Real-time PCR
Real-time PCR, also known as quantitative PCR (qPCR), integrates the amplification of a targeted DNA sequence with the precise quantification of its concentration within the reaction. This PCR based technique with specific primer and fluorescence dye has the ability to quantify the amount of gene product in the sample during the PCR cycling reaction. This method facilitates the determination of the initial template concentration, so it is a commonly used DNA copy number, viral load, SNP detection. When coupled with reverse-transcription PCR, qPCR emerges as a potent method for measuring mRNA expression levels.
Real-time PCR enables continuous monitoring of the amplification process as it reaction occurs by incorporation of fluorescent probes or dyes into the reaction mixture. The instrument measures emitted fluorescence at each cycle of the PCR reaction and calculates the amount of amplified DNA target based on the bound probes or dyes. When the DNA amplification progresses at each cycle, the amount of fluorescence increases proportionally to the amount of amplified DNA, enabling precise quantification
In real-time PCR reaction, usually, it takes around 40–45 cycles to detect the slightest amount of target nucleic acid. Illustrated in below figure, the curve typically starts from a baseline fluorescence level and exhibits an exponential increase as the target nucleic acid is amplified during the early cycles of PCR, this exponential phase is followed by a plateau phase, where fluorescence growth slows. Crossing a preset threshold marks the Ct (Cycle threshold), The earlier the Ct value occurs, the higher the initial presence of target nucleic acid in the sample.
During the PCR reaction, fluorescent probes or dyes are incorporated into the reaction mixture. There are different types of fluorescent probes used in real-time PCR, such as TaqMan probes, SYBR Green dye, and molecular beacons. These probes are designed to emit fluorescence when they bind to the amplified DNA target.
SYBR Green
The widely used bye-based detection systems is SYBR Green, which is a fluorescent dye that binds to double-stranded DNA (dsDNA) specifically. During the PCR reaction, SYBR Green binds into the newly synthesized dsDNA amplicons. As more dsDNA is produced, the fluorescence signal from SYBR Green increases proportionally. The instrument continuously detects this fluorescence signal at each PCR cycle. The amount of fluorescence detected correlates with the amount of amplified DNA present in the reaction at each cycle. SYBR Green-based real-time PCR assays are versatile, cost-effective, and suitable for a wide range of applications, including gene expression analysis, genotyping, pathogen detection, and quantitative PCR. Overall, SYBR Green-based real-time PCR is a powerful and widely used tool in molecular biology research and diagnostics.
AICON-BIO offers SYBR Green-based Master Mix reagents that are commonly used in laboratories for real-time PCR experiments.
GS AntiQ qPCR SYBR Green Master Mix
GS AntiQ qPCR SYBR Green Fast Mix(Universal)
GS AntiQ qPCR SYBR Green Fast Mix(Low ROX Premixed)
TaqMan probes
Real-time PCR assays also utilize probe-based detection systems, with one of the most commonly employed methods being TaqMan probes. Unlike SYBR Green, the probe specifically binds to the target DNA sequence in the sample and These probes utilize a fluorophore at one end of the DNA oligonucleotide and a quencher at the other (see figure below). During the PCR cycle, the Taq polymerase enzyme degrades the probe through its 5′ to 3′ exonuclease activity, so the probe will separate from the quencher, when the probe release to the solution, it has fluorescence signal. This signal is proportional to the amount of target DNA present in the reaction at each PCR cycle. TaqMan probes offer high specificity and sensitivity, as they only fluoresce when bound to the target sequence. In addition, they enable the simultaneous detection of multiple targets within the same reaction. TaqMan probe-based real-time PCR assays are widely used in various applications, including gene expression analysis, SNP genotyping, pathogen detection, and viral load quantification. They provide reliable and accurate quantification of nucleic acids, making them an essential tool in molecular biology research and clinical diagnostics.
AICON-BIO offers qPCR Probe Master Mix reagents that are commonly used in laboratories for real-time PCR experiments.
GS AntiQ qPCR Probe Master Mix
AICON-BIO UnionScript First-strand cDNA Synthesis Mix for qPCR
Additional Important Considerations
Before conducting any PCR experiment, proper DNA extraction is vital to ensure reliable and accurate results. This process involves breaking down cellular barriers to release DNA or RNA, followed by purification to remove contaminants. AICON-BIO offers DNA extraction kits designed to streamline this process, ensuring high-quality DNA/RNA isolation for accurate PCR/qPCR analysis. These kits are widely used in research, diagnostics, and biotechnology applications, providing researchers with the confidence they need for successful PCR experiments.
AICON-BIO™ Bacteria Genomic DNA Extraction Kit
AICON-BIO™ Animal Tissue/Cell Total RNA Extraction Kit
AICON-BIO™ Animal Tissue/Cell/Blood DNA Extraction Kit
AICON-BIO™ Plant Total RNA Extraction Kit
AICON-BIO™ Plant DNA Extraction Kit
AICON-BIO™ Endotoxin-Free Plasmid Mass Extraction Kit