Sequencing technology of DNA sequencing

High-throughput sequencing, also known as the "next generation sequencing technology", is characterized by being able to measure hundreds of thousands to millions of DNA molecules in parallel at one time, and generally has a short reading length.

According to the development history, influence and different sequencing principles and technologies, there are mainly the following types: massive parallel signature sequencing, MPSS, Polony sequencing, 454 pyrophosphate sequencing, Illumina (Solexa) sequencing, ABI solid-state sequencing, ionic semiconductor sequencing, DNA nanosphere sequencing and other gene analyzers (DNA sequencers) use capillary electrophoresis technology instead of traditional polyacrylamide plate electrophoresis, and use ddNTP 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, with a difference of 1 base, so that the sequencing PCR products of the four fluorescent dyes can be electrophoresed in a capillary tube, avoiding swimming lanes. Due to the different molecular sizes, the mobility in capillary electrophoresis is also 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.

I. Preparatory work

The main reagent of 1 The BigDye 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 0.2 g/L, kit matching reagents.

3.M 13(-2 1) primer TGTAAAACGACGGCCAGT, 3.2 μmol/L, 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 0.2 g/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 3.2 μmol/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 separate from 0.5ml PCR tube cover.

8.3 mol/L sodium acetate (pH5.2) Weigh 40.8g NaAc·3H2O, dissolve it in 70 ml distilled water, adjust the pH to 5.2 with glacial acetic acid, make the volume to 100 ml, and then pack it after autoclaving.

9.70% ethanol and anhydrous ethanol.

10.NAAC/ ethanol mixture can be stored at room temperature 10. NaAc/ By mixing 37.5 ml absolute ethanol and 2.5 ml 3 mol/L NaAc.

1 1.POP 6 sequencing gel.

12. template inhibitor (TSR).

13. 10× electrophoresis buffer.

14. Automatic DNA Sequencer.

15.PCR instrument

16. Desktop freezing high-speed centrifuge.

17. Desktop high-speed centrifuge or pocket centrifuge.

Second, PCR sequencing reaction

1. Take 0.2 ml PCR tubes, mark them with markers, insert the tubes into granular ice, and add reagents according to the following table:

Standard control tube for measuring template tube with added reagent.

BigDye mixture 1 microliter 1 microliter

Plasmid DNA 1 μ l-

PGEM-3Zf (+) double-stranded DNA- 1 μ l

Forward primer 1 μ l-

M 13(-2 1) primer-1μ l

Sterile deionized water 2 microliters 2 microliters

The total reaction volume is 5 μl, light mineral oil or paraffin oil is not added, the PCR tube is tightly covered, evenly mixed with finger elastic tube, and slightly centrifuged.

2. Put the PCR tube on the 9600 or 2400 PCR instrument for amplification. After denaturation at 98℃ for 2 min, the PCR cycle parameters were 96℃ 10 s, 50℃ 50℃ 5 s, 60℃ for 60℃4 min, and the temperature after amplification was set at 4℃.

3. Purification of PCR products by sodium acetate/ethanol method.

1. Centrifuge the mixture, and transfer the amplified product to 1.5 ml EP tube.

2. Add 25 μl sodium acetate/ethanol mixed solution, shake well, and put it on ice 10 min to precipitate DNA. Centrifuge at 4℃ at 12 000 rpm for 30 minutes, and carefully discard the supernatant.

3. Add 50μ l of 70% (v/v) ethanol and wash the precipitate twice. Centrifuge at 4℃ 12000 r/min for 5 min, carefully discard the supernatant and liquid beads on the tube wall, and vacuum dry the precipitate 10 ~ 15 min.

Fourthly, the treatment of sequencing PCR products before electrophoresis

1. Add 12 μl TSR to the centrifuge tube, shake vigorously, let it fully dissolve the DNA precipitate, and centrifuge slightly.

2. Transfer the solution to a 0.2 ml PCR tube separated from the cover and centrifuge slightly.

3. Perform thermal denaturation on PCR instrument (95℃ for 2 minutes), and then quench in ice for later use.

Verb (abbreviation of verb) is operated on 1 computer. Install the capillary tube according to the operating instructions of the instrument, correct the position of the capillary tube, fill the glue manually, and establish the running sequence file. 2. The instrument will automatically inject the glue into the capillary, pre-electrophorese for 5 minutes at 1.2 kV, automatically inject the sample according to the programmed sequence, pre-electrophorese for 20 minutes at 1.2 kV, and electrophorese for 2 hours at 7.5kV. 3. After the electrophoresis, the instrument will automatically clean, fill the glue, and enter the next sample for pre-electrophoresis and electrophoresis. 4. The total electrophoresis time of each sample is 2.5 hours ... 5. After electrophoresis, the instrument will automatically analyze or print out the color sequencing map.

