Principles and methods of DNA sequencing

DNA sequencing is divided into manual sequencing and automatic sequencing. Manual sequencing includes Sanger dideoxy chain termination method and Maxam-Gilbert chemical degradation method. Automated sequencing has actually become the mainstream of today's DNA sequence analysis. American PE ABI has produced 373, 377, 310, 3700 and 3100 DNA sequencers, among which 310 is the most used model in clinical testing laboratories. This experiment introduces the sequencing principles and operating procedures of the ABI PRISM 310 DNA sequencer.

【principle】
ABI PRISM 310 gene analyzer (ie DNA sequencer) uses capillary electrophoresis technology instead of traditional polyacrylamide plate electrophoresis, using the company's patented four-color fluorescent dye-labeled ddNTP (labeled terminator method), so through a single primer PCR sequencing reaction, the resulting PCR product is a single-stranded DNA mixture with 4 different fluorescent dyes at the 3 ends that differ by 1 base, so that the sequencing PCR products of the four fluorescent dyes can be electrophoresed in a capillary, thus avoiding The effect of differences in mobility between lanes greatly improves the accuracy of sequencing. Due to the different molecular size, the mobility in capillary electrophoresis is also different. When it passes through the capillary reading window, the CCD (charge-coupled device) camera detector in the laser detector window can detect the fluorescent molecules one by one and excite The fluorescence is split by the grating to distinguish different colors of fluorescence representing different base information, and synchronous imaging on the CCD camera, the analysis software can automatically convert different fluorescence into DNA sequence, so as to achieve the purpose of DNA sequencing. The analysis results can be exported in a variety of forms such as gel electrophoresis patterns, fluorescence absorption peaks, or base sequence.

It is a high-end precision instrument that can automatically fill glue, automatic sampling, automatic data collection and analysis and other fully-automatic computer-controlled determination of the sequence or size and quantification of DNA fragments. PE also provides gel polymer, including DNA sequencing gel (POP 6) and GeneScan gel (POP 4). The pore size of these gel particles is uniform, which avoids the effect of inconsistent gel matching conditions on sequencing accuracy. It is mainly composed of capillary electrophoresis device, Macintosh computer, color printer 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, PCR fragment size analysis and quantitative analysis for 10 ~ 40min.
Because the instrument has functions such as DNA sequencing, PCR fragment size analysis and quantitative analysis, it can perform DNA sequencing, heterozygosity analysis, single-strand conformation polymorphism analysis (SSCP), microsatellite sequence analysis, long fragment PCR, RT-PCR (Quantitative PCR) analysis, in addition to routine DNA sequencing, single nucleotide polymorphism (SNP) analysis, gene mutation detection, HLA matching, forensic paternity and individual identification, microbiology and Virus typing and identification.

【Reagents and equipment】
1. The main reagent of the BigDye sequencing reaction kit is BigDye Mix, which contains PE patent four-color fluorescent labeled ddNTP and ordinary dNTP, AmpliTaq DNA polymerase FS, reaction buffer, etc.
2. pGEM-3Zf (+) double-stranded DNA control template 0.2g / L, kit matching reagents.
3. M13 (-21) primer TGTAAAACGACGGCCAGT, 3.2μmol / L, namely 3.2pmol / μl, kit matching reagents.
4. DNA sequencing templates can be PCR products, single-stranded DNA and plasmid DNA. The template concentration should be adjusted to take 1μl during PCR reaction. The plasmid DNA determined in this experiment was 0.2g / L, or 200ng / μl.
5. The primers need to design forward or reverse primers according to the DNA fragments to be measured, and are formulated into 3.2μmol / L, or 3.2pmol / μl. For example, if the recombinant plasmid contains a universal primer sequence, a universal primer such as M13 (-21) primer, T7 primer, etc. can also be used.
6. Sterilize deionized or triple distilled water.
7. Separation of 0.2ml or 0.5ml PCR tube covers, products of PE company.
8.3mol / L sodium acetate (pH5.2) Weigh 40.8g NaAc · 3H2O and dissolve in 70ml distilled water, adjust the pH to 5.2 with glacial acetic acid, and bring the volume to 100ml, and then separate it after autoclaving.
9. 70% ethanol and absolute ethanol.
10. Mix 37.5ml of absolute ethanol and 2.5ml of 3mol / L NaAc in the NaAc / ethanol mixture, and store at room temperature for 1 year.
11. POP 6 sequencing gel ABI product.
12. Template inhibition reagent (TSR) ABI product.
13.10 × ABI product of electrophoresis buffer.
14. ABI PRISM 310 automatic DNA sequencer.
15. Model 2400 or 9600 PCR instrument.
16. Desktop refrigerated high-speed centrifuge.
17. Desktop high-speed centrifuge or pocket centrifuge.

