CN113924363B - Method for extracting heavy oil genome deoxyribonucleic acid, kit and application - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
Abstract
The invention provides a method for extracting heavy oil genome deoxyribonucleic acid, a kit and application thereof. The kit comprises: snailase, proteinase K, DNA extract containing triton X-100, decontamination solution, prion precipitation solution and adsorption column. The method is mainly aimed at high-density and high-viscosity oil products, and is used for combining snailase, extract containing triton X-100 and SDC solution. The kit can be applied to the extraction of the heavy oil genome DNA.
Description
Technical Field
The invention belongs to the technical field of microbiological geology, and relates to a method for extracting heavy oil genome deoxyribonucleic acid (DNA), a kit and application thereof.
Background
International general useThe degree API is 20-30 (>0.8654-0.9340) and 10-20 (A.sub.f.)>0.9340-1) and->Respectively light crude oil, medium crude oil, heavy crude oil and extra heavy crude oil. With reference to the oil reservoir Classification (SY/T6169-1995) of the Chinese Petroleum and Natural gas industry Standard, the viscosity is more than 50-10000 mPa.s, and the relative density is more than 0.9200 g.cm -3 Crude oil (20 ℃) is common thick oil; viscosity is more than 10000-50000, relative density is more than 0.9500g cm -3 Crude oil (20 ℃) is extra thick oil; viscosity is more than 50000, relative density is more than 0.9800g cm -3 Crude oil (20 ℃) is super heavy oil. High viscosity and high density are the most important characteristics of thick oil and are also the main indexes for distinguishing common thin oil. Because the heavy oil has high contents of non-hydrocarbon, asphaltene and other components, the collection of biomass and the interference of subsequent DNA extraction in the pretreatment process are affected, so that the DNA extraction difficulty is increased, and the selection of a more targeted pretreatment mode and a cell lysis mode is very important. At present, extraction methods of DNA of oil reservoir samples are reported, but no report on thick oil is found.
Disclosure of Invention
In order to overcome the defects in the prior art and further enhance the DNA extraction effect of the thickened oil, the invention aims to provide a kit for extracting the genomic deoxyribonucleic acid of the thickened oil and a method for extracting the genomic deoxyribonucleic acid of the thickened oil by using the kit; the invention also aims to provide the application of the kit in the extraction of the heavy oil genome deoxyribonucleic acid. The invention improves the components of cell lysis and DNA extract, and increases the effectiveness of heavy oil DNA extraction; simplifying and shortening partial extraction steps and time of oscillation, thermal reaction and the like, and enhancing DNA extraction efficiency; meanwhile, the environmental protection property of the pretreatment process is improved, and the green reagent is used for replacing the conventional petroleum ether, isooctane and other organic reagents.
The aim of the invention is achieved by the following technical scheme:
in one aspect, the invention provides a method for extracting heavy oil genomic deoxyribonucleic acid, comprising the following steps:
step one, adding snailase, proteinase K, DNA extracting solution containing Triton X-100 (Triton X-100) and decontaminating solution into thick oil to lyse cells;
step two, standing, centrifuging to remove sediment to obtain upper layer liquid, adding prion sediment liquid into the upper layer liquid to remove organic macromolecular components, centrifuging and collecting the upper layer liquid;
transferring the supernatant collected by centrifugation into an adsorption column for adsorption, rinsing by a rinsing liquid and eluting by a DNA eluent, and collecting to obtain the heavy oil genome deoxyribonucleic acid.
In the method, the method adopts the triton X-100 which can effectively dissolve cell membranes and improve the quality of DNA amplified by polymerase chain reaction after DNA extraction; because the thick oil system is complex and the thick oil system is extracted into the total genome, the snailase is more beneficial to the disruption of fungal cells.
In the above method, preferably, the first step further includes a step of pretreating the thick oil, where a pretreatment reagent is added to the thick oil for treatment, to obtain pretreated thick oil.
