Basic principles and processes of DNA sequencing
In 1977, Sanger et al. developed a DNA sequencing method.
Fundamental:
Dideoxy chain end termination method <br> The dideoxy chain end termination method uses a chain terminator - 2',3'-dideoxynucleoside triphosphate (ddNTP) similar to normal dNTP to specifically extend the extended DNA strand termination. The reaction system also includes a single-stranded template, primers, four dNTPs, and a DNA polymerase. It is divided into four groups, each group is added with a certain ratio (ddNTP), which can randomly infiltrate into the synthetic DNA strand, and once it is infiltrated into the synthesis, the terminal nucleotides of different size fragments must be the nucleotides. The nucleotide sequence of the DNA can be read directly from the autoradiograph.
The dideoxy chain end termination method not only has the advantages of chemical sequencing, but is also more suitable for large-scale sequencing. But it requires a pure single-stranded DNA template and a specific primer for the synthesis reaction. Therefore, early work was limited to single-stranded phage as a template, and several different specific restriction endonuclease fragments were used as primers to direct synthesis at different parts of the template strand to determine the nucleic acid sequence of the phage DNA. However, for a large number of natural double-stranded DNA molecules, the degree of application of the dideoxy chain end termination method is limited because there is no good technique for preparing a yield and a high quality separation chain.
Improvements in classical dodeoxy chain end-stop sequencing technology <br> Improvements in markers: Due to the dangers of radioisotopes to humans, many laboratories have begun to use non-radioactive materials to replace radioisotopes. Such as biotin, digoxin, silver staining, fluorescent markers such as chemiluminescent biotin, digoxin, silver staining, fluorescent markers and other chemiluminescent substances. In the case of the dideoxy sequencing, they serve as markers to trace the products of the sequencing reaction, and finally show the results of the sequencing by an enzyme color reaction or a fluorescent signal. These methods are easier, safer, faster, and less expensive to detect than traditional radioisotope methods.
Improvement of the classical dideoxy chain end-stop sequencing technology <br> Improvement of electrophoresis method: The improvement of electrophoresis method mainly adopts capillary electrophoresis. Capillary electrophoresis is the electrophoresis of a separated substance in a capillary. In 1992, Matnies et al first proposed an exhibition capillary electrophoresis method using a laser focused fluorescence scanning detection device. 25 capillaries were electrophoresed in parallel, and each capillary was able to read 350 bp in 1.5 hours. DNA sequence analysis efficiency can reach 6000bp/h.
Sequencing process: DNA Sequencing with GenomeLab TM
1. Isolation and purification of template DNA
2. Quantitative analysis of DNA template 3. Sequencing reaction 4. Purification after sequencing reaction 5. On-line denaturation 6. Capillary electrophoresis and detection 7. Data analysis
Isolation and purification of template DNA
1. Using a plasmid extraction kit or a gelatinization purification method to obtain a corresponding plasmid template or PCR product;
2. Store the DNA in sterile water such as ddH2O, 18.2MH2O ultrapure water;
Note: Do not use water treated with DEPC. There should be no EDTA in the water, otherwise the sequencing reaction will be inhibited. 3. The DNA template should be quantified by UV spectrophotometer. The concentration of DNA template should be > 0.1ug/ul;
4. Check the quality of the DNA template by agarose gel electrophoresis.
Purification after sequencing reaction <br> Removal of dNTP, ddNTP and salt ethanol in the reaction product (using a 96-well plate centrifuge)
Purification afforded <br> sequencing DNA sequencing DNA sequencing reaction product cytometry <br> fluorescently labeled DNA strand in ascending order by capillary electrophoresis.
The laser-induced fluorescence terminator is recognized by the detector and reads the nucleotide sequence of the DNA directly. Typical Sequencing Result
Welcome to visit China Microbial Species Enquiry Network , which is affiliated to Beijing Baiou Bowei Biotechnology Co., Ltd. The unit now provides microbiological strains and their related products for inquiries, consultation, ordering and after-sales service! With a number of domestic and foreign research and development units, biomedicine, third-party testing institutions, research institutes have a good and stable long-term cooperative relationship!
