ABI 310 sequencer DNA-sequencing of the principles and rules

DNA sequencing at manual and automatic sequencing sequencing, including the Sanger sequencing hand-dideoxy chain termination method and Maxam-Gilbert chemical degradation. In fact automated sequencing of DNA has become the mainstream of sequence analysis. U.S. PE ABI has produced 373-377-310-3700 and 3100, such as DNA-sequencing device, which is a 310-clinical testing laboratories used in most models. This experiment is described in the ABI PRISM 310-DNA sequencer sequencing of the principles and rules.

】 【Principle ABI PRISM
310-gene analysis (that is, DNA sequencer), using capillary electrophoresis to replace traditional flat-polyacrylamide gel electrophoresis, the company's patent application of the four-color fluorescent dyes labeled ddNTP (marking the termination method), through the Single-primer sequencing of PCR reaction, PCR product generated is the difference between a base of 3'''''''' for the end of the 4 different fluorescent dye mixture of single-stranded DNA, making four fluorescent dyes sequencing of PCR Can be a product of a capillary electrophoresis, thus avoiding the inter-lane mobility differences, greatly enhanced the accuracy of the sequencing. Due to the size of the different elements in the capillary electrophoresis mobility is also different from when reading through the capillary window above, the laser detector in the window CCD (charge-coupled device) camera detector of fluorescent molecules can be tested one by one, Excited fluorescence spectrometry by the grating in order to distinguish between different base of information on behalf of the different colors of fluorescent, and CCD imaging cameras simultaneously, the software will automatically change to a different fluorescent DNA sequence, so as to achieve the purpose of DNA sequencing. The results can gel electrophoresis, fluorescence peaks base map or order forms, such as the output.

It is automatically a plastic irrigation, automatic injection, automatic data collection, analysis, computer-controlled automatic determination of the sequence of base pairs of DNA fragments, or the size of the quantitative and high-end precision instruments. PE also offers gel polymers, including DNA sequencing gel (POP 6) and GeneScan plastic (POP 4). These gel particles uniform size, equipped with plastic to avoid the inconsistency of the conditions of sequencing accuracy. It mainly by capillary electrophoresis device, Macintosh computers, color printers, such as electrophoresis and the composition of the annex. Computers, including data collection, analysis and operation of equipment such as software. It uses the latest CCD camera detector, so that the DNA sequence has been shortened to 2.5h, PCR fragment size analysis and quantitative analysis for 10 ~ 40min.

As the DNA sequencing instruments have, PCR fragment size analysis and quantitative analysis functions, it can be carried out DNA sequencing, heterozygote analysis, single strand conformation polymorphism (SSCP), microsatellite analysis, long fragment PCR, RT -PCR (quantitative PCR) analysis, and so on, in addition to clinical routine DNA sequencing, can also carry out single nucleotide polymorphisms (SNP) analysis, gene mutations, HLA typing, on the forensic identification of individual and family , Micro-organisms and viruses such as typing and identification.

【Reagents and equipment】
1. BigDye sequencing reaction reagent kit is the main BigDye Mix, containing PE Patent four-color fluorescent ddNTP and the general dNTP, AmpliTaq DNA
polymerase FS, reaction buffer, and so on.
2. pGEM-3Zf (+) double-stranded DNA templates were 0.2g / L, matching reagent kit.
3. M13 (-21) primer TGTAAAACGACGGCCAGT, 3.2μmol / L, that is, 3.2pmol/μl, matching reagent kit.
4. DNA sequencing template
PCR can be a product of single-stranded DNA and the DNA plasmid, and so on. Template concentration should be adjusted in the PCR reaction volume of 1μl get better. Experimental determination of the plasmid DNA, concentration of 0.2g / L, that is, 200ng/μl.
5. Primer
On the basis of the determination to take the DNA fragments are designed or reverse primer, preparation 3.2μmol / L, that is, 3.2pmol/μl. If the recombinant plasmid containing universal primer sequences can also be used universal primer, such as the M13 (-21) primer, T7 primers, and so on.
6. Sterilization deionized water or distilled water three.
7.0.2ml or PCR tubes and 0.5ml of isolation cover, PE products.
8.3mol / L sodium acetate (pH5.2) that take 40.8g NaAc? 3H2O dissolved in 70ml distilled water, glacial acetic acid pH adjusted to 5.2, the volume to 100ml, hours after the high-pressure sterilization equipment.
9.70 percent ethanol and ethanol.
10. NaAc / ethanol mixture of ethanol and 37.5ml take 2.5ml 3mol / L NaAc blending, to save room temperature for 1 year.
11. POP 6 sequencing ABI plastic products.
12. Template suppression reagent (TSR) ABI products.
13.10 × electrophoresis buffer ABI products.
14. ABI PRISM 310 automatic DNA sequencer.
15.2400-9600 or PCR-based instrument.
16. Frozen high-speed desktop centrifuge.
17. High-speed desktop centrifuge or centrifuges pocket.

Key steps】
1. Sequencing of PCR reaction
(1) 0.2ml take control of the PCR, marker number tube will be inserted in the ice particles in the table below plus reagent:
The increase in the standard template reagent measured in the control tube
BigDye Mix 1μl 1μl
The test plasmid DNA 1μl --
pGEM-3Zf (+) double-stranded DNA - 1μl
DNA test positive primer 1μl --
M13 (-21) primer - 1μl
Deionized water sterilization 2μl 2μl
The total volume of reaction 5μl, no light mineral oil or paraffin oil, Gaijin PCR tube, pipe bombs mixing fingers, slightly centrifuge.