In the method, gamma-valerolactone is adopted as a pretreatment reagent, and is different from isooctane, so that the gamma-valerolactone is a green and nontoxic chemical, can reduce the viscosity of oil products, and is easy for collection of microorganisms.
In the above method, preferably, the method specifically includes the following steps:
adding 10-20 mu L of snailase into every 300-500mg of thick oil, oscillating for 20-30min at 30-37 ℃ and 90-120rpm, sequentially adding 10-20 mu L of proteinase K and 1-2mL of DNA extracting solution containing triton X-100, oscillating for 20-30min at 30-37 ℃ and 90-120rpm, finally adding 100-200 mu L of decontamination solution, and standing for 30-60min at 50-65 ℃;
or adding 10-20 μl of snailase, 10-20 μl of proteinase K, and 1-2mL of decontamination solution containing triton X-100 and 100-200 μl into the thick oil, and standing at 50-65deg.C for 30-60min;
centrifuging at room temperature and 8000rpm (7870.72 Xg) for 2-3min to remove precipitate and obtain supernatant, transferring the supernatant into a new centrifuge tube, adding prion precipitation with the same volume as the supernatant into the supernatant, centrifuging at 8000-10000rpm for 2-3min, transferring the supernatant into the new centrifuge tube, repeatedly adding prion precipitation, centrifuging and collecting the supernatant;
transferring the centrifugally collected upper layer liquid into an adsorption column for adsorption, putting the adsorption column into a collecting pipe, centrifuging for 1-2min under the condition of 10000-12000rpm (12298-17709.12 Xg) to remove waste liquid, then adding precooled rinsing liquid into the adsorption column for rinsing, centrifuging for 1min under the condition of 10000-12000rpm to remove waste liquid, and repeating the rinsing step once; placing the adsorption column at room temperature for 10-20min to air-dry the residual rinsing liquid in the adsorption film in the adsorption column; finally, suspending and dripping DNA eluent into the middle part of the adsorption film in the adsorption column, standing for 5-10min at room temperature, centrifuging for 2-3min at 10000-12000rpm, and collecting the supernatant after centrifugation to obtain heavy oil genome deoxyribonucleic acid.
In the above method, preferably, the first step further includes a step of pretreating the thick oil, wherein the pretreatment is to add 150-250 μl of pretreatment reagent into each 300-500mg thick oil, and remove the lower layer solution after vortex mixing uniformly to obtain pretreated thick oil.
In the above method, preferably, the concentration of the helicase by mass and volume is 10-20mg/mL (aqueous solution of helicase).
In the above method, preferably, the pretreatment reagent is gamma valerolactone.
In the above method, preferably, the mass volume concentration of the proteinase K is 5-20mg/mL (aqueous solution of proteinase K).
In the above method, preferably, the DNA extract containing triton X-100 comprises 50-200mmol of tris hydrochloride, 50-200mmol of triton X-100, and 50-200mmol of Na per liter of DNA extract 3 PO 4 And 0.5-2mol NaCl.
In the above method, preferably, the decontamination solution is a Sodium Deoxycholate (SDC) solution with a mass volume concentration of 50-100mg/mL (aqueous solution of sodium deoxycholate).
In the method, a Sodium Deoxycholate (SDC) solution is adopted as a decontamination solution, the sodium deoxycholate does not have a polar head end group, and the polar groups are distributed on each part of a molecular chain.
In the above method, preferably, the prion precipitation solution is a mixed solution of phenol, chloroform and isoamyl alcohol, and the mixed volume ratio of phenol, chloroform and isoamyl alcohol is (24-26): (23-25): (0.5-1.5).
In the above method, preferably, the rinse solution is chromatographic grade ethanol, and the volume concentration of the ethanol is 70%.
In the above method, preferably, the DNA eluent is sterile ultrapure water.
In the above method, preferably, the adsorption film used in the adsorption column is a silica gel film.
