What is DNA Sequencing?

DNA sequencing ( DNA sequencing , or translated DNA sequencing ) refers to the analysis of the base sequence of specific DNA fragments, that is, the adenine (A), thymine (T), cytosine (C) and guanine (G) arrangement the way. The advent of rapid DNA sequencing methods has greatly promoted research and discovery in biology and medicine.

DNA sequencing

Determine the direction and structure of recombinant DNA, locate, identify and compare mutations

In the late 1970s, Walter Gilbert invented the chemical method and Frederick Sanger invented the dideoxy termination method for manual sequencing and isotope labeling.

In the mid-1980s, automatic sequencers (using the principle of dideoxy termination), fluorescence instead of isotopes, and computer image recognition

In the mid-1990s, major improvements in sequencers and clustered capillary electrophoresis replaced gel electrophoresis

Human genome framework completed in 2001

The Human Genome Project, Gene Chips, Personalized Molecular Diagnostics, Biological Cloud Computing ... These popular words that have attracted countless eyeballs in the first decade of the 21st century all have their origins in an industry-DNA sequencing. Biotechnology and information technology blend in this creative new world. If you use a poem to describe the DNA sequencing industry escorted by two major technologies, it is that natural beauty is hard to give up.

In the eyes of people in the industry, DNA sequencing is a noble person. It cracks the genetic code (that is, the base sequence), combines genomics with IT technology, and develops a new discipline-bioinformatics. Gene technology represented by it has overturned traditional biological technology and led the future development trend of life science. The genetic engineering represented by it has played an important role in hot fields such as medical health, environmental protection, new energy, new materials, and modern agriculture.

In the eyes of people outside the industry, DNA sequencing is high-tech enough, and it can be called a model of "a new technology generates a new industry." One can accurately describe its blueprint ten years later. In the face of the ever-changing DNA sequencing technology, any prediction may seem conservative.

The high-tech field is where such a legend is born. DNA sequencing has quickly spread from a cutting-edge cutting-edge technology to routine life science technology. The cost of DNA sequencing costs has fallen almost as fast as the computing power of computer chips has increased. The development of DNA sequencing is not only reflected in the reduction of costs, but also in the high-throughput sequencing which has greatly improved the work efficiency, which paved the way for the industrialization of DNA sequencing.

Under the tide of commercialization of DNA sequencing, China's "Twelfth Five-Year Plan" for the development of biological industry proposes to complete the sequencing of the genomes of 10,000 microorganisms and 100 animal and plant species, discover about 500 new functional genes, and transform more than 5 applications into significant Gene or protein of economic value. According to the cost of sequencing "genome complete map" for each microorganism is 300,000 to 500,000 yuan, the market capacity brought by DNA sequencing reaches 100 billion yuan, which is just the tip of the iceberg in the commercial application market of DNA sequencing. ^[1]

Chemical modification sequencing principle

Several groups of radiolabeled oligonucleotides are generated independently of each other. Each group of oligonucleotides has a fixed starting point, but ends randomly at a specific residue or residues.

Because each base on the DNA has an equal chance of appearing at the variable termination, each of the above-mentioned products is a mixture of oligonucleotides. The length of these oligonucleotides is determined by the specific base in the entire DNA On the position.

Under the condition that different DNA molecules with a difference of only one nucleotide in length can be distinguished, electrophoretic analysis of each group of oligonucleotides is performed, as long as several groups of oligonucleotides are loaded into several adjacent lanes in the sequencing gel The sequence of nucleotides on the DNA can be read directly from the film's radiation from the film.

High-throughput sequencing, also known as "Next-generation" sequencing technology, enables sequencing and general reading of hundreds of thousands to millions of DNA molecules in parallel at one time Longer and shorter are the signs.

According to the development history, influence, sequencing principles and technologies, there are the following: Massively Parallel Signature Sequencing (MPSS), Polony Sequencing, 454 pyrosequencing , Illumina (Solexa) sequencing, ABI SOLiD sequencing, Ion semiconductor sequencing, DNA nanoball sequencing, etc.

