Quantitative Polymerase Chain Reaction (qPCR) has revolutionized the field of genetic analysis by providing a method to selectively amplify and quantitatively detect specific regions of DNA or complementary DNA (cDNA). This technique, sometimes known as quantitative real-time PCR, offers improved sensitivity and specificity compared to traditional PCR, allowing for accurate quantification of target sequences. In this article, we will explore how qPCR works, its experimental requirements, data analysis methods, and various applications of this powerful technique.
What is qPCR?
The qPCR technique relies on oligonucleotide primers that flank the region of interest to amplify the sequence using a DNA polymerase enzyme. Through repeated cycles of amplification, the number of copies of the target region exponentially increases. The amplification process is tracked using an intercalating dye or a sequence-specific probe that emits fluorescence detected by a qPCR machine. This information is then plotted on an output graph for analysis.
What’s the difference between qPCR and RT-qPCR?
RT-qPCR, or quantitative reverse transcription polymerase chain reaction, differs from qPCR in that it uses RNA as the starting material instead of DNA. This necessitates a reverse transcription step to convert RNA to cDNA before proceeding with the regular qPCR amplification process. RT-qPCR can be conducted in either one-step or two-step protocols depending on whether reverse transcription occurs in the same tube as amplification or separately.
How does a qPCR machine work?
A typical qPCR machine consists of a heated block with a lid to facilitate rapid temperature transitions enabling DNA template amplification. Additionally, a fluorescent source and fluorometer are included in the apparatus, which excites fluorophores and detects fluorescence emitted during cycles of qPCR amplification. The data collected is recorded by a computer connected to the machine and interpreted using specialized software based on user-defined settings.
Example of a qPCR protocol
A standard qPCR reaction includes DNA or cDNA as the template for amplification, sequence-specific primers flanking the target region, DNA polymerase enzyme for synthesizing new copies, deoxynucleoside triphosphates (dNTPs) as nucleotide building blocks, probes or dyes for detection, and buffers for optimizing reaction conditions.
1) A master mix containing all components except the template is prepared and aliquoted into wells.
2) Information about well contents, dye/probe chemistry used, standards/controls included, and cycling conditions is provided to the computer.
3) The template is denatured by heating to produce single-stranded DNA (ssDNA).
4) Sequence-specific primers bind to their target sequences (annealing), followed by primer extension where complimentary bases are added.
5) Fluorescence generated during amplification is detected and recorded.
6) Melt curve analysis can be performed after completion of amplification to assess product dissociation characteristics.
How to design primers for qPCR
Several key considerations need to be taken into account when designing primers for qPCR:
– Primers should flank the target region specifically.
– Primer sequences must be unique to avoid off-target binding.
– Melting temperatures should ideally range between 60-64°C for efficient amplification.
– GC content can impact primer specificity and melting temperature.
– Avoid secondary structures or dimerization in primer sequences.
Dye-based qPCR vs Probes
Dye-based assays use intercalating dyes that emit fluorescence when bound to dsDNA but lack specificity compared to probe-based assays. Probes offer higher specificity through sequence-specific binding mechanisms such as hydrolysis probes or molecular beacons which release fluorescence in presence of target sequences.
qPCR controls
Appropriate controls including no template control (NTC), negative control lacking target regions, positive control containing known targets are essential for interpreting results accurately. An endogenous control can also provide normalization data across samples.
qPCR Analysis – Ct value, melt curve analysis, standard curve
Critical terms such as Baseline, Normalized Reporter (Rn), Threshold Cycle (Ct), Delta Rn (ΔRn), Threshold are vital in understanding qPCR output data. Amplification plots depict fluorescence intensity over cycle numbers per well. Absolute and relative quantitation methods are used depending on the nature of the experiment.
In conclusion, qPCR has diverse applications ranging from gene expression studies and diagnostics in infectious diseases to NGS library quantifications and contaminant detection. With its sensitivity, accuracy, and ability for absolute quantitation across various samples, qPCR continues to be at the forefront of genetic research techniques.