On the other hand, the invention also provides a kit for extracting heavy oil genome deoxyribonucleic acid, which comprises the following components: snailase, proteinase K, DNA extract containing triton X-100, decontamination solution, prion precipitation solution and adsorption column.
In the above kit, preferably, the kit further comprises a pretreatment reagent, wherein the pretreatment reagent is gamma valerolactone.
In the above kit, preferably, the kit further comprises a rinse solution and a DNA eluent.
In the above kit, preferably, the kit further comprises glass beads, wherein the glass beads are monodisperse glass beads having a particle size of 1.5-2.0 mm.
In still another aspect, the invention also provides application of the kit in heavy oil genome deoxyribonucleic acid extraction.
The invention has the beneficial effects that:
the method for extracting the heavy oil genome DNA has more advantages for extracting the heavy oil, firstly, the method can directly extract the heavy oil as is, and can also select gamma-valerolactone as a pretreatment reagent according to the condition of oil products, and the pretreatment reagent is a green reagent which is more environment-friendly than pretreatment by adopting reagents such as isooctane, petroleum ether and the like. Secondly, the invention has pertinence to a thick oil sample, and has better cell lysis and decontamination effects on microorganisms in high-density and viscosity oil products by jointly applying snailase, an extracting solution containing triton X-100 and an SDC solution. Finally, the invention simplifies the extraction steps, shortens the extraction time, can complete the whole extraction process within 1 half hour at the highest speed, has higher extraction concentration, and has extremely small dimer production, and the subsequent qPCR detection, high-throughput sequencing, metagenome analysis and the like. The kit simplifies the operation method, and can be conveniently applied to the extraction of the genomic DNA of the thick oil so as to achieve the purpose of efficiently extracting the total deoxyribonucleic acid in the thick oil. Compared with the traditional genomic DNA extraction method (ZL 201710116846.5) for common crude oil, the method provided by the invention has the advantages of shorter time (saving more than 100 minutes in a comparable way), better effect, environmental friendliness, economy and the like.
Drawings
FIG. 1 is an electropherogram of PCR product amplification for genomic DNA extraction in example 1 of the present invention (wherein 1 to 4 are parallel samples of the same oil sample respectively; M is a molecular weight Marker);
FIG. 2 shows the electropherograms of PCR product amplification for genomic DNA extracted in examples 2 and 3 of the present invention (wherein 5 to 6 are parallel samples of the same oil sample of example 2; 7-8 are parallel samples of the same oil sample of example 3; M is a molecular weight Marker).
FIG. 3 is a comparison of the electropherograms of PCR product amplification of extracted genomic DNA in example 4 of the present invention (a, 9 to 10 in FIG. 3 are parallel samples of the same oil sample as in example 4, respectively) and of PCR product amplification of extracted genomic DNA in comparative example 2 of the present invention (b, 11 to 12 in FIG. 3 are parallel samples of the same oil sample as in comparative example 2, respectively; M is a molecular weight Marker).
Detailed Description
The technical solution of the present invention will be described in detail below for a clearer understanding of technical features, objects and advantageous effects of the present invention, but should not be construed as limiting the scope of the present invention.
The concentration and purity (A260/A280) of the DNA extract were measured by a nucleic acid prion detector, and the concentration was measured by the amount of binding site bits (Qubit).
All the thick oil samples involved in the examples were taken from Liaohe oil field, L2YHC23, density 0.994g/cm 3 The viscosity is 5860 mPa.s, the content of colloid and asphaltene is 46.8%, the heavy crude oil belongs to common thick oil according to Chinese standard SY/T6169-1995 and belongs to heavy crude oil according to an international common degree API classification mode.
The materials and reagents used in the examples were commercially available unless otherwise specified.