Six, the sequence analyzer will automatically sequence analysis, and can be compared according to user requirements. If the sequencing sequence is known, different bases can be marked with asterisks through sequence alignment to improve work efficiency.

Seven, instrument cleaning and sequencing according to the instrument operation procedures for instrument cleaning and maintenance.

Eight. calculate

1. Calculation formula of sequencing reaction accuracy: 100%- number of differential bases (excluding N number) /650× 100%.

2. Differential base refers to the base whose determined DNA sequence is different from the known standard DNA sequence, and N is the base that cannot be read by the instrument. SILVER SEQUENCETM DNA sequencing system is a non-radioactive sequence analysis system, which detects bands in gel by sensitive silver staining method. Silver staining provides a faster and cheaper alternative to radioactivity or fluorescence. Sequencing results can be obtained on the same day; The sequence can be read 90 minutes after electrophoresis, which is impossible for conventional radioactive sequencing. In addition, the SILVER SEQUENCETM system uses unmodified 5'OH oligonucleotides as primers, which reduces the cost of specially modified oligonucleotides. The system does not need to handle isotopes carefully in radioactive methods, nor does it need expensive fluorescent or chemiluminescence reagents. In addition, there is no need to use instruments to detect sequence bands like most fluorescence methods.

Taq DNA polymerase has strong thermal stability at 95℃. The sequencing-grade Taq DNA polymerase used in this system is an improved product of Taq DNA polymerase, which has a very good effect on double-stranded DNA templates, high accuracy, uniform bands and low background.

The SILVER SEQUENCETM system contains a modified nucleotide mixture, such as 7- deazadgtp (7-deazadgtp, or dITP) instead of dgtp, which can eliminate the band compression caused by GC-rich regions.

Annealing temperature is the most important factor in thermal cycle sequencing. High annealing temperature can reduce the secondary structure of the template. Improve the rigor of primer-template pairing. Chain annealing and the secondary structure of the template limit that primers with 50% content of small fragment PCR products (:24mer GC can get the best results.

Compared with conventional sequencing methods, this system has the following advantages: (1). In this method, the linear amplification of template DNA produces enough products to enable silver staining technology to detect sequence bands. The sequencing reaction needs 0.03 ~ 2 pmol of template DNA, which depends on the template type. (2) The high temperature in each denaturation cycle can replace the alkali denaturation and ethanol precipitation of double-stranded DNA(dsDNA) templates, and the denaturation cycle can also help to eliminate the problems caused by the rapid annealing of linear dsDNA templates (such as PCR reaction products). (3) High temperature polymerase reaction weakens the secondary structure of DNA template, allowing polymerase to pass through the highly secondary structure region.

First, reagent preparation

1. silver sequencing DNA sequencing kit.

2. Acrylamide and methylene bisacrylamide stock solution (38% acrylamide W/V, 2% methylene bisacrylamide w/v): 95 g acrylamide, 5 g methylene bisacrylamide dissolved in 140 ml double distilled water, constant volume to 250 ml, filtered with a 0.45 mm filter, stored in a brown bottle and stored in a refrigerator at 4℃ for 2 weeks.

3. 10% ammonium persulfate, dissolve 0.5 g ammonium persulfate in 4 ml water, and make the volume to 5 ml, which should be a new use.

4. 10×TBE buffer (1 mol/L Tris, 0.83mol/L boric acid,10 mmol/l EDTA):121.1g Tris, 5.

5.TBE electrode buffer: dilute 10×TBE buffer to 1×TBE for later use.

6. Temed

7. Fixed/stopped solution: 2 liters 10% glacial acetic acid (V/V) for standby.

8. Dyeing solution: 2 g silver nitrate and 3 ml formaldehyde, dissolved in 2 liters of ultrapure water for later use.

9. Developer: dissolve 60 g of sodium carbonate (Na2CO3) in 2 liters of ultrapure water, and add 3 ml of 37% 37% formaldehyde and 40 ml of sodium thiosulfate solution (10 mg/ml) before use.

