But I have to say one thing, at present, DNA sequencing relies on or Sanger's principle, just combined with fluorescent dyes to improve the sensitivity.
Principles and Methods of DNA Sequencing
DNA sequencing is divided into manual sequencing and automated sequencing, and manual sequencing includes the Sanger double deoxy chain termination method and the Maxam-Gilbert chemical degradation method. Automated sequencing has actually become the mainstream of DNA sequence analysis today. PE ABI has produced DNA sequencers such as Model 373, Model 377, Model 310, Model 3700 and Model 3100, among which Model 310 is one of the most used models in clinical testing laboratories. This experiment describes the sequencing principles and operating procedures of the ABI PRISM Model 310 DNA sequencer.
Principle ABI PRISM Model 310 Genetic Analyzer (i.e., DNA sequencer), using capillary electrophoresis technology instead of the traditional polyacrylamide plate electrophoresis, the application of the company's patented four-color fluorescent dyes labeled with ddNTP (labeled terminator method), and therefore through the single-primer PCR sequencing reaction, the generated PCR products are then the end of the 3 '''' differing by one base! for a mixture of single-stranded DNA with four different fluorescent dyes, which allows the sequencing PCR products of the four fluorescent dyes to be electrophoresed in a single capillary tube, thus avoiding the effect of mobility differences between lanes and greatly improving the accuracy of sequencing. Due to the different sizes of molecules, the mobility in capillary electrophoresis is also different, when it passes through the capillary readout window segment, the CCD (charge-coupled device) camera detector in the laser detector window can detect the fluorescent molecules one by one, and the excitation of fluorescence is spectroscopically separated by the grating in order to distinguish between different colors of fluorescence representing the information of different bases and be imaged simultaneously on the CCD The CCD camera is used to image the fluorescent molecules synchronously, and the analysis software automatically converts the different fluorescent colors into DNA sequences for the purpose of DNA sequencing. The analysis results can be output in the form of gel electrophoresis profile, fluorescence absorption peak graph or base alignment sequence.
It is a fully automated computer-controlled precision instrument for the determination of base order or size and quantification of DNA fragments with automatic gel filling, automatic sampling, and automatic data collection and analysis.
PE also provides gel polymers, including DNA sequencing gel (POP 6) and GeneScan gel (POP 4). These gel particles have uniform pore sizes, avoiding the effects of inconsistent gel dispensing conditions on sequencing accuracy. It mainly consists of a capillary electrophoresis unit, a Macintosh computer, a color printer, and accessories such as electrophoresis. The computer includes software for data collection, analysis and instrument operation. It uses the latest CCD camera detector to shorten DNA sequencing to 2.5h, and PCR fragment size analysis and quantitative analysis to 10-40min.
Because the instrument has the functions of DNA sequencing, PCR fragment size analysis, and quantitative analysis, it can carry out DNA sequencing, heterozygote analysis, single-stranded conformation polymorphism (SSCP), microsatellite sequence analysis, long-fragment PCR, and long-fragment PCR. Therefore, it can perform DNA sequencing, heterozygote analysis, single-strand conformation polymorphism analysis (SSCP), microsatellite sequence analysis, long fragment PCR, RT-PCR (quantitative PCR) and other analyses, and clinically, in addition to routine DNA sequencing, it can also be used for single-nucleotide polymorphism (SNP) analysis, mutation detection, HLA mating, forensic identification of paternity and individuality, typing and identification of microorganisms and viruses, and so on.
Reagents and equipment
1. BigDye Sequencing Reaction Kit The main reagent is BigDye Mix, which contains PE patented four-color fluorescent-labeled ddNTP and ordinary dNTP, AmpliTaq DNA polymerase FS, reaction buffer and so on.
2. pGEM-3Zf (+) Double-stranded DNA Control Template 0.2g/L, kit companion reagent.
3. M13(-21) primer TGTAAAACGACGGCCAGT, 3.2 μmol/L, i.e. 3.2 pmol/μl, kit supporting reagents.
4. DNA sequencing templates can be PCR products, single-stranded DNA and plasmid DNA, etc.. The template concentration should be adjusted in the PCR reaction to take a volume of 1μl is appropriate. The concentration of plasmid DNA in this experiment is 0.2g/L, i.e. 200ng/μl.
5. Primers: Forward or reverse primers should be designed according to the DNA fragments to be measured, and formulated as 3.2μmol/L, i.e. 3.2pmol/μl. If the recombinant plasmid contains universal primer sequences, we can use universal primers, e.g. M13(-21) primer, T7 primer, etc.
6.
6. Sterilize deionized water or triple-distilled water.
7. 0.2 ml or 0.5 ml PCR tubes Caps are separated, PE products.
8. 3mol/L sodium acetate (pH5.2) Weigh 40.8g NaAc-3H2O dissolved in 70ml distilled water, glacial acetic acid to adjust the pH to 5.2, fixed volume to 100ml, autoclave sterilization and then divided.
9. 70% ethanol and anhydrous ethanol.
10. NaAc/ethanol mixture Take 37.5 ml of anhydrous ethanol and 2.5 ml of 3mol/L NaAc and mix well, can be stored at room temperature for 1 year.
11. POP 6 sequencing gel ABI product.
12. Template suppressor reagent (TSR) ABI product.
13. 10× Electrophoresis Buffer ABI product.