Example 1
The embodiment provides a kit for extracting heavy oil genome deoxyribonucleic acid, which comprises: pretreatment reagent, glass beads, snailase, proteinase K, DNA extract containing triton X-100, decontamination solution, prion precipitation solution, rinsing solution, DNA eluent, adsorption column, collecting pipe, centrifuge tube and other components and materials, wherein the content and the materials of the components are as follows:
pretreatment reagent: gamma valerolactone;
glass beads: monodisperse glass beads 20 particles with the particle size of 1.5 mm;
snailase: the mass volume concentration is 20mg/mL;
protease K: the mass volume concentration is 20mg/mL;
extract (ph 8.0): each liter of the extract contains 100mmol of Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), 100mmol of Triton X-100 (Triton-X), and 100mmol of Na 3 PO 4 1.5mol NaCl;
decontamination solution: a mass volume concentration of 100mg/mL SDC solution;
prion precipitation: the volume ratio is 25:24:1 phenol/chloroform/isoamyl alcohol;
rinsing liquid: 70% ethanol by volume of the chromatographic grade;
DNA eluent: sterile ultrapure water;
adsorption column: contains silica gel adsorption film;
collection tube and centrifuge tube: EP tube.
The extraction method described in the embodiment can be conveniently applied by adopting the kit, so as to achieve the purpose of efficiently extracting the total deoxyribonucleic acid in the thick oil.
The implementation also provides a method for extracting heavy oil genome deoxyribonucleic acid, which comprises the following steps:
step one, taking 500mg of thick oil, adding 200-300 mu L of gamma-valerolactone, uniformly mixing by vortex, and removing the lower layer liquid to obtain pretreated thick oil;
adding 10-20 mu L of snailase into a centrifuge tube, oscillating for 30min at 37 ℃ and 120rpm, sequentially adding 10-20 mu L of proteinase K and 1-2mL of DNA extracting solution containing triton X-100, oscillating for 30min at 37 ℃ and 120rpm, finally adding 100-200 mu L of decontaminating solution, standing for 1h at 65 ℃, centrifuging for 3min at room temperature and 6000-12000rpm to remove sediment to obtain upper layer liquid, transferring the upper layer liquid into a new centrifuge tube, adding prion precipitating liquid with the same volume as the upper layer liquid into the upper layer liquid, centrifuging for 3min at 6000-12000rpm, transferring the upper layer liquid into the new centrifuge tube, repeatedly adding prion precipitating liquid into the new centrifuge tube for one time, and centrifuging to collect the upper layer liquid;
transferring 500-1000 mu L of the centrifugally collected supernatant into an adsorption column for adsorption, placing the adsorption column into a collecting pipe, centrifuging for 1min under the condition of 6000-12000rpm to remove waste liquid, repeating the step until most of the supernatant is filtered by the adsorption column (note that the supernatant cannot be absorbed into a prion precipitation layer when the supernatant is absorbed, so that the supernatant with the thickness of 3-5mm above the prion precipitation layer is reserved), then adding 500-1000 mu L of low-temperature precooled rinsing liquid into the adsorption column for rinsing, centrifuging for 1min under the condition of 6000-12000rpm to remove waste liquid, and repeating the rinsing step; placing the adsorption column at room temperature for 10min to air-dry the residual rinsing liquid in the adsorption film in the adsorption column; finally, suspending and dripping 110 mu L of DNA eluent into the middle part of the adsorption film in the adsorption column, standing for 8min at room temperature, centrifuging for 2min at 10000-12000rpm, and collecting the supernatant after centrifugation to obtain heavy oil genome deoxyribonucleic acid (DNA).
The purity of the heavy oil genomic DNA extracted in the embodiment is verified by PCR amplification, and the specific steps are as follows:
the forward Primer (Primer 1) was 515F:5'-GTGYCAGCMGCCGCGGTAA-3',
the reverse Primer (Primer 2) was 806R:5'-GGACTACVSGGGTATCTAAT-3'.
Wherein: y, M, V, S is a degenerate base.