10.95% ethanol.

1 1.0.5% glacial acetic acid.

12. Sigma Cote (smart cat. #SL-2).

Second, sequencing reaction.

1. For each sequencing reaction, label four 0.5 ml eppendorf tubes (g, a, t, c). Add 2 ml of the appropriate d/ddNTP mixture (d/ddNTP mixture) to each test tube. Add 1 drop (about 20 μl) of mineral oil, cover and store on ice or at 4℃.

2. For each group of four sequencing reactions, mix the following reagents in the eppendorf test tube:

(1) sample reaction:

Plasmid template DNA :2. 1 pmol

5 times sequencing buffer: 5 ml.

Primer: 4.5 pmol

The final volume of sterile ddH2O is16ml.

(2) controlling the reaction

PGEM-3Zf(+) control DNA (4 mg): 4.0 ml.

5 times sequencing buffer: 5 ml.

PUC/M 13 forward primer (4.5 pmol): 3.6 ml.

3. Add 1.0 ml sequencing-grade Taq DNA polymerase (5 μ/ml) to the primer/template mixture (step 2 above). Suck several times with a pipette and mix well.

4. Extract 4 ml from the enzyme/primer/template mixture in step 3 and add it to the test tube of each d/ddNTP mixture.

5. Centrifuge in a micro centrifuge so that all solutions are at the bottom of the eppendorf tube.

6. Put the reaction tube into a thermal cycler preheated to 95℃, and start the cycle program according to the intermediate cycle mode. The optimum annealing temperature must be selected for each primer/template combination. The following procedure can generally read the length of 350 bases from primers.

7. After the thermal cycling procedure is completed, add 3 μlDNA sequencing termination solution into each small tube and rotate it in a micro centrifuge to terminate the reaction.

pay attention to

The amount of template DNA used for (1) sequencing is usually added according to the following requirements:

Template Type/Length Number of Templates

200 base pairs (PCR product): 16 ng (120 fmol)

3000~5 000 bp (supercoiled plasmid DNA): 4 mg (2 pmol)

48 000 base pairs (λ, cosmid DNA): 1 mg (3 1 fmol)

Because the signal generated by supercoiled plasmid is weaker than that of relaxed linear double-stranded DNA, the amount of supercoiled plasmid used as template is larger than that of other templates.

(2) The following general formula can be used to calculate the nanogram number of primers equivalent to 4.5 pmol:

4.5 pmol= 1.5 ng×n, where n is the number of primer bases.

The following general formula can be used to calculate the microgram number of primers equivalent to 1p mol:

Dsdna:1pmol = (6.6×10 mg )× n, where n is the logarithm of the template.

Ssdna:1pmol = (3.3×10 mg )× n, where n is the number of template bases.

(3) In order to prevent Taq DNA polymerase from extending nonspecific annealing primers, the thermal cycler must be preheated to 95℃. The temperature should change as quickly as possible. The following cycle time does not include temperature change time. If you are not sure which mode to use, it is recommended to start with mode 1.

Mode 1: Suitable for primer.

95℃ for 2 minutes, then: 95℃ for 30 seconds (denaturation), 42℃ for 30 seconds (annealing), 70℃ 1 minute (extension).

Mode 2: Suitable for primers with GC content ≥24 bases or slightly shorter than 50%.

95℃ for 2 minutes, then: 95℃ for 30 seconds (denaturation) and 70℃ for 30 seconds (annealing/extension).

(4) After the termination solution is added, the sample can stay overnight at 4℃.

3. Preparation of sequencing gel plate

1. Treatment of glass plate

Silver stained glass plates must be very clean. Generally, it is washed with warm water and detergent first, then with deionized water to remove the residual detergent, and finally with ethanol. The detergent microfilm left on the glass plate may lead to high background (brown) during gel dyeing. The gel can be chemically crosslinked on short glass plates after being treated with adhesive solution. This step is very important to prevent the gel from tearing during the silver staining operation.

Treatment of (1) short glass plate

① Add 5ml of adhesive silane into 1 ml of 95% ethanol and 0.5% glacial acetic acid to make a new adhesive solution.

