TPrinciples of qPCR
qPCR works on the basic principle of traditional PCR, where a specific segment of DNA is exponentially amplified using DNA polymerase and primers. However, in qPCR, the amplification process is monitored in real-time using fluorescent dyes or probes that emit fluorescence in proportion to the amount of DNA generated.
There are two primary types of fluorescence-based detection methods in qPCR:
- DNA-binding dyes (e.g., SYBR Green): These EBay Number dyes bind to double-stranded DNA and emit fluorescence upon binding. The fluorescence intensity increases as the amount of double-stranded DNA increases during the PCR cycles.
- Fluorescently labeled probes (e.g., TaqMan probes): These probes are oligonucleotides labeled with a fluorescent reporter dye and a quencher. When the probe hybridizes to the target DNA and is cleaved by the DNA polymerase during the PCR extension phase, the reporter dye is released from the quencher, emitting fluorescence.
Experimental Setup and Protocol
A typical qPCR experiment involves the following steps:
- Sample Preparation: Extract and purify nucleic acids from the biological samples. The quality and quantity of the extracted nucleic acids are Turkey Phone Number crucial for accurate qPCR results.
- Reaction Setup: Prepare the qPCR reaction mix, which includes the DNA template, primers, DNA polymerase, dNTPs, buffer, and the fluorescence dye or probe.
- Thermal Cycling: Load the reaction mix into a qPCR machine, which performs the thermal cycling. The machine records the fluorescence intensity at the end of each cycle.
- Data Collection: The qPCR machine collects and displays the fluorescence data in real-time, producing amplification curves for each sample.