Y represents a base selected from C or T;
m represents a base selected from A or C;
v represents a base selected from A, C or G;
s represents a base selected from C or G;
thus, the base sequence of the forward primer may be selected from one of the following sequences:
5'-GTGCCAGCAGCCGCGGTAA-3' (SEQ ID NO: 1);
5'-GTGCCAGCCGCCGCGGTAA-3' (SEQ ID NO: 2);
5'-GTGTCAGCAGCCGCGGTAA-3' (SEQ ID NO: 3);
5'-GTGTCAGCCGCCGCGGTAA-3' (SEQ ID NO: 4).
The base sequence of the reverse primer may be selected from one of the following sequences:
5'-GGACTACACGGGTATCTAAT-3' (SEQ ID NO: 5);
5'-GGACTACCCGGGTATCTAAT-3' (SEQ ID NO: 6);
5'-GGACTACGCGGGTATCTAAT-3' (SEQ ID NO: 7);
5'-GGACTACAGGGGTATCTAAT-3' (SEQ ID NO: 8);
5'-GGACTACCGGGGTATCTAAT-3' (SEQ ID NO: 9);
5'-GGACTACGGGGGTATCTAAT-3' (SEQ ID NO: 10).
The amplified products were electrophoresed on a 1% agarose gel at 100V for 45min and photographed by an ultraviolet gel imaging system. L2YHC23-1 and L2YHC23-2 are a set of parallel samples, and L2YHC23p is the mean and standard deviation of the oil samples (Table 1). The DNA extracted in this example 1 was relatively pure, with an A260/A280 ratio of about 1.6, and a relatively high concentration (Table 1). Electrophoresis Nos. 1 and 2 (FIG. 1) show that the DNA amplified product has single and clear bands, and thus, the DNA extracted by the invention has better effect.
TABLE 1 concentration and purity of genomic DNA of heavy oil extracted in this example 1
Comparative example 1
500mg of thick oil is taken, the pretreatment method is the same as in example 1, 200-300 mu L of gamma-valerolactone is added, vortex mixing is carried out, the lower layer liquid is removed to obtain pretreatment liquid, and the rest extraction steps are carried out according to the QIAGEN kit method. L2YHC23-3 and L2YHC23-4 are a set of parallel samples, and L2YHC23p is the mean and standard deviation of the oil samples (Table 2). The QIAGEN kit method (Table 2) extracted heavy oil DNA at a lower purity and concentration than the method of the invention (example 1), with less variation, but subsequent sequencing showed that only the method of the invention could be used for subsequent experiments.
TABLE 2 comparative example 1 extraction of the concentration and purity of genomic DNA of heavy oil
Example 2
The only difference between this example and example 1 is the absence of step one, i.e. the extraction was carried out directly with thick oil without the use of pretreatment reagent (gamma valerolactone), the rest of the procedure being completely identical. L2YHC23-5 and L2YHC23-6 are a set of parallel samples, and L2YHC23p is the mean and standard deviation of the oil samples (Table 3). The extraction result and the verification result show that the DNA concentration extracted by the invention is higher (Table 3), and the electrophoresis bands of No. 5 and No. 6 (FIG. 2) are single and clear, which shows that the DNA extraction effect of the heavy oil is better.
TABLE 3 concentration and purity of genomic DNA from heavy oil extracted in this example 2
Example 3
Compared with the embodiment 1, the embodiment omits the first step, directly uses the thick oil for extraction, and specifically comprises the following steps: adding 500mg of thick oil into a centrifuge tube, adding 10-20 mu L of snailase, sequentially adding 10-20 mu L of proteinase K and 1-2mL of DNA extracting solution containing triton X-100, finally adding 100-200 mu L of decontaminating solution, standing at 65 ℃ for 1h, centrifuging at room temperature and 8000rpm for 3min to remove sediment to obtain upper layer liquid, transferring the upper layer liquid into a new centrifuge tube, adding prion precipitating liquid with the same volume as the upper layer liquid into the upper layer liquid, centrifuging for 3min at 8000rpm, transferring the upper layer liquid into the new centrifuge tube, repeatedly adding the prion precipitating liquid once, centrifuging and collecting the upper layer liquid; the subsequent operation is exactly the same as in example 1.
