The genetic diversity of a species is fundamentally reflected in the variation of its DNA's primary sequence. In recent years, with the rapid advancement and widespread use of DNA sequencing technologies, this technique has become an essential tool in the study of genetic diversity. This chapter outlines both a manual and a fully automated method for double-stranded DNA sequencing that are commonly used in genetic diversity research.
1. Preparation of DNA Template
In genetic diversity studies, where large sample sizes are common, the speed of DNA sequencing has become a critical factor. As a result, purified PCR double-stranded products are often directly used for sequencing without the need for cloning techniques. This section describes the methods typically used in our laboratory to prepare sequencing templates from PCR products, specifically using low melting point gel recovery.
(1) Prepare a 1.5%-2.0% agarose gel. Once solidified, cut a strip about 1 cm wide at approximately 5 cm from the loading well and pour pre-melted low melting point agarose into the groove.
(2) After the low melting point gel solidifies, load the PCR reaction mixture onto the gel and run it at a constant voltage of around 100 V until the amplified fragment migrates into the middle of the low melting point gel. Cut out the band under UV light (360 nm), and place it into a 1.5 ml centrifuge tube.
(3) Centrifuge the tube, press the gel block to the bottom, and add 500 μl of TE buffer. Melt the gel in a 68°C water bath, then quickly add an equal volume of water-saturated phenol and mix thoroughly.
(4) Shake for 10 minutes at room temperature, then centrifuge at 12,000 rpm for 10 minutes. Take the supernatant and extract it with chloroform:isoamyl alcohol (24:1) for 5 minutes.
(5) Centrifuge again at 12,000 rpm for 10 minutes, collect the supernatant, add 1/10 volume of 10 M NH4Ac and 2 volumes of absolute ethanol, and incubate at 70°C for over 30 minutes.
(6) Centrifuge for another 10 minutes, wash the pellet with 70% cold ethanol, briefly centrifuge, carefully remove the ethanol, and dry the pellet. Finally, dissolve the DNA in 20–50 μl of TE buffer or sterile deionized water to prepare the sequencing template.
There are also commercial kits available, such as the Qiagen PCR Product Purification Kit, which can be more efficient but come at a higher cost.
2. Manual DNA Sequencing Technology
2.1 Preparation of Sequencing Gel
Prepare the sequencing gel using the following formula:
- 6% polyacrylamide working solution: 70 ml
- 10% ammonium persulfate (APS): 70 μl (freshly prepared)
- TEMED: 70 μl
Mix the components and pour them into a pre-prepared rubber mold. Insert the "shark tooth" comb upside down to a depth of about 0.5–1.0 cm and allow it to polymerize at room temperature for approximately one hour.
2.2 Sequencing Reaction
The sequencing kit was purchased from USB Corporation, USA, and the enzyme used was Sequenase Version 2.0. α35S-dATP was obtained from DuPont (1 mCi/100 μl). The sequencing reaction was performed according to the manufacturer’s instructions:
(1) DNA template mixture (in a 0.5 ml Eppendorf tube):
- DNA template: 7 μl
- Sequencing primer: 1 μl
- 5× Reaction buffer: 2 μl
- NP40: 1 μl
(2) Labeling mixture (per reaction):
- DTT (0.1 M): 1 μl
- 1/5 dGTP labeling mix: 2 μl
- Enzyme dilution buffer: 1.75 μl
- NP40: 0.55 μl
- Deionized water: 0.375 μl
- Sequencing enzyme: 0.125 μl
- 35S-dATP: 0.5 μl
(3) ddNTP (ddA, ddG, ddC, ddT): 2.5 μl
(4) Annealing: Heat the DNA template mixture for 3 minutes, then rapidly cool it in an ice-ethanol bath for about 30 seconds. If no dry ice is available, freeze at -20°C to -70°C. Keep the reaction solution on dry ice or crushed ice before proceeding.
(5) Labeling: Remove the DNA template from the ice, let it thaw in your hand, and add 5.5 μl of the labeling mixture. Centrifuge for 10–15 seconds, then leave at room temperature for 1 minute.
(6) Chain termination: Add 3.3 μl of ddNTP previously heated at 37°C to the mixture and incubate at 37°C for 4–5 minutes.
(7) Add 4 μl of stop solution to each tube.
2.3 Electrophoresis
Use 1× TBE buffer (pH 8.0). Pre-electrophorese for 30–60 minutes. Use a constant temperature mode with an aluminum thermostat set to ~50°C. Run the gel at constant power (90–110 W) for 2.5–5 hours.
