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Introduction of dyes and probes for fluorescence quantitative PCR

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Introduction of dyes and probes for fluorescence quantitative PCR

What is PCR?

PCR is a kind of in vitro DNA amplification technology, the DNA templates, primers and the presence of the four DNA nucleotides, depend on the DNA polymerase of an enzymatic reaction, the purpose of DNA fragments from its side chain of oligonucleotide primers complementary through a series of reaction cycle for many times, in a relatively short period of time to obtain the required specific gene fragment.

Reaction components: template DNA, a pair of primers complementary to both ends of template DNA, DNA polymerase, and dNTP

Principle of PCR: After 1 cycle, we can get 2 amplification products; After 2 cycles, we have 4 amplification products; And so on, after 40 cycles, we could theoretically get 1×240 amplification products.

 Introduction of dyes and probes for fluorescence quantitative PCR

Dyes and probes for fluorescence quantitative PCR

01 Dye method

An excess of fluorescent dye was added to the PCR amplification system, which was only bound to double-stranded DNA furrows, not single-stranded DNA strands, and did not emit light in the free state, but only when bound to double-stranded DNA furrows. Therefore, with the exponential amplification of PCR products, the fluorescence signal intensity was positively correlated with the number of PCR products.

02 Probe method

During PCR amplification, a specific fluorescent probe was added in addition to the primer. The probe is a linear oligonucleotide with a fluorescent reporter group and a fluorescent quench group attached to both ends. When the fluorescent probe is in the free state, the signal emitted by the reporter group is absorbed by the quenched group, and the probe does not emit light. When the PCR amplification was in the extension stage, the Taq enzyme digested and degraded the probe, promoted the separation of the reporter group and quenched group, and the probe emitted light. In addition, each DNA strand was amplified to form a fluorescent molecule, which realized the complete synchronization of fluorescence signal accumulation and product generation.

Advantages of dye method: easy to use, can be used for different templates, cheap, sensitive; Disadvantages: Easy to bind to non-specific products.

The advantages of the probe method are as follows: it has high specificity for target sequence and is suitable for SNP detection; Cons: Expensive and only applicable to specific target genes.

 

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