Procedures for automated sequencing in DNA sequencing methods <br> Gene analyzers (ie, DNA sequencers) use capillary electrophoresis
instead of traditional polyacrylamide plate electrophoresis, using the company's patented four-color fluorescent dye-labeled ddNTP (marker) Termination method), therefore, by single-primer PCR sequencing reaction, the generated PCR product is a single-stranded DNA mixture of 4 different fluorescent dyes at the 3' end of 1 base, so that the sequencing PCR products of the four fluorescent dyes can be Electrophoresis in a capillary tube avoids the influence of differences in mobility between lanes and greatly improves the accuracy of sequencing. Due to the different molecular sizes, the mobility in capillary electrophoresis is also different. When passing through the capillary reading window segment, the CCD (charge-coupled device) camera detector in the laser detector window can detect the fluorescent molecules one by one. The fluorescence is spectrally split to distinguish the fluorescence of different colors representing different base information, and is simultaneously imaged on a CCD camera. The analysis software can automatically convert different fluorescence into DNA sequences for DNA sequencing purposes. The analysis results can be output in various forms such as gel electrophoresis pattern, fluorescence absorption peak map or base arrangement order.
It is a high-end precision instrument that can measure the base sequence or size and quantitative of DNA fragments by automatic computerized automatic filling, automatic injection, automatic data collection and analysis. PE also offers gel polymers, including DNA sequencing gel (POP 6) and GeneScan gel (POP 4). These gel particles have a uniform pore size, which avoids the influence of inconsistent rubber mixing conditions on sequencing accuracy. It consists mainly of capillary electrophoresis devices, Macintosh computers, color printers, and electrophoresis accessories. 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 for 10 to 40 minutes.
Because the instrument has the functions of DNA sequencing, PCR fragment size analysis and quantitative analysis, it can perform DNA sequencing, heterozygous analysis, single strand conformation polymorphism analysis (SSCP), microsatellite sequence analysis, long fragment PCR, RT-PCR. (Quantitative PCR) and other analyses, in addition to routine DNA sequencing, can also perform single nucleotide polymorphism (SNP) analysis, gene mutation detection, HLA matching, forensic paternity and individual identification, microbial and Classification and identification of viruses.
First, preparation work 1. BigDye sequencing reaction kit The main reagent is BigDye Mix, which contains PE patent four-color fluorescent labeled ddNTP and common dNTP, AmpliTaq DNA polymerase FS, reaction buffer and so on.
2. pGEM-3Zf (+) double-stranded DNA control template 0.2 g/L, kit kit reagent.
3. M13 (-21) primer TGTAAAACGACGGCCAGT, 3.2 μmol/L, ie 3.2 pmol/μl, reagent kit.
4. The DNA sequencing template may be a PCR product, single-stranded DNA, and plasmid DNA. The template concentration should be adjusted to take 1 μl in the PCR reaction. The plasmid DNA determined in this experiment was 0.2 g/L, i.e., 200 ng/μl.
5. Primers should be designed with either forward or reverse primers based on the DNA fragment to be assayed and formulated into 3.2 μmol/L, or 3.2 pmol/μl. For example, a universal primer such as M13 (-21) primer, T7 primer or the like can be used as the universal primer sequence in the recombinant plasmid.
6. Sterilize deionized or triple distilled water.
7. Separate 0.2 ml or 0.5 ml PCR tube cap.
8. 3 mol/L sodium acetate (pH 5.2) Weigh 40.8 g NaAc·3H2O dissolved in 70 ml of distilled water, adjust the pH to 5.2 with glacial acetic acid, dilute to 100 ml, and disperse after autoclaving.
9. 70% ethanol and absolute ethanol.
10. NaAc/ethanol mixture was mixed with 37.5 ml of absolute ethanol and 2.5 ml of 3 mol/L NaAc, and stored at room temperature for 1 year.
11. POP 6 sequencing glue.
12. Template Inhibitory Reagent (TSR).
13. 10× electrophoresis buffer.
14. Fully automatic DNA sequencer.
15. PCR instrument.
16. Benchtop Freezer High Speed ​​Centrifuge.
17. Benchtop high speed centrifuge or pocket centrifuge.
Second, PCR sequencing reaction 1. Take a 0.2 ml PCR tube, mark it with a marker, insert the tube into the pellet ice, and add the reagent according to the following table:
Standard reagent tube
BigDye Mix 1 μl 1 μl
Plasmid DNA to be tested 1 μl -
pGEM-3Zf (+) double stranded DNA - 1 μl
Forward primer for the DNA to be tested 1 μl -
M13(-21) Primer - 1 μl
Sterilized deionized water 2 μl 2 μl
The total reaction volume is 5 μl. Add no light mineral oil or paraffin oil, cover the PCR tube, mix with a finger tube, and centrifuge slightly.
2. Place the PCR tube on a 9600 or 2400 type PCR machine for amplification. The PCR cycle was carried out after denaturation at 98 °C for 2 min. The PCR cycle parameters were 96 °C for 10 s, 50 °C for 5 s, 60 °C for 4 min, 25 cycles, and 4 °C incubation was performed after the amplification.
3. Purification of PCR products by sodium acetate/ethanol method 1. Centrifuge the mixture and transfer the amplified product to a 1.5 ml EP tube.
2. Add 25 μl of sodium acetate/ethanol mixture, shake well, and place on ice for 10 min to precipitate DNA. Centrifuge at 12 000 r/min for 30 min at 4 ° C and carefully discard the supernatant.
3. Wash the pellet twice with 70% (V/V) ethanol 50 μl. Centrifuge at 12 000 r/min for 5 min at 4 °C, carefully discard the supernatant and the liquid droplets on the tube wall, and dry the precipitate in vacuum for 10-15 min.
4. Processing of sequencing PCR products before electrophoresis 1. Add 12 μl of TSR to the centrifuge tube and shake vigorously to allow it to fully dissolve the DNA pellet and centrifuge slightly.
2. Transfer the solution to a 0.2 ml PCR tube separated by a cover and centrifuge slightly.
3. Thermal denaturation on the PCR machine (95 ° C for 2 min), quenching in ice, waiting to be on the machine.
Five, on the machine operation 1. Install the capillary according to the instrument operating instructions, perform capillary position correction, manually fill the glue and create a running sequencing sequence file.
2. The instrument will automatically fill the capillary to the capillary, pre-electrophoresis for 1.2 min at 1.2 kV, auto-inject in the programmed order, pre-electrophoresis (1.2 kV, 20 min), and electrophoresis at 7.5 kV for 2 h.
3. After the electrophoresis is finished, the instrument will automatically clean, fill the gel, enter the next sample, pre-electrophoresis and electrophoresis.
4. The total electrophoresis time of each sample is 2.5 h.
5. After the electrophoresis is finished, the instrument will automatically analyze or print out the color sequencing map.
Sixth, the instrument will automatically <br>
sequence analysis Sequence analysis and sequence comparison based on user requirements. If the sequencing sequence is known, the difference bases can be marked with an asterisk by sequence comparison to improve work efficiency.
Seven, instrument cleaning <br> be completed sequencing instrument by instrument cleaning and maintenance procedures.
Eight, calculation 1. Sequencing reaction accuracy calculation formula: 100% - number of differential bases (excluding N number) / 650 × 100%.
2. The difference base is the base in which the determined DNA sequence is different from the known standard DNA sequence, and N is the base that the instrument cannot read.
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