MPSS

Massively Parallel Signature Sequencing, developed by Lynx Therapeutics in the 1990s, MPSS technology is a pioneer in the development of "next generation" sequencing technology. MPSS is a complex technology based on the connection and decoding of beads and adapters. The measurement results are short and are mostly used for

Automatic sequencing

The gene analyzer (ie DNA sequencer) uses capillary electrophoresis technology to replace the traditional polyacrylamide plate electrophoresis and uses the company's patented four-color fluorescent dye labeled ddNTP (labeled terminator method), so the reaction is sequenced by single primer PCR The generated PCR product is a single-stranded DNA mixture with 4 different fluorescent dyes at the 3 end of 1 base. The sequencing PCR products of the four fluorescent dyes can be electrophoresed in one capillary, thereby avoiding migration between lanes. The effect of the rate difference greatly improves the accuracy of sequencing. Due to different molecular sizes, 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 fluorescent molecules one by one and excite The fluorescence is separated by a grating to distinguish different colors of fluorescence that represent different base information, and the images are synchronized on a CCD camera. The analysis software can automatically convert different fluorescences into DNA sequences to achieve the purpose of DNA sequencing. The analysis results can be output in various forms such as gel electrophoresis, fluorescence absorption peaks, or base sequence.

It is a high-end precision instrument that can automatically determine the base sequence or size and quantification of DNA fragments, such as automatic glue injection, automatic injection, automatic data collection and analysis. PE company also provides gel polymer, including DNA sequencing gel (POP 6) and GeneScan gel (POP 4). These gel particles have a uniform pore size, which avoids the effect of inconsistent dispensing conditions on the accuracy of sequencing. It is mainly composed of accessories such as capillary electrophoresis device, Macintosh computer, color printer and electrophoresis. 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 the PCR fragment size analysis and quantitative analysis is 10 to 40 minutes.

Because the instrument has functions such as DNA sequencing, PCR fragment size analysis, and quantitative analysis, it can perform DNA sequencing, heterozygous analysis, single-strand conformation polymorphism analysis (SSCP), microsatellite sequence analysis, long fragment PCR, RT-PCR (Quantitative PCR) and other analysis, in addition to routine DNA sequencing, single nucleotide polymorphism (SNP) analysis, genetic mutation detection, HLA matching, parent-child and individual identification in forensics, microorganisms and Typing and identification of viruses.

I. Preparation

1. The main reagents of the BigDye sequencing reaction kit are BigDye Mix, which contains PE patented four-color fluorescently labeled ddNTP and ordinary dNTP, AmpliTaq DNA polymerase FS, reaction buffer, etc.

2. pGEM-3Zf (+) double-stranded DNA control template 0.2 g / L, the kit comes with reagents.

3. M13 (-21) primer TGTAAAACGACGGCCAGT, 3.2 mol / L, that is 3.2 pmol / l, the kit comes with reagents.

4. DNA sequencing templates can be PCR products, single-stranded DNA, and plasmid DNA. The template concentration should be adjusted to 1 l during the PCR reaction. The concentration of plasmid DNA determined in this experiment was 0.2 g / L, that is, 200 ng / l.

5. Primers need to be designed according to the DNA fragments to be determined, forward or reverse primers, formulated to 3.2 mol / L, that is 3.2 pmol / l. If the recombinant plasmid contains a universal primer sequence, universal primers can also be used, such as M13 (-21) primer, T7 primer and so on.

6. Sterilize deionized or triple distilled water.

7. Separate 0.2 ml or 0.5 ml PCR tube covers.

8. 3 mol / L sodium acetate (pH5.2) Weigh 40.8 g of NaAc · 3H2O and dissolve it in 70 ml of distilled water. Adjust the pH to 5.2 with glacial acetic acid.

9. 70% ethanol and absolute ethanol.

10. NaAc / ethanol mixed solution: 37.5 ml of absolute ethanol and 2.5 ml of 3 mol / L NaAc are mixed and stored at room temperature for 1 year.

11. POP 6 sequencing gel.

12. Template Inhibitory Reagent (TSR).

13. 10 × running buffer.

14. Fully automatic DNA sequencer.

15. PCR instrument.

16. Desktop refrigerated high-speed centrifuge.

17. Desktop high-speed centrifuge or pocket centrifuge.

Second, PCR sequencing reaction

1. Take a 0.2 ml PCR tube, number it with a marker, insert the tube in pellet ice, and add reagents according to the following table:

Standard reagent control tube

BigDye Mix 1 l 1 l

1 l of plasmid DNA to be tested-

pGEM-3Zf (+) Double-stranded DNA-1 l

1 l of the forward primer of the DNA to be tested-

M13 (-21) Primer-1 l

Sterile deionized water 2 l 2 l

The total reaction volume is 5 l. Without adding light mineral oil or paraffin oil, cover the PCR tube tightly, use the finger to bounce the tube to mix, and centrifuge slightly.