② Wipe the carefully cleaned and naturally dried glass plate with absorbent cotton paper soaked with newly prepared glue solution, and the whole plate surface must be wiped.

③ After 4 ~ 5 minutes, wipe the glass plate with 95% ethanol in one direction, and then wipe it vertically with a little force. Repeat this cleaning process three times, removing the excess adhesive solution with clean paper each time.

pay attention to

① When wiping the glass plate with 95% ethanol in one direction, excessive force will bring too much adhesive silane, which will make the gel not adhere well.

② Replace gloves before preparing the long glass plate to prevent silane adhesion and adhesion.

③ It is very important to prevent the glue from polluting the long glass plate, otherwise the gel will tear.

(2) Treatment of long glass plate:

① Wipe the clean long glass plate with cotton paper soaked in Sigmacote solution.

② After 5 ~10 minutes, wipe the glass plate with absorbent cotton paper to remove excess Sigmacote solution.

pay attention to

① You can soak the used gel in water and scrape it off with a shaving blade or a plastic scraper. The glass plate must be thoroughly cleaned with detergent. Or remove the gel after soaking in 10%NaOH. In order to prevent cross-contamination, tools for cleaning short glass sheets must be separated from tools for cleaning long glass sheets. If cross contamination occurs, the gel prepared later may tear or become loose.

2. Preparation of gel

(1) The glass plate can be fixed after being treated with adhesive silica gel and Sigmacote. In this method, 0.2 mm or 0.4 mm thick edge strips are placed on the left and right sides of the glass plate, and another glass plate is pressed on it. Insert the flat edge of the shark tooth into one side of the long glass plate and fix it with a clip.

(2) According to the required gel concentration, prepare sequencing gel according to the following table. Generally, the gel concentration of 6% ~ 8% can achieve good results. In the preparation process, urea is dissolved in proper amount of double distilled water, and then ACR & amp； Bis and 10×TBE buffer, then adjust the final volume to 99.2 ml with double distilled water, filter with 0.45 mm filter membrane, and then add ammonium persulfate and TEMED. No heating is needed to dissolve urea. If heating is really needed, TEMED and ammonium persulfate can only be added after the solution is completely cooled. Generally, polymerization begins 4 ~ 6 minutes after pouring. If the polymerization is not good, high concentration of TEMED and ammonium persulfate should be used.

(3) After the glue is prepared, the glue board can be poured. Generally, the gel is slowly poured into the groove of the glass plate along the edge of delamination, and then allowed to stand for complete polymerization.

pay attention to

(1) When fixing the glass plate with a fixture, it is best to use a fixture with a little strength to prevent the glue from leaking due to insufficient strength during the glue pouring process.

(2) Bubble generation should be strictly prevented during glue filling, otherwise the sequencing results will be affected.

Fourthly, electrophoresis.

1. Preelectrophoresis

(1) After the gel polymerization is completed, pull out the shark tooth comb, invert the comb, and insert the tooth end into the gel to form a sample adding hole.

(2) Fix the gel plate in the sequencing gel tank immediately. Usually, the upper and lower wells of sequencing gel wells are separated, so the buffer can only be added after the gel plate is fixed.

(3) Dilute the buffer solution of 10×TBE to 1×TBE, add the buffer solution into the upper and lower electrophoresis tanks, remove the generated bubbles, and turn on the power supply to prepare for pre-electrophoresis.

(4) Some electrophoresis tanks, such as Macrophor of LKB, are heated by water bath, so the water bath should be heated to 55℃ before pre-electrophoresis. Some do not use water bath heating, but rely on the heat generated by themselves during electrophoresis, such as the sequencing electrophoresis tank produced by Shanghai Precision Plexiglass Instrument. This tank needs to be sandwiched with two heat dissipation aluminum plates to make the temperature of the whole gel plate consistent.

(5) Pre-electrophoresis at a voltage of 30 V/cm for 20-30 minutes. The process of pre-electrophoresis is to remove impurity ions in the gel and make the gel plate reach the required temperature at the same time. High temperature electrophoresis can prevent the formation of hairpin structure in GC-rich regions and affect the sequencing results.

pay attention to

(1) When making the sample adding hole with shark tooth comb, it is necessary to pay attention to that the tooth tip should be inserted into the glue for about 0.5mm, and the sample adding hole should not be leaked, otherwise the correct result will not be obtained.

