DNA sequence determination 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 DNA sequence analysis today. PE ABI Company of the United States has produced DNA sequencers such as 373, 377, 310, 3700 and 3100. Among them, 310 is the most commonly 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
The 310 genetic analyzer (i.e. DNA sequencer) uses capillary electrophoresis technology to replace the traditional polyacrylamide plate electrophoresis and applies the company's patented four-color fluorescence Dye-labeled ddNTP (labeled terminator method), so through a single-primer PCR sequencing reaction, the PCR product generated 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 four fluorescent dyes can be electrophoresed in a capillary tube, thus avoiding the influence of mobility differences between lanes and greatly improving the accuracy of sequencing. Since molecules have different sizes, their mobility in capillary electrophoresis is also different. When they pass 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 them. The fluorescence is separated by a grating to distinguish different colors of fluorescence representing different base information, and is simultaneously imaged on a CCD camera. The analysis software can automatically convert the different fluorescence into DNA sequences to achieve the purpose of DNA sequencing. The analysis results can be output in various forms such as gel electrophoresis patterns, fluorescence absorption peak diagrams or base arrangement sequences.
It is a high-end precision instrument capable of automatic gel filling, automatic sample introduction, automatic data collection and analysis and other fully automatic computer-controlled determination of the base sequence or size and quantification of DNA fragments. PE Company also provides gel polymers, including DNA sequencing gel (POP 6) and GeneScan gel (POP 4). The pore sizes of these gel particles are uniform, which avoids the impact of inconsistent gel preparation conditions on sequencing accuracy. It mainly consists of capillary electrophoresis device, Macintosh computer, color printer and electrophoresis and other accessories. The computer includes software for data collection, analysis, and instrument operation. It uses the latest CCD camera detector to shorten DNA sequencing to 2.5 hours, and PCR fragment size analysis and quantitative analysis to 10 to 40 minutes.
Since this instrument has functions such as DNA sequencing, PCR fragment size analysis and quantitative analysis, it can perform DNA sequencing, heterozygote analysis, single-stranded conformation polymorphism analysis (SSCP), microsatellite sequence analysis, Long-segment PCR, RT-PCR (quantitative PCR) and other analyses, in addition to conventional DNA sequencing, can also be used for single nucleotide polymorphism (SNP) analysis, gene mutation detection, HLA matching, and forensic medicine in clinical practice. Paternity and individual testing, typing and identification of microorganisms and viruses, etc.
Reagents and equipment
1. BigDye Sequencing Reaction Kit The main reagent is BigDye Mix, which contains PE patented four-color fluorescently labeled ddNTPs and ordinary dNTPs, AmpliTaq DNA
polymerase FS, reaction buffer, etc.
2. pGEM-3Zf (+) double-stranded DNA control template 0.2g/L, kit supporting reagents.
3. M13(-21) primer TGTAAAACGACGGCCAGT, 3.2μmol/L, i.e. 3.2pmol/μl, supporting reagent of the kit.
4. DNA sequencing templates
can be PCR products, single-stranded DNA, plasmid DNA, etc. The template concentration should be adjusted to 1 μl during PCR reaction. The concentration of plasmid DNA measured in this experiment is 0.2g/L, that is, 200ng/μl.
5. Primers
It is necessary to design forward or reverse primers according to the DNA fragment to be measured, and prepare them to 3.2μmol/L, that is, 3.2pmol/μl.
If the recombinant plasmid contains a universal primer sequence, universal primers can also be used, such as M13(-21) primer, T7 primer, etc.
6． Sterilized deionized water or triple distilled water.
7. 0.2ml or separate from the 0.5ml PCR tube cap, product of PE company.
8.3mol/L sodium acetate (pH5.2) Weigh 40.8g NaAc?3H2O and dissolve it in 70ml distilled water. Adjust the pH to 5.2 with glacial acetic acid, adjust the volume to 100ml, and autoclave it before packaging. .
9. 70% ethanol and absolute ethanol.
10． NaAc/ethanol mixture: Take 37.5ml of absolute ethanol and 2.5ml of 3mol/L NaAc and mix well. It can be stored at room temperature for 1 year.
11. POP 6 sequencing gel ABI product.
12． Template Suppression Reagent (TSR) ABI Products.
13. 10× electrophoresis buffer ABI product.
14. ABI PRISM 310 fully automatic DNA sequencer.
15. 2400 or 9600 PCR instrument.
16． Benchtop refrigerated high-speed centrifuge.