2. Place the PCR tube on a 9600 or 2400 PCR instrument for amplification. The PCR cycle was performed after denaturing at 98 ° C for 2 minutes. The PCR cycle parameters were 96 ° C for 10 s, 50 ° C for 5 s, and 60 ° C for 4 minutes. After 25 cycles, 4 ° C incubation was set after the amplification was completed.

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

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

2. Add 25 l sodium acetate / ethanol mixture, shake thoroughly, and place on ice for 10 min to precipitate DNA. Centrifuge at 12 000 r / min for 30 min at 4 ° C. Discard the supernatant carefully.

3. Wash the pellet twice with 50 l of 70% (V / V) ethanol. Centrifuge at 12 000 r / min for 5 minutes at 4 ° C. Carefully discard the supernatant and the beads on the tube wall, and dry the pellet under vacuum for 10-15 minutes.

Processing of sequencing PCR products before electrophoresis

1. Add 12 l of TSR to a centrifuge tube, shake it vigorously to fully dissolve the DNA pellet, and centrifuge slightly.

2. Transfer the solution to a 0.2 ml PCR tube with a lid and centrifuge a little.

3. Perform thermal denaturation on the PCR instrument (95 ° C for 2 min), quench in ice, and wait for the machine to run.

V. Operation on the machine 1. Install the capillary according to the instrument operation manual, correct the position of the capillary, manually fill the gel and establish a running sequencing file.
2. The instrument will automatically fill the gel to the capillary, 1.2 kV pre-electrophoresis for 5 min, auto-inject according to the programmed sequence, and then pre-electrophoresis (1.2 kV, 20 min), and electrophoresis at 7.5 kV for 2 h.
3. After the electrophoresis is completed, the instrument will automatically clean, fill the gel, and advance the next sample, pre-electrophoresis and electrophoresis.
4. The total electrophoresis time for each sample is 2.5 h.
5. After the electrophoresis is finished, the instrument will automatically analyze or print the color sequencing map.

6. Sequence analysis < br The instrument will automatically perform sequence analysis and perform sequence comparison according to user requirements. If the sequencing sequence is known, the difference bases can be marked with an asterisk through sequence comparison to improve work efficiency.

VII. Instrument cleaning < br After sequencing, perform instrument cleaning and maintenance according to the instrument operating procedures.

Calculation

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

2. Different bases are different bases compared with known standard DNA sequences. N is a base that cannot be read by the instrument.

Non-isotopic silver staining

The SILVER SEQUENCETM DNA Sequencing System is a non-radioactive sequence analysis system that detects bands in gels using a sensitive silver staining method. Silver staining provides a faster, cheaper alternative to radioactive or fluorescent methods. Sequencing results can be obtained on the same day; the sequence can be read within 90 minutes after electrophoresis is completed, which is not possible with conventional radiosequencing. In addition, the SILVER SEQUENCETM system uses unmodified 5'OH oligonucleotides as primers, reducing the cost of specially modified oligonucleotides. This system does not require the careful handling of isotopes in radioactive methods, nor does it require expensive reagents for fluorescence or chemiluminescence techniques. In addition, instrumentation is not required to detect sequence bands, as is the case with most fluorescence methods.

Taq DNA polymerase is extremely thermostable at 95 ° C. The sequencing-grade Taq DNA polymerase used by this system is a modified product of Taq DNA polymerase, which has very good effects on double-stranded DNA templates, has high accuracy, can produce uniform bands, and has a low background.

The SILVER SEQUENCETM system contains a mixture of modified nucleotides such as 7-deaza dGTP (7-deaza dGTP, or dITP) instead of dGTP to eliminate band compression caused by GC-rich regions.