14. ABI PRISM 310 fully automated DNA sequencer.
15. 2400 or 9600 PCR instrument.
16. Benchtop freezing high-speed centrifuge.
17. Benchtop high-speed centrifuge or pocket centrifuge.
Operating steps
1. PCR sequencing reaction
(1) Take 0.2 ml PCR tubes, number them with a marker pen, insert the tubes in pellet ice, and add reagents according to the following table:
Reagents added Measurement template tubes Standard control tubes
BigDye Mix 1 μl 1 μl
Plasmid DNA to be measured 1μl -
pGEM-3Zf (+) double-stranded DNA - 1μl
Forward primer for DNA to be tested 1μl -
M13(-21) primer - 1μl
Sterilization Deionized water 2 μl 2 μl
Total reaction volume 5 μl without light mineral or paraffin oil, cap the PCR tube tightly, mix by flicking the tube with your finger and centrifuge slightly.
(2) The PCR tube was placed on a 9600 or 2400 PCR instrument for amplification. 98℃ denaturation for 2min followed by PCR cycling, PCR cycling parameters for 96℃ 10s, 50℃ 5s, 60℃ 4min, 25 cycles, set 4℃ insulation after the end of amplification.
2. Purification of PCR products by sodium acetate/ethanol method
(1) The mixture was centrifuged, and the amplification products were transferred to 1.5 ml EP tubes.
(2) Add 25 μl of sodium acetate/ethanol mixture, shake well, and place on ice for 10 min to precipitate the DNA. centrifuge at 12,000 r/min for 30 min at 4°C, and carefully discard the supernatant.
(3) Add 70% (V/V) ethanol 50μl to wash the precipitation twice. 12 000r/min centrifugation at 4℃ for 5min, carefully discard the supernatant and the liquid beads on the wall of the tube, and vacuum dry the precipitation for 10-15min.
3. Processing of PCR products for pre-electrophoresis sequencing.
(1) Add 12 μl of TSR to the centrifuge tube, shake vigorously, let it fully dissolve the DNA precipitate, and centrifuge slightly.
(2) Transfer the solution to a capped body separated 0.2 ml PCR tube and centrifuge slightly.
(3) Perform thermal denaturation (95°C for 2 min) on a PCR machine, quench in ice, and leave on the machine.
4. On-machine operation according to the instrument operating instructions to install the capillary, capillary position correction, manual manual gel filling and the establishment of the running sequencing sequence file. The instrument will automatically fill the capillary with gel, pre-electrophoresis at 1.2kV for 5min, feed the sample automatically according to the programmed sequence, then pre-electrophoresis (1.2kV, 20min), and electrophoresis at 7.5kV for 2h. After the end of electrophoresis, the instrument will automatically clean, fill the capillary with gel, and then feed the next sample, pre-electrophoresis and electrophoresis. The total electrophoresis time for each sample is 2.5 h. After electrophoresis, the instrument will automatically analyze or print out the color sequencing map.
5. The instrument will automatically analyze the sequence and can perform sequence comparison according to user requirements. If the sequencing sequence is known, the difference bases can be marked with an asterisk by sequence comparison to improve work efficiency.
6. After sequencing, the instrument will be cleaned and maintained according to the instrument operating procedures.
Calculation
The formula for calculating the accuracy of the sequencing reaction: 100% - the number of differential bases (excluding the number of N)/650×100%
Differential bases are the bases that are different between the measured DNA sequences and the known standard DNA sequences, and the N is the bases that the instrument cannot read.
Precautions and Evaluation
1. The ABI PRISM 310 Genetic Analyzer is a high-grade precision instrument that requires dedicated personnel for operation, management and maintenance.
2. The total volume of the sequencing PCR reaction in this experiment was 5 μl, and it was not covered with mineral oil, so the sealing of the PCR tube cap is very important, in addition to tightening the cap of the PCR tube after adding reagents, and it is best to use the PCR tube of PE company. If the PCR solution is less than 4~4.5μl after PCR, this PCR reaction may fail, and it is not necessary to carry out purification and sampling.
3. As a sequencing user, you only need to provide purified DNA samples and primers, a sequencing PCR reaction using different templates, the amount of DNA required is different, PCR sequencing requires a small amount of templates, the general PCR product requires 30 to 90ng, single-stranded DNA requires 50 to 100ng, double-stranded DNA requires 200 to 500ng, the purity of DNA is generally A260nm. The purity of DNA is generally A260nm/A280nm 1.6~2.0, it is best to use deionized water or triple-distilled water to dissolve DNA, not TE buffer. It is better to use deionized water or triple-distilled water to make 3.2 pmol/μl for primers.
4. The sequencing kit used in this experiment is BigDye Fluorescent Labeling Termination Substrate Cycle Sequencing Kit, and the length of DNA can be measured is about 650bp. The DNA sequencing accuracy of this instrument is (98.5±0.5) %, the instrument can not read the base N <2%, the length of the measurement needs to be more than 650bp, then you need to design another primer. To ensure more accurate sequencing, reverse primers can be designed to sequence the same template and corroborate each other. For N bases manual checking can be done and sometimes it can be read out. In order to improve the accuracy of sequencing, according to the location of the asterisk, the color map can be manually analyzed, and the bases can be further checked.