L2YHC23-7 and L2YHC23-8 are a set of parallel samples, and L2YHC23p is the mean and standard deviation of the oil samples (Table 4). The extraction result and the verification result show that the DNA concentration extracted by the method is higher (Table 4), and the electrophoresis bands of No. 7 and No. 8 (FIG. 2) are the clearest and bright, which shows that the DNA extraction effect of the thickened oil is better.
TABLE 4 concentration and purity of genomic DNA from heavy oil extracted in this example 3
Example 4
This example uses the same thickened oil (different storage conditions after collection) as example 3, and the amounts and experimental procedure are exactly the same. L2YHC23-9 and L2YHC23-10 are a set of parallel samples, and L2YHC23p is the mean and standard deviation of the oil samples (Table 5). The extraction result and the verification result show that the concentration and the purity of the DNA extracted by the method are higher (Table 5), and the electrophoresis bands (a in figure 3) of No. 9 and No. 10 are clearer and brighter, thus indicating that the DNA extraction effect of the thickened oil is better.
TABLE 5 concentration and purity of genomic DNA from heavy oil extracted according to this example 4
Comparative example 2
In comparative example 2, the same thick oil was used as in example 4, and the extraction method was performed according to the method of patent No. ZL 201710116846.5. L2YHC23-11 and L2YHC23-12 are a set of parallel samples, L2YHC23p is the average value and standard deviation (Table 6) of the oil samples, and the extraction result and verification result show that the concentration and purity of DNA extracted by the method are lower (Table 6) than those of the method (example 4) of the invention, and the electrophoresis bands (b in FIG. 3) of No. 11 and No. 12 are not bright (a in FIG. 3) of the method of the invention, thus indicating that the extraction effect of the invention is better. In addition, compared with the prior invention, the method has the advantages that the time for extracting the DNA is shorter, the DNA can be completed within 90min at the highest speed, and at least 100min can be saved; meanwhile, the pretreatment process of the invention does not use organic reagents such as isooctane and the like, and has more environmental protection than the prior invention.
TABLE 6 concentration and purity of genomic DNA extracted from heavy oil according to comparative example 2
The genome DNA extracted by the method can meet the analysis requirements of subsequent qPCR detection, high-throughput sequencing, metagenome and the like.
Sequence listing
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Claims (15)
1. A method for extracting heavy oil genomic deoxyribonucleic acid, comprising the following steps:
step one, adding snailase, proteinase K, DNA extracting solution containing triton X-100 and decontamination solution into thick oil which is not pretreated or thick oil after pretreatment for cell lysis, wherein gamma-valerolactone is added into thick oil for treatment;
step two, standing, centrifuging to remove sediment to obtain upper layer liquid, adding prion sediment liquid into the upper layer liquid to remove organic macromolecular components, centrifuging and collecting the upper layer liquid;
transferring the supernatant collected by centrifugation into an adsorption column for adsorption, rinsing by a rinsing liquid and eluting by a DNA eluent, and collecting to obtain the heavy oil genome deoxyribonucleic acid.