Annealing temperature is the most important factor in thermal cycle sequencing. High annealing temperatures can reduce template secondary structure. Improve the rigor of primer-template binding. Chain reannealing and template secondary structure limit the ability of small fragment PCR products (<500bp) to obtain clear sequence data. Primer extension begins at the annealing stage of each cycle. At lower temperatures, the polymerase may encounter strong secondary structure regions that can cause the polymerase to dissociate. In all four electrophoretic channels, there are bands with the same relative position. For these reasons, the highest possible annealing temperature should be used. For templates with strong secondary structure, it is recommended to use a cycle mode of denaturation at 95 ° C and annealing / extension at 70 ° C. Generally, longer primers and primers with high GC content can get stronger signals. The experimental results show that primers with a GC content of> 24mer of about 50% can give the best results.

Because this system uses a thermal cycling device, it has the following advantages compared to conventional sequencing methods: (1). This method linearly amplifies template DNA to produce enough products to enable silver staining technology to detect sequence bands. The sequencing reaction requires 0.03 ~ 2pmol of template DNA, depending on the type of template. (2). The high temperature in each denaturation cycle can replace the alkaline denaturation and ethanol precipitation of the double-stranded DNA (dsDNA) template. The denaturation cycle also helps to eliminate the rapid reannealing of linear dsDNA templates (such as PCR reaction products). Caused by the problem. (3). The high temperature polymerase reaction weakens the secondary structure of the DNA template, allowing the polymerase to pass through highly secondary structured regions.

First, reagent preparation

1. SILVER SEQUENCETM DNA Sequencing Kit.

2. Acrylamide and methylene bisacrylamide stock solution (38% acrylamide W / V, 2% methylene bisacrylamide W / V): 95 g of acrylamide, 5 g of methylene bisacrylamide in 140 ml of double Make up to 250 ml in distilled water. After filtering through a 0.45 mm filter, store in a brown bottle and store in a refrigerator at 4 ° C for 2 weeks.

3. 10% ammonium persulfate, 0.5 g of ammonium persulfate is dissolved in 4 ml of water, and the volume is adjusted to 5 ml.

4. 10 × TBE buffer (1 mol / L Tris, 0.83mol / L boric acid, 10 mmol / L EDTA): 121.1 g Tris, 51.35 g boric acid, 3.72 g Na2EDTA · 2H2O, dissolved in double distilled water to make up to 1 volume It can be stored at 4 ° C for 2 weeks, and its pH is about 8.3.

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

6. TEMED

7. Fixation / stop solution: 10% glacial acetic acid (V / V) is prepared for 2 liters.

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

9. Developing solution: 60 g of sodium carbonate (Na2CO3) is dissolved in 2 liters of ultrapure water, and 3 ml of 37% formaldehyde and 40 ml of sodium thiosulfate solution (10 mg / ml) are added before use.

10. 95% ethanol.

11. 0.5% glacial acetic acid.

12. Sigmacote (Sigma CAT. # SL-2).

Second, the sequencing reaction

1. For each set of sequencing reactions, label four 0.5 ml eppendorf tubes (G, A, T, C). Add 2 ml of the appropriate d / ddNTP Mix (d / ddNTP Mix) to each tube. Add 1 drop (approximately 20 l) of mineral oil each, cover with a lid and store on ice or at 4 ° C until use.

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

(1) Sample reaction:

Plasmid template DNA: 2.1 pmol

5 × sequencing buffer: 5 ml

Primer: 4.5 pmol

Sterile ddH2O to a final volume of 16 ml

(2) Control reaction

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

5 × sequencing buffer: 5 ml

pUC / M13 forward primer (4.5 pmol): 3.6 ml

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

4. Pipet 4 ml of the enzyme / primer / template mixture from step 3 into each d / ddNTP mixture tube.

5. Centrifuge in a microcentrifuge so that all the solution is at the bottom of the eppendorf tube.

6. Place the reaction tube in a thermal cycler preheated to 95 ° C, and start the cycle program based on the circulation mode in [Caution]. The optimal annealing temperature must be selected for each primer / template combination. The following procedure generally reads 350 bases from the start of the primer.

7. After the thermal cycling procedure is completed, add 3 l of DNA sequencing stop solution to each vial, and slightly rotate in a microcentrifuge to stop the reaction.

note

(1) The amount of template DNA used for sequencing is generally added as follows:

Template type / length template amount

200 bp (PCR product): 16 ng (120 fmol)

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

48 000 bp (, cosmid DNA): 1 mg (31 fmol)

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

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

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

Calculate the number of micrograms of primers equivalent to 1p mol using the following general formula:

dsDNA: 1 pmol = (6.6 × 10mg) × n, where n is the number of template base pairs

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

(3) To prevent Taq DNA polymerase from extending non-specific annealing primers, the thermal cycler must be preheated to 95 ° C. The faster the temperature, the better. The following cycle times do not include the temperature change time. If you are not sure which mode to use, it is recommended to start with Mode 1.