17. Benchtop high speed centrifuge or pocket centrifuge.
Operation steps
1. PCR sequencing reaction
(1) Take a 0.2ml PCR tube, number it with a marker, insert the tube into granular ice, and add reagents according to the following table:
The added reagents are measured Template tube standard control tube
BigDye Mix 1μl 1μl
Plasmid DNA to be tested 1μl -
pGEM-3Zf (+) double-stranded DNA - 1μl
p>Forward primer of DNA to be tested 1μl -
M13(-21) primer - 1μl
Sterilized deionized water 2μl 2μl
The total reaction volume is 5 μl. Do not add light mineral oil or paraffin oil. Cap the PCR tube tightly, mix by flicking the tube with your finger, and centrifuge slightly.
(2) Place the PCR tube on the 9600 or 2400 PCR machine for amplification. After denaturing at 98°C for 2 minutes, perform a PCR cycle. The PCR cycle parameters are 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes, for 25 cycles. After amplification, set the temperature to 4°C for incubation.
2． Purification of PCR products by sodium acetate/ethanol method
(1) Centrifuge the mixture and transfer the amplification products to a 1.5ml EP tube.
(2) Add 25μl sodium acetate/ethanol mixture, shake thoroughly, and place on ice for 10 minutes to precipitate DNA. Centrifuge at 12 000 r/min for 30 min at 4°C, and carefully discard the supernatant.
(3) Add 50μl of 70% (V/V) ethanol to wash the pellet twice. Centrifuge at 12 000 r/min for 5 minutes at 4°C. Carefully discard the supernatant and liquid beads on the tube wall, and vacuum dry the precipitate for 10 to 15 minutes.
3． Processing of sequencing PCR products before electrophoresis.
(1) Add 12μl of TSR to the centrifuge tube, shake vigorously to fully dissolve the DNA precipitate, and centrifuge briefly.
(2) Transfer the solution to a 0.2ml PCR tube with a separated lid, and centrifuge slightly.
(3) Perform thermal denaturation on a PCR machine (95°C for 2 minutes), quench in ice, and wait for use on the machine.
4. Operation on the machine
Install the capillary according to the instrument operating instructions, correct the position of the capillary, manually fill the gel and establish the sequencing sequence file for the run. The instrument will automatically fill the gel into the capillary, perform pre-electrophoresis at 1.2kV for 5 minutes, automatically inject samples according to the programmed sequence, then pre-electrophores (1.2kV, 20min), and electrophores at 7.5kV for 2 hours. After electrophoresis, the instrument will automatically clean, fill gel, enter the next sample, pre-electrophoresis and electrophoresis. The total electrophoresis time for each sample was 2.5h. After electrophoresis, the instrument will automatically analyze or print out a color sequencing map.
5． The instrument will automatically perform sequence analysis and can perform sequence comparison according to user requirements. If the sequencing sequence is known, the difference bases can be marked with asterisks through sequence comparison to improve work efficiency.
6． After sequencing, clean and maintain the instrument according to the instrument operating procedures.
Calculation
Sequencing reaction accuracy calculation formula: 100% - number of difference bases (excluding N number)/650×100%
Difference bases That is, the measured DNA sequence is different from the known standard DNA sequence, and N is the base that cannot be read by the instrument.
Notes and comments
1. ABI PRISM 310 genetic analyzer is a high-end precision instrument that requires dedicated personnel to operate, manage and maintain.
2． The total volume of the sequencing PCR reaction in this experiment is 5 μl, and it is not covered with mineral oil, so the sealing of the PCR tube cover is very important. In addition to tightening the PCR tube cover after adding reagents, it is best to use PCR tubes from PE companies. 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 loading are not necessary.
3． As a sequencing user, you only need to provide purified DNA samples and primers. Different templates are used in a sequencing PCR reaction, and the amount of DNA required is also different. The amount of template required for PCR sequencing is smaller. Generally, the PCR product needs 30~ 90ng, single-stranded DNA requires 50-100ng, double-stranded DNA requires 200-500ng. The purity of DNA is generally A260nm/A280nm is 1.6-2.0. It is best to dissolve DNA in deionized water or triple distilled water instead of TE buffer. It is better to use deionized water or triple distilled water to prepare the primer at 3.2 pmol/μl.
4. The sequencing kit used in this experiment is the BigDye Fluorescent Labeled Termination Substrate Cycle Sequencing Kit, which can generally measure DNA lengths of about 650bp. The DNA sequencing accuracy of this instrument is (98.5±0.5)
%. The number of bases that cannot be read by the instrument is less than 2%. If the length required exceeds 650bp, additional primers need to be designed. To ensure more accurate sequencing, reverse primers can be designed to sequence the same template for mutual confirmation. The N base can be manually checked and sometimes can be read. In order to improve the accuracy of sequencing, according to the position indicated by the asterisk, the color map can be manually analyzed to further check the bases.
The specific configuration method can be found in the product manual you get, and most laboratories have relevant formulas.