for 2 to 10 minutes. This high heat separates any existing double-stranded template DNA and activates the hot-start DNA polymerase.
The Cycling Stage follows, consisting of 40 repeated cycles. Each cycle starts with a brief Denaturation step at 95°C for 5 to 30 seconds to separate all double-stranded DNA products. The temperature then drops to the Annealing step, usually between 55°C and 65°C for about 30 seconds, allowing primers to bind to their complementary sequences.
The final step is the Extension phase, where the DNA polymerase synthesizes new DNA strands starting from the annealed primers. Many protocols use a two-step cycling method where Annealing and Extension are combined at 60°C for 30 to 60 seconds. Data Acquisition—measuring SYBR Green fluorescence—is performed during or immediately after this extension phase, when the maximum amount of new dsDNA product is present.
Interpreting Quantification and Specificity
After thermal cycling, the data is analyzed to determine the quantity and specificity of the amplified product. The primary metric for quantification is the Quantification Cycle, or Ct value. This represents the cycle number at which the fluorescence signal crosses a predetermined threshold set above the background level. Samples with a higher initial amount of template DNA reach this threshold earlier, resulting in a lower Ct value, compared to samples with less starting material.
To translate Ct values into quantifiable amounts, a standard curve is often generated using known concentrations of the target DNA. This allows for either absolute quantification (determining the exact copy number) or relative quantification (comparing target DNA amounts between samples). Because SYBR Green binds to any double-stranded DNA, a mandatory post-run check for product quality is required.
This quality control step is the Dissociation Curve, or Melt Curve, analysis. The instrument slowly increases the temperature from approximately 60°C to 95°C while monitoring the decrease in fluorescence. As the dsDNA product denatures into single strands, the SYBR Green is released, causing a sharp drop in fluorescence. Plotting the rate of this fluorescence change against temperature results in a peak representing the melting temperature ($\text{T}_\text{m}$) of the amplicon. A single, sharp peak confirms that only the intended target product was amplified; multiple or irregular peaks indicate non-specific products or primer-dimers.