Mode 1: Suitable for primers <24 bases or GC content <50%

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

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

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

(4) After adding the stop solution, the sample can be stored at 4 ° C overnight.

Preparation of sequencing gel plates

Glass plate treatment

The glass plate for silver staining sequencing must be very clean. Generally, the glass plate is washed with warm water and detergent, and then the glass plate is washed with deionized water to remove the residual detergent, and the glass plate is finally washed with ethanol. Detergent microfilm remaining on the glass plate may cause a high background (brown) when the gel is stained. The short glass plate is chemically cross-linked to the glass plate by treatment with a binding solution. This step is essential to prevent gel tearing during the silver staining operation.

(1) Handling of short glass plates

Add 5 ml of Bind Silane to 1 ml of 95% ethanol and 0.5% glacial acetic acid to prepare a fresh binding solution.

Wipe the carefully cleaned and naturally dried glass plate with absorbent tissue paper soaked with the newly prepared adhesive solution. The entire surface must be wiped.

After 4 to 5 minutes, wipe the glass plate with 95% ethanol in one direction, and then wipe it slightly in the vertical direction. This cleaning process was repeated three times, each time using clean paper to remove excess adhesive solution.

note

Excessive force when wiping the glass plate with 95% ethanol in one direction will take away too much adhesive silane, making the gel not adhere well.

Change gloves before preparing long glass plates to prevent sticking silane.

It is important to prevent the adhesive solution from contaminating the long glass, otherwise it will cause the gel to tear.

(2) Handling of long glass plates:

Wipe cleaned long glass plates with tissue paper saturated with Sigmacote solution.

After 5-10 minutes, wipe the glass plate with absorbent tissue to remove excess Sigmacote solution.

note

The used gel can be soaked in water and then scraped off with a razor blade or a plastic spatula. The glass plate must be completely cleaned with detergent. Or the gel was removed after soaking in 10% NaOH. To prevent cross-contamination, the tool used to clean short glass plates must be separated from the tool used to clean long glass plates. If cross-contamination occurs, gels prepared later may tear or become loose.

2. Preparation of gel

(1) After the glass plate has been treated with bonded silica gel and Sigmacote, the glass plate can be fixed. In this method, a 0.2 mm or 0.4 mm thick edge strip is 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 comb on one side of the long glass plate and fix it with a clip.

(2) According to the required gel concentration, prepare the sequencing gel according to the following table. Generally, a gel concentration of 6% to 8% can obtain better results. In the preparation process, firstly dissolve urea with an appropriate amount of double-distilled water, then add Acr & Bis and 10 × TBE buffer, and then adjust the final volume to 99.2 ml with double-distilled water, and filter through a 0.45 mm filter membrane, then add ammonium persulfate and TEMED. . No heating is necessary to dissolve urea. If heating is indeed required, the solution should be completely cooled before adding TEMED and ammonium persulfate. Generally, polymerization starts 4 to 6 minutes after the glue is poured. If the polymerization is not good, high concentrations of TEMED and ammonium persulfate should be used.

(3) After the glue is prepared, the rubber sheet can be poured. Generally, the gel is slowly poured into the groove of the glass plate along the edge of the bead. After pouring, the gel is allowed to stand still to complete the polymerization.

note

When using a clip to fix the glass plate, it is best to use a slightly stronger clip to prevent leakage of glue during the filling process due to insufficient strength.

It is necessary to prevent the generation of air bubbles during the process of gel casting, otherwise it will affect the sequencing results.

Fourth, electrophoresis

Pre-electrophoresis

(1) When the gel is completely polymerized, pull out the shark tooth comb, turn the comb over, and insert the toothed end into the gel to form a sample hole.

(2) Immediately fix the gel plate in the sequencing gel tank. Generally, the upper and lower tanks of the sequencing gel tank are separated, so the TBE buffer can only be added after the gel plate is fixed.

(3) Dilute 10 × TBE buffer to 1 × TBE, add this buffer to the upper and lower electrophoresis tanks, remove the generated air bubbles, and connect to the power supply to prepare for pre-electrophoresis.