(2) always pay attention to whether the buffer in the upper electrophoresis tank leaks, otherwise it is easy to cause short circuit and damage the electrophoresis instrument.

2. Preparation of samples

During pre-electrophoresis, samples can be prepared, and the reacted samples can be heated in boiling water bath for 1 ~ 3 minutes and immediately placed on ice. If the sample is not used for a long time, it should be reprocessed. 4 ~ 6% polyacrylamide gel with a thickness of 0.4 mm can be used. Glue with a thickness less than 0.4 mm may cause the signal to be too weak. When adding samples, it is not necessary to suck the mineral oil covered by the upper layer, but carefully suck the blue sample under the mineral oil.

3. Sample loading and electrophoresis

Turn off the electrophoresis apparatus, use a pipette to suck the buffer to clean the sample hole, remove the urea diffused during pre-electrophoresis, and then immediately suck the sample with a capillary sampler and add it to the sample hole. The sequence of sample addition is generally G, A, T and C, and electrophoresis is performed immediately after sample addition. At the beginning, electrophoresis can be carried out at 30 V/cm, and it can be increased to 40 ~ 60 v/cm after 5 minutes, and the constant pressure is maintained. Generally speaking, a gel plate with a length of 55 cm and a thickness of 0.2 mm can reach the bottom after electrophoresis for 2 hours at a constant voltage of 2500 V, and the current can be steadily reduced from 28 mA to 25 mA during electrophoresis. To read a longer sequence, two or more rounds of loading can be used.

pay attention to

① When loading the sample for electrophoresis, be sure to pay attention to whether the temperature of the gel plate reaches about 55℃. If not, you should wait until the temperature reaches.

② Generally speaking, it is not advisable to use too high voltage in electrophoresis, because too high voltage will reduce the resolution of gel and make the bands spread. Electrophoresis can use constant power electrophoresis.

V silver staining of sequencing gel

The dyeing process needs to immerse the gel in a plastic dish. Therefore, at least two plates similar in size to glass plates are used. Wash dishes with high quality water before adding fresh solution to them.

1. After electrophoresis, carefully separate the two plates with a plastic sheet, and the gel should be firmly attached to the short glass plate.

2. Gel fixation: put the gel (with glass plate) into a plastic tray, immerse it in the fixing/stopping solution, and fully shake it for 20 minutes or until the dye in the sample completely disappears. The gel can be stored in the fixed/terminated solution overnight (without shaking). The fix/stop solution is used to terminate the color reaction.

3. Glue washing: Wash the glue with ultrapure water for 3 times, 2 minutes each time. Take it out of the water, hold the edge of the rubber plate when transferring to the next solution, and let it stand for 10 ~ 20 seconds to make the water flow out.

4. Gel dyeing: transfer the gel into the dyeing solution and shake it fully for 30 minutes.

5. Gel development

(1) formaldehyde (3 ml) and sodium thiosulfate solution (400 μl) were added to the developer to complete the preparation of the developer.

(2) Take out the gel from the dyeing solution, and put it in a dish filled with ultrapure water for soaking and washing for 5- 10 second. Note that the total time of gel transfer from ultrapure water to developer should not exceed 5 ~ 10 second. Soaking for too long will lead to weak signal or signal loss. If the soaking time is too long, you can repeat the fifth step and soak it with dye solution.

(3) Immediately transfer the gel into 1L (half of the total amount) precooled developer, fully shake until the template bands begin to appear or the first batch of bands begin to appear, and transfer the gel into the remaining 1L developer to continue developing for 2-3 minutes or until all bands appear.

6. Fixing glue: directly add the same volume of fixing/stopping liquid into the developer. Stop the development reaction and repair the gel.

7. Soak the gel in ultrapure water twice for 2 minutes each time. Pay attention to put on gloves and hold the edge of the glue board to avoid printing fingerprints on the glue during this operation.

8. Dry the gel at room temperature, or dry the gel by pumping and heating. Observe the gel on a visible light box or a bright white and yellow background (such as paper). If permanent recording is needed, the experimental results can be saved with EDF film.

pay attention to

Silver staining of sequencing products is a new method to reveal sequence information, and the success or failure of this system is influenced by many factors.

① Water quality is extremely important for the success of dyeing. Ultra-pure water (NANOpureR or Milli-QR water) or double distilled water can achieve good results. If there are impurities in the water, low molecular weight bands may not appear.

