The genetic diversity of a species is essentially the diversity of the primary sequence of DNA. In recent years, with the rapid development and increasing popularity of DNA sequencing technology, DNA sequencing is playing an increasingly important role in the study of genetic diversity. This chapter describes a manual and a fully automated double-stranded DNA sequencing method currently used in genetic diversity research.
1 Preparation of DNA template
In the study of genetic diversity, the speed of DNA sequencing has become a critical factor due to the large sample size. Therefore, purified PCR double-stranded products are often directly assayed in such studies without the use of cloning techniques. This section describes the methods commonly used in this laboratory to prepare sequencing templates from PCR products, ie, low melting point gel recovery methods.
(1) Prepare a 1.5%-2.0% agarose gel. After it is fully solidified, cut a strip of about 1 cm wide at about 5 cm from the spotting line, and pour the pre-boiled low in the cut trough. Melting point agarose gel.
(2) After the low melting point gel is solidified, the PCR reaction solution to be purified is completely spotted, and electrophoresis is performed at a constant pressure of about 100 V until the amplified fragment enters the middle of the low melting point gel. The strip that had entered the low melting point gel was cut under 360 nm ultraviolet light and placed in a 1.5 ml centrifuge tube.
(3) Centrifuge, compress the rubber block to the bottom of the tube, and then add TE to 500 μl. The low melting point gel was melted in a 68 ° C water bath, and then an equal volume of water-saturated phenol was quickly added and mixed.
(4) Shake for 10 minutes at room temperature and centrifuge for 10 minutes at 12 000 r/min. The supernatant was taken and extracted with chloroform-isoamyl alcohol (24:1) for 5 minutes.
(5) Centrifuge at 12 000 r/min for 10 minutes, take the supernatant, add 1/10 volume of 10 mol/dm3 NH4Ac and 2 volumes of absolute ethanol, and precipitate at 70 ° C for more than half an hour.
(6) Centrifuge for another 10 minutes at 12 000 r/min, and wash the pellet with 70% cold ethanol; again, after briefly centrifuging, carefully pour off the ethanol solution. After the precipitate is dried, it is dissolved in 20-50 μl of TE buffer or sterile deionized water to prepare a prepared DNA template.
There are also some very effective commercial kits available for purification of PCR products, such as the Oiagen PCR Product Purification Kit, but at a higher cost.
2 manual DNA sequence analysis technology
2.1 Preparation of sequencing glue
Prepare sequencing electrophoresis gel according to the following formula:
6% plastic working fluid 70ml
10% persulphate (APS) 70μl (freshly prepared)
TEMED 70μl
After the above solution is mixed, it is poured into a prepared rubber plate, and the "shark tooth" comb is reversely inserted into the rubber to a depth of about 0.5 to 1.0 cm. Polymerize at room temperature for about 1 hour.
12.2.2 Sequencing reaction
The sequencing kit was purchased from USB Corporation of the United States, and the sequencing enzyme was Sequenase Version 2.0; α35S-dATP was purchased from the United States.
DuPont (1mCui/100μl). The sequencing reaction was performed as recommended by USB:
(1) DNA template mixture (0.5 ml Eppendorf tube):
DNA template 7μl
Sequencing primer 1μl
5 times Reaction buffer 2μl
NP4O 1μl
(2) Labeling the mixture (the amount of each reaction):
DTT (0.1mol/dm3) 1μl
1/5dGTP labelling mix 2μl
Enzyme dilution buffer 1.75μl
NP4O 0.55μl
Deionized water 0.375μl
Sequencing enzyme 0.125μl
35S-dATP 0.5μl
(3) ddNTP (ddA, ddG, ddC, ddT) 2.5μl
(4) Annealing: The DNA template mixture is boiled for 3 minutes, and quickly placed in a mixed ice bath of ethanol and dry ice for about 30 seconds (if there is no dry ice, it is frozen at -20 ° C ~ -70 ° C The anhydrous ethanol is taken out from the refrigerator before use. The reaction solution is stored in dry ice or crushed ice before the labeling reaction.
(5) Labelling: The annealed DNA template mixture was taken out from dry ice or ice, allowed to melt in the hands, and then 5.5 μl of the labeled mixture was added. After centrifugation for 10 to 15 seconds on a microcentrifuge, set to room temperature for 1 minute. (6) Chain termination: The mixture prepared in the previous step was charged into a tube with ddNTP previously heated at 37 ° C in a tube of 3.3 μl per tube, and reacted at 37 ° C for 4 to 5 minute.
(7) Add 4 μl of stop solution to each tube.
2.3 Electrophoresis
Electrode buffer 1 times TBE (pH 8.0)
Pre-electrophoresis for 30 minutes to 1 hour
Constant temperature mode sequencing rubber plate clamp aluminum thermostat
Electrophoresis temperature is around 50 °C
Electrophoresis conditions constant power 90 ~ 110W
Electrophoresis time 2.5 hours to 5 hours
2.4 Dry glue preparation and tableting
After the electrophoresis is finished, the rubber plate is removed, and the glass plate is opened with a tool to leave the glue on one of the plates. The cut Xinhua No. 3 filter paper is adhered to the glue to make it adhere to the filter paper, and then the glue is placed in the dry glue machine to prepare the dry glue for 1 to 1.5 hours. The prepared dry glue is placed in a tablet box, placed in a dark room, and the X-ray film is pressed onto the glue for exposure for 2 to 3 days.