(4) Some electrophoresis tanks, such as LKB's Macrophor, are heated with a water bath. The water bath should be heated to 55 ° C before pre-electrophoresis. Some do not use a water bath for heating, and rely on the heat generated during the electrophoresis for thermal insulation. For example, the sequencing electrophoresis tank produced by Shanghai Qiujing Plexiglas Instruments, this tank requires two heat-dissipating aluminum plates to make the temperature of the entire gel plate consistent .

(5) Pre-electrophoresis at a voltage of 30 V / cm for 20 to 30 minutes. The process of pre-electrophoresis is to remove the impurity ions of the gel while bringing the gel plate to the required temperature. High-temperature electrophoresis can prevent hairpin-like structures formed in GC-rich regions and affect sequencing results.

note

When using a shark tooth comb to make a sample hole, be careful to insert the tip of the tooth into the glue about 0.5mm. Be careful not to leak the sample hole, otherwise you will not get the correct result.

Pay attention to whether the buffer solution in the electrophoresis tank leaks at all times, otherwise it will easily cause short circuit and damage the electrophoresis instrument.

2. Sample preparation

When pre-electrophoresis, the sample can be prepared, and the reaction sample can be heated in a boiling water bath for 1 to 3 minutes, and then placed on ice immediately. If the sample is not used for a long time, it should be reprocessed. 4 to 6% polyacrylamide gel can be used with a thickness of 0.4 mm. Adhesives less than 0.4 mm thick may cause weak signals. It is not necessary to remove the mineral oil covered by the upper layer when applying, but be careful to suck the blue sample under the mineral oil.

3. Loading and electrophoresis

Turn off the electrophoresis instrument, wash the sample wells with a pipette suction buffer, remove the urea diffused during the pre-electrophoresis, and then immediately use the capillary sampler to aspirate the samples and add them to the sample wells. The loading sequence is generally G, A, T, C. Immediately after adding the sample, electrophoresis was performed. Electrophoresis can be started at 30 V / cm. After 5 minutes, it can be increased to 40-60 V / cm and maintained at a constant voltage. In general, a 55 cm long, 0.2 mm thick gel plate can reach the bottom under 2 hours of electrophoresis under a constant voltage of 2500 V. At the same time, the current can be stably reduced from 28 mA to 25 mA during electrophoresis . In order to read longer sequences, two or more rounds of loading can be used.

note

When loading electrophoresis, be sure to pay attention to whether the temperature of the gel plate has reached about 55 ° C. If it has not been reached, wait until the temperature has reached before loading the gel.

In general, it is not appropriate to use too high voltage during electrophoresis, because too high voltage will reduce the resolution of the gel and make the band diffuse. Constant power electrophoresis can be performed during electrophoresis.

Five, silver staining of sequencing gels

The staining process requires the gel to be immersed in a plastic dish. Therefore use at least two plates, similar in size to glass plates. Wash the dishes with high-quality water before adding fresh solutions to the dishes.

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

2. Fix the gel: Put the gel (with the glass plate) in a plastic plate, immerse it with the fixation / stop solution, and shake it for 20 minutes or until the dye disappears completely in the sample. oscillation). Retain the fixation / stop solution for stopping the development reaction.

3. Washing gel: Wash the gel with ultrapure water 3 times for 2 minutes each. Remove from the water, hold the edge of the rubber plate for 10 to 20 seconds when transferring to the next solution, and let the water run out.

4. Gel staining: Move the gel to the staining solution and shake thoroughly for 30 minutes.

5. Gel development

(1) Add formaldehyde (3 ml) and sodium thiosulfate solution (400 l) to the developing solution to complete the preparation of the developing solution.

(2) Take out the gel from the dyeing solution and put it in a tray filled with ultrapure water for 5-10 seconds. Note that the total time to transfer the gel from ultrapure water to the developing solution cannot be longer than 5 to 10 seconds. Prolonged soaking time results in weak or lost signal. If the soaking time is too long, repeat the fifth step to soak with the staining solution.

(3) Immediately transfer the gel to 1 liter (half of the total amount) of the pre-cooled developer solution and fully shake it until the template band starts to appear or the first bands start to appear. Continue to develop for 2 to 3 minutes or until all bands appear.

6. Fixing gel: Add an equal volume of fixing / stopping solution directly to the developer. Stop the development reaction and fix the gel.

