The genetic diversity of a species is fundamentally reflected in the variation of its DNA's primary sequence. Over the past few years, with the rapid advancement and widespread use of DNA sequencing technologies, this technique has become an essential tool in studying genetic variation. This chapter introduces both manual and fully automated methods for double-stranded DNA sequencing that are commonly applied in genetic diversity research.
1. Preparation of DNA Template
In genetic diversity studies, where large sample sizes are often involved, the speed of DNA sequencing has become a critical factor. As a result, purified PCR double-stranded products are frequently used directly for sequencing without the need for cloning. This section outlines the common laboratory method used to prepare sequencing templates from PCR products—low melting point gel recovery.
(1) Prepare a 1.5%-2.0% agarose gel. Once it has fully solidified, cut a 1 cm wide strip about 5 cm from the spotting line and pour pre-melted low melting point agarose into the cut groove.
(2) After the low melting point gel has solidified, load the PCR reaction mixture onto the gel and perform electrophoresis at a constant voltage of approximately 100 V until the amplified fragment enters the middle of the gel. Under UV light (360 nm), cut out the band containing the fragment and transfer it into a 1.5 ml centrifuge tube.
(3) Centrifuge the tube, compress 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 the mixture at room temperature for 10 minutes and centrifuge at 12,000 rpm for 10 minutes. Transfer the supernatant to a new tube and extract with chloroform-isoamyl alcohol (24:1) for 5 minutes.
(5) Centrifuge again at 12,000 rpm for 10 minutes, take the supernatant, and add 1/10 volume of 10 M NH4Ac and 2 volumes of absolute ethanol. Precipitate the DNA by incubating at 70°C for more than 30 minutes.
(6) Centrifuge for another 10 minutes at 12,000 rpm, wash the pellet with 70% cold ethanol, briefly centrifuge, and carefully remove the ethanol. Dry the pellet and dissolve it in 20–50 μl of TE buffer or sterile deionized water to prepare the DNA template.
There are also commercial kits available, such as the Qiagen PCR Product Purification Kit, which offer high efficiency but come at a higher cost.
2. Manual DNA Sequence Analysis Technology
2.1 Preparation of Sequencing Gel
To prepare the sequencing gel, follow this formula:
- 6% polyacrylamide working solution: 70 ml
- 10% APS: 70 μl (freshly prepared)
- TEMED: 70 μl
Mix the above ingredients, pour them into a pre-assembled rubber plate, and insert a "shark tooth" comb to a depth of about 0.5–1.0 cm. Allow the gel to polymerize at room temperature for about 1 hour.
2.2 Sequencing Reaction
The sequencing kit was purchased from USB Corporation, USA, using Sequenase Version 2.0 as the sequencing enzyme, and α35S-dATP was obtained from DuPont (1 mCi/100 μl). The sequencing reaction was carried out according to USB's recommendations:
(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 mol/dm³): 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: Boil the DNA template mixture for 3 minutes, then quickly place it in an ice bath made of ethanol and dry ice for about 30 seconds. If dry ice is unavailable, freeze at -20°C to -70°C. Ensure the anhydrous ethanol is taken from the refrigerator before use. Keep the reaction solution on dry ice or crushed ice until the labeling step.
(5) Labeling: Remove the DNA template mixture from the dry ice or ice, allow it to melt in your hands, and add 5.5 μl of the labeled mixture. Centrifuge for 10–15 seconds, then let it sit 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
- Electrode buffer: 1× TBE (pH 8.0)
- Pre-electrophoresis: 30–60 minutes
- Temperature: ~50°C
- Power: 90–110 W
- Time: 2.5–5 hours
2.4 Dry Gel Preparation and Film Exposure
After electrophoresis, remove the rubber plate and open the glass plate with a tool, leaving the gel on one side. Attach Xinhua No. 3 filter paper to the gel to help it adhere, then place it in a dry gel machine for 1–1.5 hours. Once dried, place the gel in a tablet box, store it in a dark room, and expose it to X-ray film for 2–3 days.
2.5 X-ray Film Development
Develop the exposed X-ray film for 4–8 minutes, fix it for 5–10 minutes, and then rinse it to read the DNA sequence.