In 1977, Sanger et al. developed a DNA sequencing method.
Fundamental:
Dideoxy chain end termination method <br> The dideoxy chain end termination method uses a chain terminator - 2',3'-dideoxynucleoside triphosphate (ddNTP) similar to normal dNTP to specifically extend the extended DNA strand termination. The reaction system also includes a single-stranded template, primers, four dNTPs, and a DNA polymerase. It is divided into four groups, each group is added with a certain ratio (ddNTP), which can randomly infiltrate into the synthetic DNA strand, and once it is infiltrated into the synthesis, the terminal nucleotides of different size fragments must be the nucleotides. The nucleotide sequence of the DNA can be read directly from the autoradiograph.
The dideoxy chain end termination method not only has the advantages of chemical sequencing, but is also more suitable for large-scale sequencing. But it requires a pure single-stranded DNA template and a specific primer for the synthesis reaction. Therefore, early work was limited to single-stranded phage as a template, and several different specific restriction endonuclease fragments were used as primers to direct synthesis at different parts of the template strand to determine the nucleic acid sequence of the phage DNA. However, for a large number of natural double-stranded DNA molecules, the degree of application of the dideoxy chain end termination method is limited because there is no good technique for preparing a yield and a high quality separation chain.
Improvements in classical dodeoxy chain end-stop sequencing technology <br> Improvements in markers: Due to the dangers of radioisotopes to humans, many laboratories have begun to use non-radioactive materials to replace radioisotopes. Such as biotin, digoxin, silver staining, fluorescent markers such as chemiluminescent biotin, digoxin, silver staining, fluorescent markers and other chemiluminescent substances. In the case of the dideoxy sequencing, they serve as markers to trace the products of the sequencing reaction, and finally show the results of the sequencing by an enzyme color reaction or a fluorescent signal. These methods are easier, safer, faster, and less expensive to detect than traditional radioisotope methods.
Improvement of the classical dideoxy chain end-stop sequencing technology <br> Improvement of electrophoresis method: The improvement of electrophoresis method mainly adopts capillary electrophoresis. Capillary electrophoresis is the electrophoresis of a separated substance in a capillary. In 1992, Matnies et al first proposed an exhibition capillary electrophoresis method using a laser focused fluorescence scanning detection device. 25 capillaries were electrophoresed in parallel, and each capillary was able to read 350 bp in 1.5 hours. DNA sequence analysis efficiency can reach 6000bp/h.
Sequencing process: DNA Sequencing with GenomeLab TM
1. Isolation and purification of template DNA
2. Quantitative analysis of DNA template 3. Sequencing reaction 4. Purification after sequencing reaction 5. On-line denaturation 6. Capillary electrophoresis and detection 7. Data analysis
Isolation and purification of template DNA
1. Using a plasmid extraction kit or a gelatinization purification method to obtain a corresponding plasmid template or PCR product;
2. Store the DNA in sterile water such as ddH2O, 18.2MH2O ultrapure water;
Note: Do not use water treated with DEPC. There should be no EDTA in the water, otherwise the sequencing reaction will be inhibited. 3. The DNA template should be quantified by UV spectrophotometer. The concentration of DNA template should be > 0.1ug/ul;
4. Check the quality of the DNA template by agarose gel electrophoresis.
Purification after sequencing reaction <br> Removal of dNTP, ddNTP and salt ethanol in the reaction product (using a 96-well plate centrifuge)
Purification afforded <br> sequencing DNA sequencing DNA sequencing reaction product cytometry <br> fluorescently labeled DNA strand in ascending order by capillary electrophoresis.
The laser-induced fluorescence terminator is recognized by the detector and reads the nucleotide sequence of the DNA directly. Typical Sequencing Result
Welcome to visit China Microbial Species Enquiry Network , which is affiliated to Beijing Baiou Bowei Biotechnology Co., Ltd. The unit now provides microbiological strains and their related products for inquiries, consultation, ordering and after-sales service! With a number of domestic and foreign research and development units, biomedicine, third-party testing institutions, research institutes have a good and stable long-term cooperative relationship!
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