7. Immerse the gel twice in ultrapure water for 2 minutes each time. Pay attention to wearing gloves and holding the edge of the rubber plate to avoid printing fingerprints on the glue.

8. Allow the gel to dry at room temperature or dry by suction heating. Observe the gel in a visible light box or on a bright white, yellow background (such as paper). If you need to save the record permanently, you can use EDF film to retain the experimental results.

note

The silver staining of sequencing products is a new method for visualizing sequence information. The success of the system is affected by several factors.

The quality of water is extremely important for the success of dyeing. Ultrapure water (water of NANOpureR or Milli-QR) or double-distilled water can get better results. If there are impurities in the water, low molecular weight bands may not appear.

Sodium carbonate is also very important. Use fresh, American Chemical Society-grade sodium carbonate, such as Fisher and Kodak ACS reagent grade sodium carbonate (Fisher Cat # S263-500 or S262-3, or Kodak Cat # 109-1990), and generally get better results .

The washing step after coloring is very critical. If the gel is washed too long, the silver particles can detach from the DNA, producing little or no sequence signals. If the washing time is too long, the staining step can be repeated.

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

Perform all steps at room temperature, except for the development reaction. The developing solution must be pre-cooled to 10-12 ° C to reduce background noise. Note: Add formaldehyde and sodium thiosulfate to the developing solution just before use. Use a new dyeing and developing solution. Do not reuse any solution.

Six, EDF film development

The use of EDF film can enhance the contrast of the sequencing bands. If the bands on the sequencing gel are very light, we recommend that the data be transferred to the EDF film. The silver stained gel can enhance the readability of the band after the image is transferred to the EDF film.

1. In a dark room, place the stained gel on the glass plate (with the glue side up) on a fluorescent light box. If a suitable diffuser is used, a white light box can also be used. In order to ensure the exposure time, a small strip of EDF film is used to expose at different times, and different exposure intensities are checked. Generally, good results are obtained by exposure for 20 to 40 seconds.

2. Find the notched corner of the EDF film under the red light, and then place the film on the gel so that the gap is at the upper left corner. Because the EDF film is single-sided, you must ensure that the notch is in the upper left corner.

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

4. To develop the EDF film manually by developing the autoradiographic film, use the following procedure:

(1) Develop in Kodak GBX developer for 1-5 minutes;

(2) Wash for 1 minute;

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

(4) Wash with water for 1 minute.

note

The gel must be completely dry before developing EDF film. Wear gloves to avoid fingerprints. Also note that EDF films cannot be used with automatic film processors.

The optimal exposure time for different light sources may be different. Select the best exposure time for your light source by exposing a small strip of EDF film at different times, see the film manual.

If the exposure time is short, the EDF film image will be darker, and if the exposure time is long, it will help to weaken the background.

Shotgun

The "shotgun method" is a method of extracting a gene of interest from a biological genome. First, physical methods (such as shear force, ultrasound, etc.) or enzymatic methods (such as restriction endonucleases) are used to cut the chromosomal DNA of biological cells into many fragments at the gene level. Recombinant DNA is transferred to the recipient bacteria and amplified to obtain a gene library of asexual reproduction. Combined with screening methods, a strain containing a certain gene is selected from a large number of transformant strains, and the recombinant DNA is separated and recovered.

This method is the use of genetic engineering technology to isolate the target gene, which is characterized by bypassing the difficulty of direct gene isolation and screening the target gene in a genomic DNA library. It can be said that this is to "hit" a gene by using the principle of "scatter shot". Because the gene of interest is too small and too small in the entire genome, it still depends on "luck" to a considerable extent, so people call this method the "shotgun method" or "shotgun" experimental method.

1. A large insert DNA of the target fragment was cut with a restriction enzyme and recovered by Agarose gel electrophoresis.

Second, the recovered large fragment DNA is cut by physical methods (such as ultrasound, etc.), and then T4DNAPolymerase is used to smooth the end of the small fragment DNA.

3. Perform Agarose electrophoresis and cut the gel to recover small fragments of 1kb ~ 2kbp DNA. Then use BcaBestDNAPolymerase to add an A base to the 3 'end of the DNA.

4. A-tail DNA fragments are ligated into T-Vector, then transformed, and clones are selected.

5. DNA sequencing of positive clones (containing 1kbp ~ 2kbpInsert).

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