quantitative PCR

Quantitative PCR (qPCR) is a method that uses DNA polymerase chain reaction (PCR) technology to quantitatively detect the amount of target DNA. This method has wide application value in the fields of medical diagnosis, gene expression research, genetic analysis, and virus titration.

1. Definition and Principle

Quantitative PCR is designed to estimate the number of original templates in a sample. Its principle is based on the regularity of PCR amplification. During the exponential phase of PCR amplification, the amount of the original template is inferred by measuring the amount of amplification product. Specifically, if you know the amount of amplified product N and the efficiency of PCR amplification e, you can use the formula to calculate the value of the original template No.

In Real-time Fluorescent Quantitative PCR (FQ-PCR), fluorescent chemicals are also introduced. As the PCR reaction proceeds, the fluorescence signal intensity increases proportionally. By setting the fluorescence threshold and measuring the cycle threshold (CT value), which is the number of cycles experienced when the fluorescence signal reaches the set threshold, the amount of starting DNA template can be further accurately calculated.

2. Experimental steps

Quantitative PCR experiments usually include the following key steps:

1. Prepare reagents: including template DNA, primers (forward primer and reverse primer), DNA polymerase, dNTP (four deoxynucleotides), buffer and MgCl2, etc.

2. Construct a reaction system: Mix template DNA, primers, DNA polymerase, dNTPs, buffer and MgCl2 to obtain a PCR reaction solution.

3. Optimize PCR amplification conditions: adjust the temperature conditions and the number of PCR cycles to ensure that the PCR reaction proceeds under optimal conditions.

4. PCR amplification: It includes three steps of denaturation, annealing and extension, and achieves exponential amplification of target DNA through multiple cycles.

5. Detection of PCR products: It can be carried out by gel electrophoresis, fluorescence quantitative PCR or real-time quantitative PCR. Real-time quantitative PCR is popular because it can monitor the progress of PCR reactions in real time and accurately measure the amount of PCR products.

3. Technical characteristics

1. High sensitivity: Quantitative PCR can detect very low concentrations of DNA templates and is suitable for the analysis of trace samples.

2. High specificity: Through the design of specific primers, it can be ensured that the PCR reaction only amplifies the target DNA sequence.

3. Accurate quantification: Combined with fluorescence quantification technology, accurate quantification of the number of starting DNA templates can be achieved.

4. Wide application: It has important applications in the fields of medical diagnosis, gene expression research, genetic analysis, and virus titration.

4. Application prospects

With the continuous development of biotechnology, quantitative PCR has increasingly broad application prospects in scientific research and clinical practice. Especially in terms of virus titration, qPCR technology has become the preferred method for many virus detections due to its characteristics of rapidness, sensitivity, low probability of contamination and reproducibility. In addition, quantitative PCR also plays an irreplaceable role in fields such as gene expression research and genetic analysis.

It can be seen from the above that quantitative PCR is an important molecular biology technology, and its application prospects in many fields are worth looking forward to. With the continuous advancement and improvement of technology, it is believed that quantitative PCR will play a more important role in future scientific research and clinical work.