② Sodium carbonate is also very important. It is best to use fresh, American Chemical Society grade sodium carbonate, such as Fisher and Kodak ACS reagent grade sodium carbonate (Fisher catalog #S263-500 or S262-3, or Kodak catalog # 109- 1990), and generally better results can be obtained.

③ The washing step after coloring is very important. If the gel washing time is too long, silver particles will be separated from DNA, resulting in little or no sequence signal. If the washing time is too long, the dyeing step can be repeated.

④ If the gel thickness exceeds 0.4 mm or the acrylamide concentration is higher than 4 ~ 6%, it is necessary to extend the fixation and dyeing time. If the gel is thinner than 0.4 mm, the washing after the dyeing reaction must be shortened to no more than 5 seconds.

⑤ All the steps were carried out at room temperature except the color reaction. The developer must be precooled to 10 ~ 12℃ to reduce the background noise. Note: Before use, add formaldehyde and sodium thiosulfate to the developer. Use the newly prepared dyeing and developing solution. Do not reuse any solution.

Sixth, the development of EDF films.

The contrast of sequencing bands can be enhanced by using EDF film. If the bands on the sequencing gel are shallow, we suggest transferring the data to EDF film. Silver-dyed glue can enhance the readability of the strip after its image is transferred to EDF film.

1. In the darkroom, put the dyed gel (with the adhesive side facing up) on the fluorescent lamp box. If there is a suitable diffuser, you can also use a white light box. In order to ensure the exposure time, a small piece of EDF film was exposed at different times and different exposure intensities were checked. Generally, exposure for 20 ~ 40 seconds can get better results.

2. Find the missing corner of EDF film under red light, and then put the film on the gel so that the gap is located in the upper left corner. Since the EDF film is single-sided, it is necessary to ensure that the gap is in the upper left corner.

3. Put a clean and dry glass plate on the EDF film and turn on the light box for about 20 seconds.

4. Develop the EDF film manually by developing the autoradiography film. The following procedures can be used:

(1) develop in Kodak GBX developer 1 ~ 5 minutes;

(2) washing with water for 65438 0 minutes;

(3) fixing in Kodak GBX fixing solution for 3 minutes;

(4) Water washing 1 min.

pay attention to

① The gel must be completely dried before the EDF film is developed. Wear gloves to avoid leaving fingerprints. At the same time, it should be noted that the automatic processor cannot be used for EDF film.

② The optimal exposure time of different light sources may be different. By exposing a small piece of EDF film for different times, choose the best exposure time for the light source you use. Please refer to the film manual.

③ Short exposure time will make the EDF film image deeper, while long exposure time will help to weaken the background. "shotgun" is a method to extract the target gene from the biological genome. Firstly, the chromosome DNA of biological cells is cut into many fragments at the gene level by physical methods (such as shear force, ultrasound, etc.). ) or enzymatic chemical methods (such as restriction endonucleases), and then these fragments are combined with appropriate vectors, and the recombinant DNA is transferred into recipient bacteria for amplification to obtain a gene library of asexual reproduction. Then, combined with screening method, strains containing a certain gene were selected from many transformant strains, and recombinant DNA was isolated and recovered from them.

This method is to isolate the target gene by genetic engineering technology, which is characterized by bypassing the difficulty of directly isolating the gene and screening the target gene from the genomic DNA library. It can be said that this is "hitting" a gene with the principle of "shotgun shooting". Because the target genes in the whole genome are too few and too small, which depends on "luck" to a great extent, people call this method "shotgun" or "shotgun" experiment.

1. The large inserted DNA of the target fragment was cut by restriction endonuclease and recovered by agarose gel electrophoresis.

Second, use physical methods (such as ultrasound, etc.) to cut off the recovered large fragments of DNA. ), and then smooth the ends of small fragments of DNA with t 4 DNA polymerase.

Thirdly, agarose electrophoresis was carried out, and the DNA fragments of 1 kbp ~ 2 kbp were recovered by gel cutting. Then add an A base to the 3' end of DNA with BcaBestDNAPolymerase.

Fourthly, the DNA fragment added with A- tail was connected to T- vector, and then transformed and cloned.

5. DNA sequencing of positive clones (including 1 kbp ~ 2 kbp insert).

Sixth, edit the data and finally connect them into a large DNA sequence.