【Steps】
1. PCR sequencing reaction
(1) Take a 0.2ml PCR tube, number it with a marker, insert the tube in the pellet ice, and add reagents according to the following table:
Standard control tube for added reagent determination template tube
BigDye Mix 1μl 1μl
1μl of plasmid DNA to be tested －
pGEM-3Zf (+) double-stranded DNA-1μl
1μl forward primer of DNA to be tested －
M13 (-21) primer-1μl
Sterile deionized water 2μl 2μl
The total reaction volume is 5μl, without adding light mineral oil or paraffin oil, close the PCR tube tightly, mix with a finger bomb tube, and centrifuge slightly.
(2) Place the PCR tube on the 9600 or 2400 type PCR instrument for amplification. After denaturation at 98 ℃ for 2min, PCR cycle was carried out. The PCR cycle parameters were 96 ℃ 10s, 50 ℃ 5s, 60 ℃ 4min, 25 cycles. After the amplification, set 4 ℃ to keep warm.
2. Purification of PCR products by sodium acetate / ethanol method
(1) Centrifuge the mixture and transfer the amplified product to a 1.5ml EP tube.
(2) Add 25μl of sodium acetate / ethanol mixture, shake thoroughly, and place on ice for 10min to precipitate DNA. Centrifuge at 12,000 r / min at 4 ° C for 30 min, and carefully discard the supernatant.
(3) Add 50% of 70% (V / V) ethanol to wash the pellet twice. Centrifuge at 12,000r / min at 4 ° C for 5min, carefully discard the supernatant and the beads on the tube wall, and dry the precipitate in vacuum for 10-15min.
3. Processing of sequencing PCR products before electrophoresis.
(1) Add 12μl of TSR to the centrifuge tube, shake vigorously to allow it to fully dissolve the DNA pellet, and centrifuge it slightly.
(2) Transfer the solution to a 0.2ml PCR tube separated by a lid and centrifuge it slightly.
(3) Perform heat denaturation (95 ° C 2min) on the PCR instrument, quench in ice, and wait for the machine to be used.
4. Install the capillary according to the operation manual of the instrument, correct the position of the capillary, manually fill the glue manually and establish the running sequence file. The instrument will automatically pour glue into the capillary, pre-electrophoresis at 1.2kV for 5min, inject samples automatically according to the programmed sequence, then pre-electrophoresis (1.2kV, 20min), and electrophorese at 7.5kV for 2h. After the electrophoresis is completed, the instrument will be automatically cleaned, filled with glue, enter the next sample, pre-electrophoresis and electrophoresis. The total electrophoresis time of each sample is 2.5h. After electrophoresis, the instrument will automatically analyze or print out the color sequencing map.
5. The instrument will automatically perform sequence analysis and sequence comparison according to user requirements. If the sequencing sequence is known, the difference base can be marked with an asterisk through sequence comparison to improve work efficiency.
6. After sequencing, perform instrument cleaning and maintenance according to the instrument operating procedures.

【Calculation】
Sequencing reaction accuracy calculation formula: 100%-difference base number (not including N number) / 650 × 100%
Differential bases are the bases where the determined DNA sequence is compared with the known standard DNA sequence. N is the base that the instrument cannot read.

【Notes and evaluation】
1. The ABI PRISM 310 Genetic Analyzer is a high-end precision instrument that requires dedicated personnel to operate, manage and maintain it.
2. In this experiment, the total volume of the PCR reaction is 5μl, and it is not covered with mineral oil, so the tightness of the PCR tube cap is very important. In addition to closing the PCR tube cap after adding the reagent, it is best to use the PCR tube of PE company. If the PCR solution is less than 4 to 4.5 μl after the PCR is completed, the PCR reaction may fail, and purification and sample loading are not necessary.
3. As a sequencing user, you only need to provide purified DNA samples and primers. The template used in a sequencing PCR reaction is different, and the amount of DNA required is also different. The amount of template required for PCR sequencing is less. Generally, the PCR product needs 30 ～ 90ng, 50-100ng for single-stranded DNA and 200-500ng for double-stranded DNA. The purity of DNA is generally A260nm / A280nm is 1.6-2.0. It is best to use deionized water or triple distilled water to dissolve the DNA without TE buffer. It is better to make the primer into 3.2pmol / μl with deionized water or three distilled water.
4. The sequencing kit used in this experiment is a BigDye fluorescent labeling termination substrate cycle sequencing kit. The DNA length can generally be measured to be about 650bp. The DNA sequencing accuracy of this instrument is (98.5 ± 0.5)%, the base N which can not be read by the instrument is less than 2%, and the length of the determination exceeds 650bp, additional primers need to be designed. In order to ensure more accurate sequencing, reverse primers can be designed to sequence the same template and confirm each other. The N bases can be checked manually and sometimes can be read out. In order to improve the accuracy of sequencing, according to the position indicated by the asterisk, the color atlas can be manually analyzed and the bases can be further checked.

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