DNA sequence analysis techniques can be used to (1) analyze the genetic diversity of species, (2) identify new species, and (3) for comparative genomics.
Operation method
DNA Sequence Analysis Technology
Principle
This section describes the methodology for double-stranded DNA sequencing using Perkin-Elmer's fully automated DNA sequencers Model 373 and 377. Thermal cycle sequencing reactions are performed using the Applied Biosystems Inc. DyeDeoy TM Terminator Cycle Sequencing Kit (#401434) or FS-DNA Sequencing Kit (#402079). The procedure was performed primarily according to the manufacturer's recommendations.
Materials and Instruments
PCR products Move 1 Mixing the reaction products (1) Take 2.5 μl of purified PCR product [relative to 2.5 μl of pGEM-3Zf(+) DNA from the sequencing kit] and perform agarose gel electrophoresis. (2) The gel was stained with ethidium bromide, decolorized by rinsing with running water, and photographed under UV light. Refer to pGEM-3Zf (x) DNA Estimate the concentration of the DNA template to be tested to determine the amount of template to be taken in the sequencing reaction. (3) In a labeled 0.5 ml Eppendorf tube, mix the following reactants: DNA template Appropriate amount 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 ice bath for 2 minutes and centrifuge briefly, then add: DyeDeoy TM Terminator Cycle Sequencing Kit, 4.75 μl. Sequencing Solution in DyeDeoy TM Terminator Cycle Sequencing Kit 4.75 μl or Sequencing Solution in FS-DNA Sequencing Kit 4.0 μl. Final volume 10μl (4) Mix the reactants thoroughly with a microsampler and cover with 20 μl of paraffin oil. 2 Thermal cycling reaction The cooling time in this reaction is critical (~1°C/s). Therefore, a Perkin-Elmer serial thermal cycler (PCR instrument, e.g., 480, 2400 or 9600) can be used. The operations described below are based on the Perkin-Elmer Model 480 Thermal Cycler. Place the reaction tube in the thermal cycler and perform the thermal cycling reaction according to the following procedure: (1) 96°C for 1 second 96°C for 30 seconds (2) 50°C 1 sec. 50°C for 1 minute (3) 60 ℃ l seconds 60℃ 4 minutes A total of 25 cycles are required. 3 Purification of Sequencing Products The best way to purify the sequencing product is to use a Centri SeP column (#CS-901) from Princeton Separations Inc. The procedure is as follows: (1) Flick the column to lift the Cephadex G-50 gel powder from the bottom of the column. (2) Remove the top cover of the column, add 800 μl of deionized water, and then cover and shake to remove air bubbles. (3) Leave at room temperature for 30 minutes to hydrate the gel column. (4) Remove the top and bottom caps and place the gel column into a configured elution tube, allowing excess liquid to drain. (5) Pour off the liquid and centrifuge the column with the elution tube at 3,000 r/min for 2 minutes. (6) Place the column into a 1.5 ml centrifuge tube and use a microsampler to remove the sequencing reaction and inject it into the center of the gel column. Be careful to avoid bringing in paraffin oil. (7) Centrifuge for 2 minutes at 3,000r/min. Note that the orientation of the column must be consistent with the first centrifugation. (8) Dry the sample in a vacuum drying centrifuge. (9) Store the dried sample at -70°C for electrophoresis. Samples stored under these conditions for up to 1 year will not burst out of fluorescence. It should be noted that the Centri-sep column can be reused several times. After each use, the column should be washed 3 times with tap water and 2 times with distilled water, then inverted onto a test tube rack to dry, and 50 mg of Cephadex G-50 (Sigma, #9048719) should be added to the column before reuse. 4 Electrophoresis Sequence data were recorded by electrophoresis using a fully automated DNA sequence analyzer model 377 from ABI. The control software was PRIM377 Collection, and the electrophoresis procedure was as follows. (1) The concentration of polypropylene phenolamine gel is 4.25%, and the preparation process is as follows: Urea 18g Amberlite ion exchange resin (Sigma, MB-lA) about 39g 40% Acry mide:Bis (19:1 ) (AMRESCO, #0496-500) 5.3ml Deionized water 25ml Stir the mixture on an electromagnetic stirrer for 10 minutes. (2) Take 5 ml of 10x TBE and filter through a 2 μm filter membrane before filtering the above gel solution. 10x TBE formula: Tris-base 108g Boric acid 55g Na2 EDTA(2H2O) 7.44g Volume to 1L (3) Volume the filtrate to 50 ml, add 35 μl of TEMED and 250 μl of 10% amine persulfate (APS), shake gently to mix, and inject the gel into the assembled glass plate with a 50 ml needleless syringe. (4) After 1 hour, the gel was mounted on a fully automated sequencer for pre-electrophoresis for 30 minutes at a constant pressure of 1 kv and the temperature was raised to 51°C. The gel was then pre-electrophoresed on a fully automated sequencer for 1 hour. (5) At the same time of pre-electrophoresis, take out 36 samples stored at -70℃, and add 5μl of loading buffer (50μl of loading buffer in the kit + 250μl of deionized formamide, prepared before use) to each sample. (6) Shake the mixture in a vortex mixer, denature for 2 min at 94°C, and centrifuge briefly after 2 min in an ice bath. (7) Take 1.5μl of each spot sample and electrophoresis for 7 hours at constant pressure 1.68kv. The machine will automatically analyze and record the sequence results. There are often cases of misrecognition of electrophoretic lanes, which should be repaired by human. Caveat The Centri-sep column can be reused several times. After each use, the column should be washed 3 times with tap water and 2 times with distilled water, then inverted onto a test tube rack to dry, and 50 mg of Cephadex G-50 (Sigma, #9048719) weighed into the column is ready for reuse. Common Problems Note the sequencing primer specificity. For more product details, please visit Aladdin Scientific website.
Agarose TE Water-saturated phenol Anhydrous ethanol 70% ethanol TEMED Sequencing enzymes Ethidium bromide
Electrophoresis apparatus Centrifuge tubes Centrifuge Ice bath Thermostat DNA sequencer