PCR Technology Service and its Application

2017-08-30

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Since its establishment in 1985, PCR technology has developed rapidly and has shown extremely wide application value. Based on the basic principles of PCR, many scholars give full play to their creative thinking, research, and improve PCR technology, so that PCR technology has not only been further matured and improved, And many new application fields and methods have been derived from this basis.

Application of PCR

PCR technology first has an irreplaceable contribution in the field of genetic engineering:

1. Get the target gene fragment.

PCR provides a simple and rapid method for obtaining the target gene fragment in the process of DNA recombination. Before the completion of the human genome project, PCR is the main method to obtain new gene fragments or genes with similar sequences from cDNA libraries or genome libraries. At present, this technology has become the main method to quickly obtain gene fragments with known sequences from various biological specimens or genetic engineering vectors.

2. microanalysis of DNA and RNA

PCR technology has high sensitivity and a low requirement on the amount of template DNA, so it is a good method for micro qualitative and quantitative analysis of DNA and RNA. Theoretically, as long as there is a template of one molecule, the target fragment can be obtained. In practice, one drop of blood, one hair, or one cell is enough to meet the needs of PCR detection, so it has a very broad application prospect in gene diagnosis.

3. DNA sequence analysis

PCR technology is introduced into DNA sequence determination, which greatly simplifies the sequencing work and improves the sequencing speed. It is the basis of high-throughput DNA sequence analysis. The DNA fragment to be detected can be cloned into a specific vector for sequence determination or directly determined.

4. Gene mutation analysis

The combination of PCR and other technologies can greatly improve the sensitivity of gene mutation detection, such as single-strand conformation polymorphism analysis, allele-specific oligonucleotide probe analysis, gene chip technology, DNA sequence analysis, etc.

5. In vitro mutation of gene

Before the use of PCR technology, it is time-consuming and laborious work to mutate genes in vitro. At present, PCR technology can be used to design primers to modify the target gene fragment in vitro, such as insertion, insertion, deletion, and point mutation.

The PCR technology provides a faster and simpler method than the original technology to modify DNA fragments according to special requirements. Because compared with recombinant DNA technology, PCR technology itself is relatively simple; and it can also change the base sequence of DNA more simply by chemically synthesizing oligonucleotide primers without restricting DNA fragments. Endonuclease and ligase operations; in addition, PCR technology may also be convenient for sequence modification, such as adding an “addition” sequence to the 5’end of the primer. In addition, the efficiency of modified products produced by PCR technology can reach almost 100%.

In addition, the principle of introducing DNA sequence variation through PCR primers is very useful. The simplest is that it can make one chain or the other chain or both chains of the PCR product specifically labeled with radioisotopes, biotin, or fluorescein. It can also specifically carry out site substitution, deletion, insertion, or recombination at any position in the DNA sequence. At the same time, it can accurately connect unrelated sequences and obtain heritable mutations.

In recent years, various new technologies based on PCR have been continuously updated, improved, and developed, such as in-situ PCR technology, ligase chain reaction (LCR), and nucleic acid sequence-based amplification technology (Nucleic acid sequence-based amplification). , NASBA), transcription-dependent amplification system (Transcript-based amplification system, TAS), labeled PCR technology (Labelled Primers, LP-PCR), color PCR technology (Color Complementation assay PCR, CCAPCR), reverse PCR technology (reverse PCR), asymmetric PCR technology (asymmetric PCR) and the current real-time fluorescence quantitative technology (real-time PCR), etc., are not only used in scientific research, but also social life such as animal and plant quarantine, food safety, forensic identification, etc. All aspects have increasingly greater application value.

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Let's introduce the application of PCR technology closely related to medical research briefly .

1. RT-PCR technique

Reverse Transcription PCR (RT-PCR) can detect less than 10 copies of specific RNA in a single cell. At present, RT-PCR is an effective method to obtain the target gene from tissues or cells, and to conduct qualitative and semi-quantitative analysis of RNA with known sequences. It is also one of the widely used PCR methods.

This technology combines RNA reverse transcription reaction with PCR reaction. Firstly, RNA is used as a template to synthesize cDNA under the action of reverse transcriptase, and then cDNA is used as a template to amplify the target gene through PCR reaction.

2. PCR technology In situ

In Situ PCR is to amplify the target gene fragment in the cell by using the whole cell as a tiny reaction system. The PCR reaction is carried out in a single cell on a tissue section or cell smear fixed in formaldehyde solution (formalin) and wrapped in paraffin. After the PCR reaction, the specific probe is used for in-situ hybridization to detect whether the DNA or RNA to be detected exists in the tissue or cell. Because the products of conventional PCR or RT-PCR can't be directly localized in tissue cells, they can't be associated with specific tissue cell phenotypes. However, in situ hybridization has a good localization effect, but its sensitivity is not high.

In-situ PCR makes up for the shortcomings of PCR and in-situ hybridization. Combining the amplification and localization of the target gene, can not only identify the cells with the sequence but also mark the position of the target sequence in the cells. It is of great practical value to study the pathogenesis and clinical process of diseases at the molecular and cellular levels.

3. Real-time PCR technology

Conventional PCR reaction detects the product content at the end of the reaction. However, during the reaction, the product increases exponentially, and the accumulation of products after repeated cycles will affect the accurate judgment of the original template content difference. Therefore, the conventional PCR reaction can only be used as a semi-quantitative means.

Real-time PCR technology eliminates the interference of product accumulation on quantitative analysis by dynamically monitoring the number of products in the reaction process, and is also called Quantitative PCR (qPCR). The quantitative analysis of mRNA, miRNA and other non-coding RNA by PCR has been realized quickly and accurately, and it has been applied to gene diagnosis in the clinic.

3.1 Application of real-time PCR

Real-time PCR has the characteristics of quantification, specificity, sensitivity, and rapidity. At present, real-time fluorescence PCR has been widely used in basic scientific research, clinical diagnosis, disease research, and drug research and development. These applications will change the phenotypic cognition and phenotypic diagnosis of diseases in the past, but recognize and diagnose diseases in essence.

(1) Application of real-time PCR in tumor field;

Real-time PCR can not only effectively detect gene mutation, rearrangement, and translocation, but also accurately detect oncogene expression. It can be used for early diagnosis, differentiation, typing, staging, treatment, and prognosis evaluation of tumors.

(2) Real-time PCR for polymorphism analysis

Real-time PCR has a good application prospect in the analysis of single nucleotide polymorphism (SNP). For example, it can be used to predict the different therapeutic responses caused by different effects of drugs in different individuals with the same disease. If drugs are used according to the characteristics of gene polymorphism, the clinical treatment will meet the individual requirements.

(3) Real-time PCR for pathogen detection

Real-time PCR can also be used to detect a variety of bacteria, viruses, mycoplasma, and chlamydia.

For example. The detection of the hepatitis B virus mainly depends on the indirect index of hepatitis B surface antigen (HBsAg). However, if HBsAg is positive or negative, it is difficult to judge whether the HBV in a patient is in the replication stage, how much the virus replicates, and whether the patient is infectious. The appearance of real-time PCR can accurately detect the copy number of HBV in samples and monitor the drug treatment effect and clinical status of patients in real-time.

Real-time PCR can not only characterize viruses, but also can conveniently, quickly, sensitively, and accurately quantify their DNA or RNA sequences, and dynamically observe the number of potential viruses in the course of the disease, because of its small differences between batches and within batches and good repeatability.