2.5 X-ray film rinse
The exposed X-ray film is developed for 4 to 8 minutes (depending on the length of time the developer is used); after fixing for 5 to 10 minutes, the DNA sequence can be read after rinsing.
3 automatic DNA sequence analysis technology
This section describes methods for double-stranded DNA sequencing using Perkin-Elmer's Models 373 and 377 automated DNA sequencers. The thermal cycle sequencing reaction was carried out using Applied Biosystems Inc.'s DyeDeoyTM Terminator Cycle Sequencing Kit (#401434) or FS-DNA Sequencing Kit (#402079). The operation process is mainly carried out according to the method recommended by the manufacturer. The specific operation method is as follows.
3.1 Mixed reactants
(1) 2.5 μl of the purified PCR product [pGEM-3Zf(+) DNA in 2.5 μl sequencing kit] was taken for agarose gel electrophoresis.
(2) The gel was stained with ethidium bromide, rinsed with running water, and photographed under ultraviolet light. Reference pGEM-3Zf
(10) 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
Primer (10pmol/ml) 0.5μl
Add water to 5.25μl or 6.0μl (FSKit)
Denaturation at 100 ° C for 2 minutes, then ice bath for 2 minutes, then briefly centrifuge, then add:
Sye solution 4.75μl in DyeDeoy TM Terminator Cycle Sequencing Kit
Or sequencing solution 4.0μl in the FS-DNA Sequencing Kit
Final volume 10μl
(4) After thoroughly mixing the reactants with a microsampler, 20 μl of paraffin oil was added thereto.
3.2 Thermal cycling reaction
The cooling time in this reaction is very critical (about 1 ° C / s). Therefore, a Perkin-Elmer sequence thermal cycler (PCR instrument such as 480, 2400 or 9600) can be used. The operations described below are based on the Perkin-Elmer Model 480 Thermal Cycler. Put the reaction tube into the thermal cycler and perform the thermal cycle reaction as follows:
(1) 96 ° C for 1 second
96 ° C 30 seconds
(2) 50 ° C for 1 second
50 ° C 1 minute
(3) 60 ° C l seconds
60 ° C 4 minutes
A total of 25 cycles are required.
3.3 Purification of sequencing products
The best way to purify the sequencing product is to use the Centri SeP column (#CS-901) from Princeton Separations Inc. The operation process is as follows:
(1) Light-column column to lift Cephadex G-50 powder from the bottom of the column.
(2) Remove the top cover, add 800 μl of deionized water, and cover with a lid to shake to remove air bubbles.
(3) Place at room temperature for 30 minutes to hydrate the gel column.
(4) Remove the column top cover and the lower cover, and place the gel column in a configured elution tube to allow excess liquid to flow out.
(5) The liquid was poured off and the column was centrifuged together with the elution tube at 3 000 r/min for 2 minutes.
(6) Place the column in a 1.5 ml centrifuge tube and take the sequencing reaction with a microsampler and place it in the center of the gel column. Take care to avoid introducing paraffin oil.
(7) Centrifuge at 3 000 r/min for 2 minutes. It should be noted that the orientation of the column must be consistent with the first centrifugation.
(8) The sample was dried in a vacuum drying centrifuge.
(9) The dried sample was stored at -70 ° C until electrophoresis. Samples that have been stored for 1 year under these conditions will not quench their fluorescence.
It should be noted that the Centri-sep cylinder can be used multiple times. After each use, wash it 3 times with tap water, wash it twice with distilled water, then put it on a test tube rack to dry it, then weigh 50mg Cephadex G-50 (Sigma, #9048719) and put it back in use. .
3.4 Electrophoresis
Electrophoresis was performed using ABI's Model 377 Automated DNA Sequence Analyzer and sequence data was recorded. The control software is the PRIM377 Collection. The electrophoresis process is as follows.
(1) The concentration of the polyacrylamide gel is 4.25%, and the preparation process is as follows:
Urea 18g
Amberlite ion exchange resin (Sigma, MB-lA) approx. 39g
40% Acry mide: Bis (19:1) (AMRESCO, #0496-500) 5.3ml
Deionized water 25ml
The mixture was stirred on a magnetic stirrer for 10 minutes.
(2) Take 5 ml of 10 times TBE, filter through a 2 μm filter, and then filter the above glue.
10 times TBE formula:
Tris-base 108g
Boric acid 55g
Na2 EDTA(2H2O) 7.44g
Constant volume to 1L
(3) The filtrate was made up to 50 ml, 35 μl of TEMED and 250 μl of 10% persulphate (APS) were added, and the mixture was gently shaken, and the gel was poured into the assembled glass plate using a 50 ml needleless syringe.
(4) After 1 hour, the glue was mounted on a fully automatic serializer for 30 minutes, with a constant voltage of 1 kV and the temperature was raised to 51 °C.
(5) At the same time as pre-electrophoresis, take 36 samples stored at -70 °C, and add 5 μl of each sample solution (50 μl of loading buffer + 250 μl of deionized formamide in the kit, prepared before use).
(6) The mixture was shaken on a vortex mixer, denatured at 94 ° C for 2 minutes, and ice-bathed for 2 minutes and then briefly centrifuged.
(7) Each sample was taken at 1.5 μl and electrophoresed at a constant pressure of 1.68 kV for 7 hours. The machine will automatically analyze and record the sequence results. There are often cases where the electrophoresis channel is identified incorrectly, and it should be artificially repaired at this time.
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