3. Automated DNA Sequence Analysis Technology
This section describes the use of Perkin-Elmer Models 373 and 377 automated sequencers for double-stranded DNA sequencing. Thermal cycle sequencing reactions were performed using the Applied Biosystems DyeDeoxy™ Terminator Cycle Sequencing Kit (#401434) or FS-DNA Sequencing Kit (#402079). The procedure followed the manufacturer’s instructions.
3.1 Mixing Reagents
(1) Take 2.5 μl of the purified PCR product [pGEM-3Zf(+) DNA in 2.5 μl sequencing kit] and run agarose gel electrophoresis.
(2) Stain the gel with ethidium bromide, rinse with running water, and photograph under UV light. Use pGEM-3Zf(+) DNA as a reference. Estimate the DNA concentration to determine the amount needed for the sequencing reaction.
(3) In a labeled 0.5 ml Eppendorf tube, mix the following components:
- DNA template
- Primer (10 pmol/ml): 0.5 μl
- Add water to 5.25 μl or 6.0 μl (FSKit)
- Denature at 100°C for 2 minutes, then cool on ice for 2 minutes, briefly centrifuge, and add:
- Sye solution: 4.75 μl (DyeDeoxy™ Terminator Cycle Sequencing Kit)
- Sequencing solution: 4.0 μl (FS-DNA Sequencing Kit)
- Final volume: 10 μl
(4) Mix thoroughly with a micropipette, then add 20 μl of mineral oil.
3.2 Thermal Cycling Reaction
Cooling is crucial in this process (about 1°C/s). A Perkin-Elmer Model 480 thermal cycler can be used. The following program is recommended:
(1) 96°C for 1 second
(2) 96°C for 30 seconds
(3) 50°C for 1 second
(4) 50°C for 1 minute
(5) 60°C for 1 second
(6) 60°C for 4 minutes
Repeat for 25 cycles.
3.3 Purification of Sequencing Products
The best method for purifying sequencing products is using the CentriSep column (#CS-901) from Princeton Separations Inc. Follow these steps:
(1) Lift the column to release Cephadex G-50 powder from the bottom.
(2) Remove the top cover, add 800 μl of deionized water, replace the lid, and shake to remove air bubbles.
(3) Let it 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, 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, being careful not to introduce paraffin oil.
(7) Centrifuge at 3,000 rpm for 2 minutes. Ensure the column orientation is consistent with the first centrifugation.
(8) Dry the sample in a vacuum centrifuge.
(9) Store the dried sample at -70°C until electrophoresis. Samples stored for up to one year under these conditions will retain their fluorescence.
Note: The CentriSep column can be reused after washing three times with tap water, twice with distilled water, and drying on a test tube rack. Then, add 50 mg of Cephadex G-50 (Sigma, #9048719) and reuse.
3.4 Electrophoresis
Electrophoresis was conducted using the ABI Model 377 Automated DNA Sequencer, with data recorded via PRIM377 software. The process is as follows:
(1) 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
Stir on a magnetic stirrer for 10 minutes.
(2) Filter 5 ml of 10× TBE through a 2 μm filter, then filter the gel mixture.
10× TBE formula:
- Tris-base: 108 g
- Boric acid: 55 g
- Na2EDTA (2H2O): 7.44 g
- Bring to 1 L with deionized water.
(3) Make up to 50 ml, add 35 μl of TEMED and 250 μl of 10% APS, gently mix, and pour into the assembled glass plates using a 50 ml syringe.
(4) After 1 hour, mount the gel on the automated sequencer and pre-electrophorese for 30 minutes at 1 kV while raising the temperature to 51°C.
(5) At the same time, take 36 samples stored at -70°C, add 5 μl of each sample solution (50 μl loading buffer + 250 μl formamide from the kit, prepared before use).
(6) Vortex the mixture, denature at 94°C for 2 minutes, then cool on ice for 2 minutes, and briefly centrifuge.
(7) Load 1.5 μl of each sample and run at a constant voltage of 1.68 kV for 7 hours. The machine will automatically analyze and record the sequence results. If any channel is misidentified, manually correct it at this stage.
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