The commonly used molecular diagnostic techniques

With the development of molecular biology and molecular genetics, more and more molecular diagnostic techniques are applied to the diagnosis of diseases, completely breaking the conventional diagnosis method, and no longer prescribing the occurrence and development of diseases based on the phenotype of diseases. Molecular diagnostic techniques, by detecting the structure or expression level of genetic material, not only found that the disease is related to the presence, transcription and expression of specific genes, but also that individual genetic polymorphisms are closely related to disease-specific medications.

In 1953, Watson and Crick proposed a DNA double helix structure model, and based on this, proposed a central rule to describe the flow of genetic information from gene to protein, marking the birth of molecular biology as an independent discipline. Throughout the 60-year history of molecular diagnosis, molecular diagnostic techniques are broadly divided into three major areas: molecular hybridization, gene amplification, and gene sequencing. Molecular diagnostics technology is characterized by automation, rapidization, and accuracy, and plays an increasingly important role in the diagnosis, treatment, and prognosis of diseases.

1. Molecular hybridization technology

Molecular hybridization refers to a process in which two nucleic acid single strands having homologous sequences form a double strand according to the principle of base complementary pairing under certain conditions. Since the 1960s, hybridization technology has developed rapidly, and it is possible to capture target sequences by probes of known gene sequences. The main techniques include colony in situ hybridization, dot blot hybridization, Southern blotting, Northern blotting, fluorescence in situ hybridization, and chip hybridization.

In situ hybridization is the use of the principle of base pairing to display the position of a target gene on a tissue, cell or chromosome. In 1975, Southern invented the Southern blotting technique, which uses DNA fragment digestion and molecular probe hybridization to ensure the specificity of detection. At present, Southern blotting is still the most classical molecular detection method in the field of probe hybridization, and it is widely used in the detection of gene mutations. In 1977, Rudkin invented fluorescence in situ hybridization using a fluorescein-labeled probe, making the in situ hybridization technique more accurate and more sensitive. In situ hybridization is mainly used in gene mapping, cancer gene detection and judgment. In 1991, Affymetrix's Fordor developed photo-etching technology and prepared the first slide-based microarray, marking the official realization of chip technology as a practical molecular biology technology. Gene chips, also known as DNA microarrays, are new tools for detecting DNA sequences by densely arranging a large number of DNA or oligonucleotide probes on the surface of a vector by nucleic acid hybridization. Gene chip technology has the characteristics of high throughput, high sensitivity, miniaturization and automation. It is used in single nucleotide polymorphism (SNP) detection, pre-diagnosis and drug screening.

2. Gene amplification technology

In 1983, Mullis of Cetus, USA, invented polymerase chain reaction technology (PCR), the technology utilizes the principle of high temperature denaturation and low temperature renaturation of DNA to successfully achieve in vitro amplification of nucleic acid fragments by denaturation, renaturation and extension of three temperature changes. PCR technology is widely used in medicine, agriculture, food testing and other fields because of its high specificity, high sensitivity, simplicity, and rapidity, and low purity requirements for specimens. There are two types of PCR technology: conventional PCR technology and real-time PCR technology. Conventional PCR technology refers to a nucleic acid amplification technique that only qualitatively or semi-quantitatively analyzes the end product of a PCR amplification reaction, cannot accurately quantify the starting template, and cannot detect the amplification reaction in real time, but the technology platform and equipment technology requires are relatively low, and the cost is relatively low. Currently, the platform is mainly used to detect qualitative items such as missing genes, mutant genes, and fusion genes. Real-time PCR technology, also known as real-time quantitative fluorescence PCR technology, refers to the technique of adding fluorescent groups to the PCR reaction system, monitoring the whole PCR process by fluorescence signal accumulation, and finally quantitatively analyzing the unknown template through the standard curve. Real-time PCR technology has the characteristics of strong specificity, high accuracy and good reproducibility. It is mainly used in nucleic acid quantification and mRNA expression level analysis in laboratory medicine. It can analyze and guide clinical drug use, monitor drug efficacy and judge disease progression. In 1977, Maxam proposed a chemical modification degradation model, which kicked off the arrival of the era of nucleic acid sequencing. In the same year, Sanger et al. invented the DNA dideoxy chain end termination method, which can detect the nucleic acid sequence of a species or cell, and then compare it with the gene pool to know the characteristics of the tested species or cells. As the most classical sequencing method, the Sanger method can read sequences long and can process repetitive sequences and multimers well. It is still a commonly used sequencing method and is widely used in the detection of multiple repeats such as genomic DNA and cDNA. The technical disadvantages are lower sensitivity and lower flux. In 1998, Ronaghi invented pyrosequencing, the basic principle of which is to use the pyrophosphate group released by the primer to excite fluorescence, and determine the number of bases matched by the peak height. Compared with the Sanger method, the sensitivity is improved, and it is widely used in SNP site detection and allelic mutation measurement. In recent years, high-throughput sequencing technology has been invented, which is a revolutionary innovation in traditional technology. The technology constructs a DNA library by DNA fragmentation, cross-links the library and the vector for amplification, and performs side-synthesis and sequencing reaction on the carrier surface to complete high-throughput sequencing of massive data. The technology has high sequencing speed and high accuracy, and can be used for large-scale sequencing detection, and is mainly applied to analysis and research of whole genome sequences, intron sequences, and exon sequences.

Edited by Suzhou Yacoo Science Co., Ltd.