At the end of 1990s, the Human Genome Project, which has the same epoch-making significance as the Manhattan Atomic Bomb Project and the Apollo Moon Landing Project, was launched. This is the first time that human beings have fully understood themselves at the molecular level. With the development of the human genome project, people have a deeper understanding of the relationship between genes and diseases, and have the opportunity to judge the risks of various diseases through the detection and analysis of gene defects, and thus derive a new health service project-gene sequencing.
After 38 years of development, Sanger sequencing has been quite perfect, and the cost has been reduced by more than 10 times. It is still the gold standard and main force of the global gene sequencing industry. Sanger sequencing is the international gold standard for all gene sequencing at present, including fluorescence quantitative PCRTaqman probe method, common PCR method, chip method, second-generation sequencing method, mass spectrometry and other methods.
Since 20 10, many new-generation sequencer products have appeared. The new generation sequencing has low cost, large data and high speed, but it needs to be mature and the accuracy is not high enough.
(1) Sanger sequencing
The base sequence of the detected DNA is read more than 800bp in turn, which is the gold standard for all sequencing in the first generation and all sequencing in the new generation. Sanger sequencing in the United States is represented by instrument ABI3730XL, which is rarely carried out in general hospitals and is carried out by companies. The human genome project, which took as long as 13 years, was finally completed by Sanger sequencing method. Sanger sequencing is to design primers for the mutation sites of known pathogenic genes, carry out PCR amplification and direct sequencing. The amplification of a single mutation point (including the amplification of some exon fragments) does not need to amplify all exons of the gene where the point is located. Therefore, Sanger sequencing can play the advantages of accuracy and low cost when detecting disease samples controlled by a single gene or part of genes.
(2) STR paternity test and forensic identification
By measuring the length and color of DNA fragments, the genetic relationship can be determined. It is a technology developed on the basis of Sanger sequencing technology. The instrument used is the same as that used in Sanger sequencing, and the detection principle is the same. One device is equipped with two sets of testing software and one instrument has three functions. The representative instruments ABI 3 130, 3 130XL and 3730 are in the United States. General Justice and Sanger Sequencing Company have instruments, but hospitals don't.
Paternity identification is to determine the genetic relationship between parents and children by detecting DNA genetic fragments.
(3) snapshot sequencing technology
SNaPshot technology, developed by American Applied Biology Company (ABI), is a fluorescence-labeled single-base extension typing technology, also known as small sequencing, which is mainly used to detect SNP mutation typing projects with medium flux. It is usually used to analyze 10~30 SNP mutation sites. It is a technology developed on the basis of Sanger sequencing technology. Like the Sanger sequencer, the detection principle is the same. One device is equipped with two sets of testing software and one instrument has three functions. Instruments ABI3 130, 3 130XL and 3730 representing the United States are generally owned by Sanger Sequencing Company.
Advantages: 1, accurate typing, 2, Qualcomm quantity, 3, fast detection speed, 4, not limited by SNP polymorphism characteristics, 5, not limited by sample number. SNaPshot technology is the gold standard for SNP mutation detection of new generation sequencing technology (including second generation sequencing and chip sequencing), and sanger sequencing technology is the gold standard for mutation detection of SNaPshot technology.
(4) Real-time fluorescence quantitative PCR
The whole PCR process is monitored in real time by the accumulation of fluorescence signals, and finally the unknown template is quantitatively analyzed by the standard curve (or compared with the internal reference). Representative instruments include ABI 7500 fluorescence quantitative PCR instrument in the United States. Generally, there are such instruments in the third-class hospitals, and they are very popular. Easy to use and maintain. 1, mutation search: quantitative PCR TaqMan probe hybridization. 2. Gene quantification: Relative fluorescence quantitative PCR is used to detect the expression of bacterial or viral RNA in medicine. Fluorescent quantitative PCR is a new quantitative experimental technique introduced by ABI company in the United States on 1996.
Application fields of real-time fluorescence quantitative PCR: clinical disease diagnosis: diagnosis and curative effect evaluation of various infectious diseases such as hepatitis, AIDS, avian influenza, tuberculosis, sexually transmitted diseases, prenatal and postnatal detection of thalassemia, hemophilia, abnormal gender development, mental retardation syndrome and fetal malformation, tumor markers and tumor gene sequencing, and genetic gene sequencing.