qPCR: Quantifying Nucleic Acids with Real-Time PCR Technology

Quantitative PCR (qPCR), also known as real-time PCR, is a cornerstone technique in molecular biology. This blog post serves as a technical resource, dissecting the principles, applications, and advancements of qPCR.

Core Principles of qPCR:

qPCR leverages the same fundamental tenets of conventional PCR: primer-directed amplification of a specific DNA fragment using Taq polymerase. However, qPCR integrates a fluorescent reporter molecule for real-time quantification of the amplicon during each cycle.

Detection Methods:

  • TaqMan Probes: These hydrolysis probes exhibit high target sequence specificity. During extension, Taq polymerase cleaves the probe, releasing a fluorescent reporter, signifying successful amplification. Fluorescence intensity directly correlates with the initial amount of target DNA.

taqman; qPCR; RTPCR; 4466334

  • SYBR Green Dye: This non-specific dye binds to all double-stranded DNA. As amplification progresses, more DNA becomes available for SYBR Green binding, resulting in a progressively stronger fluorescent signal.

Syber Green; qPCR; RTPCR ; 3355/56

Optimizing qPCR Experiments:

  • Primer Design: Stringent in silico primer design is essential for specific and efficient amplification. Utilize PrimerBLAST ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) for optimal primer design.
  • Standard Curve Generation: A standard curve, constructed using known concentrations of the target DNA, enables accurate quantification of the unknown sample based on its Ct (cycle threshold) value – the cycle at which fluorescence intensity surpasses a predefined threshold.
  • Control Implementation: Employing positive and negative controls ensures reaction specificity and absence of contamination.

Advancements in qPCR Technology:

  • Digital PCR: This innovative technique partitions the sample into numerous microscopic reactions, enabling absolute quantification of even rare target molecules.

Visualized workflow of ddPCR assay. QX200 Droplet Digital PCR System (Bio-Rad) consists of automated droplet generator, thermal cycler, and droplet reader. Positive droplets (blue or green) and negative droplets (gray) can be finally analyzed with the QuantaSoft software, where thresholds should be set between the negative and positive droplets.

  • Multiplex qPCR: This method facilitates the simultaneous amplification and detection of multiple targets within a single reaction, enhancing efficiency and reducing costs.
  • High-Resolution Melt Curve Analysis (HRM): HRM analyzes the melting behavior of PCR products, enabling the detection of single-nucleotide polymorphisms (SNPs) and mutations.

Principle of high-resolution melting curve analysis (HRM) for detection of allelic expression imbalance. A single labelled fluorescent probe is designed with complete complementarity to one allele of the exonic SNP chosen as marker, while mismatching the other allele. Following an asymmetric PCR reaction in presence of the probe, HRM analysis allows the alleles in heterozygous individuals to be distinguished by differences in their melting temperatures (Tm), with a fluorescent signal correlated to the relative abundance of each transcript. The Allele 2/Allele 1 ratio is calculated as h2/h1.

Diverse Applications of qPCR:

  • Diagnostics: qPCR plays a central role in diagnosing infectious diseases, identifying genetic disorders, and monitoring minimal residual disease (MRD) in cancer patients.
  • Gene Expression Analysis: qPCR allows for the quantification of mRNA levels, providing valuable insights into gene regulation and cellular processes.
  • Gene Quantification in Transgenics: qPCR is instrumental in quantifying the copy number of transgenes integrated into the genome of genetically modified organisms.
  • Food Science: qPCR can detect foodborne pathogens, allergens, and GMOs in food products.

An overview figure of qPCR application in different fields

The Future of qPCR:

The future of qPCR is promising, with ongoing advancements aiming to achieve even greater sensitivity, specificity, and multiplexing capabilities. Emerging technologies like microfluidics and next-generation sequencing have the potential to further revolutionize this cornerstone technique in molecular biology.

Learn more about the differences between TaqMan and SYBR Green in this video:

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qPCR: Quantifying Nucleic Acids with Real-Time PCR Technology
Gen store May 22, 2024
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