DNA sequencing can be divided into manual sequencing and automatic sequencing. Manual sequencing includes Sanger dideoxy chain termination method and maxam-Gilbert chemical degradation methods. Automated sequencing has actually become the mainstream of DNA sequence analysis. American PE ABI Company produced 373, 377, 3 10, 3700 and 3 100 DNA sequencers, among which 3 10 is the most widely used model in clinical laboratory. This experiment introduces the sequencing principle and operating rules of ABI PRISM 3 10 DNA sequencer.
Principle ABI PRISM 3 10 gene analyzer (DNA sequencer) uses capillary electrophoresis technology instead of traditional polyacrylamide plate electrophoresis, and uses ddNTP (labeled terminator method) labeled with four-color fluorescent dyes patented by the company. Therefore, through single primer PCR sequencing reaction, the generated PCR product is a single-stranded DNA mixture with four different fluorescent dyes at the 3' end. The difference is 1 base, so that the sequencing PCR products of four fluorescent dyes can be electrophoresed in capillary, which avoids the influence of the mobility difference between lanes and greatly improves the sequencing efficiency. Because of the different molecular sizes, the mobility in capillary electrophoresis is different. When molecules pass through the capillary reading window, the CCD (Charge Coupled Device) camera detector in the laser detector window can detect fluorescent molecules one by one. The excited fluorescence is divided by grating to distinguish different colors of fluorescence representing different substrate information, and the images are synchronized on CCD camera. Analysis software can automatically convert different fluorescence into DNA sequence, so as to achieve the purpose of DNA sequencing. The analysis results can be output in various forms, such as gel electrophoresis map, fluorescence absorption peak map or base arrangement order.
It is an advanced precision instrument for measuring the base sequence, size and quantification of DNA fragments by computer automatic control, such as automatic glue 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). The pore size of these gel particles is uniform, which avoids the influence of inconsistent gel preparation conditions on sequencing accuracy. It is mainly composed of capillary electrophoresis device, Macintosh computer, color printer and electrophoresis accessories. Computers include software for data collection, analysis and instrument operation. It uses the latest CCD camera detector to shorten DNA sequencing to 2.5h hours, and PCR fragment size analysis and quantitative analysis are 10 ~ 40 minutes.
Because the instrument has the functions of DNA sequencing, PCR fragment size analysis and quantitative analysis, it can perform DNA sequencing, heterozygote analysis, single strand conformation polymorphism analysis (SSCP), microsatellite sequence analysis, long fragment PCR, RT-PCR (quantitative PCR) and so on. In addition to routine DNA sequencing, it can also carry out single nucleotide polymorphism (SNP) analysis, gene mutation detection, HLA matching and forensic medicine.
Reagents and equipment
The main reagent of 1 The Big Dye sequencing reaction kit is BigDye Mix, which contains four-color fluorescent labeled ddNTP, common dNTP patented by PE, AmpliTaq DNA polymerase FS, reaction buffer, etc.
2.PGEM-3ZF (+) double-stranded DNA control template 0.2g/L, kit matching reagents.
3.M 13 (-2 1) primer TGTAAAACGACGGCCAGT, 3.2μmol/L, which is the reagent of the kit.
4.DNA sequencing templates can be PCR products, single-stranded DNA and plasmid DNA. The template concentration should be adjusted, and the PCR reaction should be1μ L. The concentration of plasmid DNA determined in this experiment is 0.2g/L, that is, 200 ng/μ L. ..
5. Primers need to design forward or reverse primers according to the DNA fragment to be detected, and prepare 3.2μmol/L, that is, 3.2 pmol/μ L. If the recombinant plasmid contains universal primer sequences, universal primers such as M 13(-2 1) primer and T7 primer can also be used.
6. Disinfect with deionized water or triple distilled water.
7.0.2ml or separated from 0.5ml PCR tube cover, a product of PE company.
8.3 mol/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, and make it constant to 100ml, then pack it after autoclaving.
9.70% ethanol and anhydrous ethanol.
10. by mixing 37.5 ml absolute ethanol and 2.5 ml 3 mol/l sodium acetate, the NaAc ethanol mixture can be stored at room temperature for 65438 0 years.
1 1.pop6 sequencing gel ABI products.
12. template inhibitor (TSR) ABI products