2.4 Dry Gel Preparation and Film Exposure
After electrophoresis, remove the gel from the rubber plate, open the glass plates, and attach Xinhua No. 3 filter paper to the gel. Place the gel in a dry gel machine for 1–1.5 hours. Then, store the dried gel in a tablet box in a dark room and expose X-ray film for 2–3 days.
2.5 X-ray Film Development
Develop the exposed X-ray film for 4–8 minutes, depending on the developer's age. Fix for 5–10 minutes, then rinse and read the DNA sequence.
3. Automated DNA Sequencing Technology
This section describes the use of Perkin-Elmer Model 373 and 377 automated sequencers for double-stranded DNA sequencing. Thermal cycle sequencing reactions were carried out using the Applied Biosystems DyeDeoxy™ Terminator Cycle Sequencing Kit (#401434) or FS-DNA Sequencing Kit (#402079). The procedure follows the manufacturer’s recommendations.
3.1 Reaction Mix
(1) Take 2.5 μl of the purified PCR product [pGEM-3Zf(+)] and run it on an agarose gel.
(2) Stain the gel with ethidium bromide, rinse with running water, and photograph under UV light. Estimate the DNA concentration to determine the amount needed for the sequencing reaction.
(3) In a labeled 0.5 ml Eppendorf tube, mix:
- DNA template
- Primer (10 pmol/ml): 0.5 μl
- Water to 5.25 μl or 6.0 μl (FSKit)
- Denature at 100°C for 2 minutes, then ice-bath for 2 minutes, briefly centrifuge, and add:
- DyeDeoxy™ Terminator Cycle Sequencing Kit solution: 4.75 μl
- Or FS-DNA Sequencing Kit solution: 4.0 μl
- Final volume: 10 μl
(4) Add 20 μl of mineral oil after mixing.
3.2 Thermal Cycling Reaction
Use a Perkin-Elmer Model 480 thermal cycler. Perform the following cycles:
- 96°C for 1 second
- 96°C for 30 seconds
- 50°C for 1 second
- 50°C for 1 minute
- 60°C for 1 second
- 60°C for 4 minutes
Repeat for 25 cycles.
3.3 Purification of Sequencing Products
Use the CentriSep column (#CS-901) from Princeton Separations Inc. for purification. Follow these steps:
(1) Lift the column to expose the Cephadex G-50 powder at the bottom.
(2) Remove the top cover, add 800 μl of deionized water, replace the lid, and shake to remove air bubbles.
(3) Let the column hydrate at room temperature for 30 minutes.
(4) Remove the top and bottom covers, place the gel column in an elution tube to drain excess liquid.
(5) Pour off the liquid and centrifuge at 3,000 rpm for 2 minutes.
(6) Place the column in a 1.5 ml centrifuge tube and add the sequencing reaction to the center of the gel column, avoiding paraffin oil.
(7) Centrifuge at 3,000 rpm for 2 minutes. Ensure the column orientation remains consistent.
(8) Dry the sample in a vacuum centrifuge.
(9) Store the dried sample at -70°C. Samples stored for up to one year will retain their fluorescence.
3.4 Electrophoresis
Use the ABI Model 377 Automated DNA Sequence Analyzer. Prepare a 4.25% polyacrylamide gel with the following components:
- Urea: 18 g
- Amberlite ion exchange resin (Sigma, MB-lA): ~39 g
- 40% Acrylamide: Bis (19:1): 5.3 ml
- Deionized water: 25 ml
Mix on a magnetic stirrer for 10 minutes.
Filter the gel through a 2 μm filter. Prepare 10× TBE:
- Tris-base: 108 g
- Boric acid: 55 g
- Na2 EDTA (2H2O): 7.44 g
Dilute to 1 L.
Add 35 μl TEMED and 250 μl 10% APS, gently mix, and pour into the assembled glass plates using a 50 ml syringe. After 1 hour, mount the gel on the automated sequencer and run at 1 kV for 30 minutes while raising the temperature to 51°C.
At the same time, take 36 samples stored at -70°C, add 5 μl of each sample (50 μl loading buffer + 250 μl formamide), vortex, denature at 94°C for 2 minutes, and ice-bath for 2 minutes. Briefly centrifuge. Load 1.5 μl of each sample and run at 1.68 kV for 7 hours. The machine will automatically analyze and record the sequence data. In some cases, the electrophoresis channels may be misidentified, so manual correction may be necessary.
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