(2) PCR tube placed in 9600 or 2400 based on PCR amplification instrument. 98 ℃ degeneration 2min after the PCR cycle, PCR parameters to Circulating 96 ℃ 10s, 50 ℃
5s, 60 ℃ 4min, 25 cycles, amplified after the end of the heat setting 4 ℃.

2. Sodium acetate / ethanol purified PCR product
(1) centrifuge mixture, amplified products will be transferred to 1.5ml EP tube.
(2) by adding 25μl sodium acetate / ethanol mixture, full oscillation, home ice 10min to precipitate DNA. 12 000r/min at 4 ℃ centrifuge 30 min, the supernatant discarded carefully.
(3) plus 70% (V / V) ethanol washing 50μl precipitation 2. 12 000r/min at 4 ℃ centrifuge 5min, care of abandoned liquid supernatants wall and beads, vacuum drying precipitation 10 ~ 15min.

3. Before electrophoresis sequencing of the PCR product of the deal.
(1) by adding 12μl of the TSR in centrifuge tubes, severe vibration, to dissolve the full DNA precipitation, centrifugal slightly.
(2) solution will be to build the separation 0.2ml PCR tube, slightly centrifuge.
(3) in the PCR instrument on thermal denaturation (95 ℃ 2min), in ice cold at first, to be on the plane.

4. Operation on
Manual operation of equipment by capillary installed, the location of the capillary correction, manually artificial plastic irrigation operation and the establishment of the sequencing of the order paper. The device will automatically filling plastic to capillary, 1.2kV electrophoresis pre-5min, according to the order of auto-injection program, and then pre-electrophoresis (1.2kV, 20min), the 7.5kV electrophoresis under 2h. After the electrophoresis apparatus will be self-cleaning, filling plastic, into the next sample, and pre-PAGE electrophoresis. Each sample of the total time for electrophoresis 2.5h. After the electrophoresis apparatus will automatically print out the color analysis or sequencing map.

5. The device will automatically sequence analysis, and based on user requirements sequence comparison. If the sequence of known sequence, the sequence can be compared with an asterisk marked difference base, and enhance efficiency.

6. Completed by sequencing equipment and cleaning procedures for equipment maintenance.

By calculating】
Sequencing reactions accuracy formula: 100% - difference in the number base (N does not include the number) / 650 × 100%
That is, differences in the determination of the base sequence of DNA known standard DNA sequence comparison of different bases, N for the equipment can not read the base.
【Notes and Evaluation】
1. ABI PRISM 310 genetic analyzer is a high-end precision instruments, to be special operations, management and maintenance.
2. In this study sequencing of the PCR reaction of the total volume was 5μl, and not covered by mineral oil, the PCR tube covered sealing is very important, with the exception of Canada finished after Gaijin PCR reagent tube cover, the best selection of the company's PE tube PCR. If the PCR after the end of the PCR solution is less than 4 ~ 4.5μl, the PCR reaction is likely to fail, do not have to carry on and the kind of purification.
3. Sequencing as users only need to provide good purification of DNA samples and primers, a sequence of the PCR reaction using the template, the need for DNA will be different amount, PCR sequencing of the required template low, generally a product of PCR To be 30 ~ 90ng, single-stranded DNA to be 50 ~ 100ng, double-stranded DNA to be 200 ~ 500ng, DNA purity A260nm/A280nm generally 1.6 to 2.0, the best deionized water or distilled water three dissolved DNA, do not have to TE buffer Liquid solution. Primer deionized water or distilled water three 3.2pmol/μl a good match.
4. Sequencing of Experimental use of this kit is a fluorescent BigDye termination of the substrate cycle sequencing kit, a general DNA test can be about 650bp in length. Of the DNA sequencing machines for accuracy (98.5 ± 0.5)
% Identified instruments can not read the base N <2%, the required determination of the length of more than 650bp, need to design another primer. In order to ensure a more accurate sequencing can be designed reverse primer on the same template for sequencing, and confirmed with each other. N for the base can be checked manually, in some cases can be read out. In order to enhance the accuracy of sequencing, according to the location of the star tips, analysis of the artificial color map of the premises, the Department of the base for further checking.

Sanger dideoxy sequencing principle

The need for DNA replication: DNA polymerase, DNA single-strand template, with a 3'-OH at the end of the single-stranded oligonucleotide primers, 4 dNTP (dATP, dGTP, dTTP and dCTP). Using polymerase template for guidance, constantly dNTP will be added to the primer 3'-OH at the end, so that the primer extension, the synthesis of new complementary DNA strand. If a particular nucleotide, double-nucleoside triphosphate (ddNTP), as a result of deoxyribose in the 3 'position of a lack of hydroxy can not be formed with the follow-up to the phosphodiester dNTP key. For example, there is ddCTP, dCTP and the other three dNTP (one mark for the α-32P), to primers, DNA polymerase template and insulation together, can form a whole has the same primer 5'- Side and ddC residues for the 3 'end at the end of a series of fragments of different lengths of the mixture. By denaturing polyacrylamide gel electrophoresis separation obtained from the radioactive zone map for developing a new synthesis of different length of the DNA chain of distribution of C to provide accurate information, so as to the location of C will be determined. A similar approach in ddATP, ddGTP and ddTTP existing conditions, can be obtained at the same time were ddA, ddG and ddT residue for the 3 'end of the head of the three short fragments of varying. The system will be a mixture of four parallel increase in the place of denaturing polyacrylamide gel electrophoresis gel electrophoresis board for each product in each of the components according to their length will be different from the separation, obtained from the radioactive Map image. Map can be obtained directly from the reading of the DNA base sequences.