2. The method according to claim 1, wherein the method comprises in particular the steps of:
adding 10-20 mu L of snailase into every 300-500mg of thick oil, oscillating for 20-30min at 30-37 ℃ and 90-120rpm, sequentially adding 10-20 mu L of proteinase K and 1-2mL of DNA extracting solution containing triton X-100, oscillating for 20-30min at 30-37 ℃ and 90-120rpm, finally adding 100-200 mu L of decontamination solution, and standing for 30-60min at 50-65 ℃;
or adding 10-20 μl of snailase, 10-20 μl of proteinase K, 1-2mL of DNA extract containing triton X-100, 100-200 μl of decontamination solution into every 300-500mg of thick oil, and standing at 50-65deg.C for 30-60min;
step two, centrifuging for 2-3min at room temperature and 8000-10000rpm to obtain an upper layer liquid, transferring the upper layer liquid into a new centrifuge tube, adding a prion precipitation liquid with the same volume as the upper layer liquid into the upper layer liquid, centrifuging for 2-3min at 8000-10000rpm, transferring the upper layer liquid into the new centrifuge tube, repeatedly adding the prion precipitation liquid, centrifuging and collecting the upper layer liquid;
transferring the centrifugally collected supernatant into an adsorption column for adsorption, putting the adsorption column into a collecting pipe, centrifuging for 1-2min under 10000-12000rpm to remove waste liquid, adding precooled rinsing liquid into the adsorption column for rinsing, centrifuging for 1-2min under 10000-12000rpm to remove waste liquid, and repeating the rinsing step; placing the adsorption column at room temperature for 10-20min to air-dry the residual rinsing liquid in the adsorption film in the adsorption column; finally, suspending and dripping DNA eluent into the middle part of the adsorption film in the adsorption column, standing for 5-10min at room temperature, centrifuging for 2-3min at 10000-12000rpm, and collecting the supernatant after centrifugation to obtain heavy oil genome deoxyribonucleic acid.
3. The method according to claim 1, wherein the pretreatment is to add 150-250 μl of gamma valerolactone to every 300-500mg of thick oil, and remove the lower layer liquid after vortex mixing to obtain pretreated thick oil.
4. A method according to any one of claims 1-3, wherein the snailase is present in a mass volume concentration of 10-20mg/mL.
5. A method according to any one of claims 1-3, wherein the mass volume concentration of proteinase K is 5-20mg/mL.
6. A method according to any one of claims 1 to 3, wherein the DNA extract containing triton X-100 comprises 50-200mmol of tris hydrochloride, 50-200mmol of triton X-100, 50-200mmol of Na per liter of DNA extract 3 PO 4 And 0.5-2mol NaCl.
7. A method according to any one of claims 1 to 3, wherein the decontamination solution is a sodium deoxycholate solution having a mass volume concentration of 50-100mg/mL.
8. A method according to any one of claims 1 to 3, wherein the prion precipitation is a mixture of phenol, chloroform and isoamyl alcohol, the mixture of phenol, chloroform and isoamyl alcohol being in a volume ratio of (24-26): (23-25): (0.5-1.5).
9. A process according to any one of claims 1 to 3, wherein the rinse solution is chromatographic grade ethanol having a volume concentration of 70%.
10. A method according to any one of claims 1 to 3, wherein the DNA eluate is sterile ultra pure water.
11. A method according to any one of claims 1 to 3, wherein the adsorption film employed in the adsorption column is a silica gel film.
12. A kit for extracting heavy oil genomic dna, wherein the kit comprises: snailase, proteinase K, DNA extracting solution containing triton X-100, decontaminating solution, prion precipitating solution and adsorption column, wherein the kit also comprises a pretreatment reagent, and the pretreatment reagent is gamma-valerolactone.
13. The kit of claim 12, wherein the kit further comprises a rinse solution and a DNA eluate.
14. The kit of claim 12, further comprising glass beads, wherein the glass beads are monodisperse glass beads having a particle size of 1.5-2.0 mm.
15. Use of the kit according to any one of claims 12-14 for heavy oil genomic dna extraction.
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| CN105624152B (en) * | 2016-03-01 | 2019-02-19 | 中国人民解放军第二军医大学 | A kind of yeast-like fungi total DNA for nucleic acid amplification is without instrument extracting method |
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| KR20050055980A (en) * | 2003-12-09 | 2005-06-14 | 주식회사 마이진 | Kit for extracting genomic dna from human blood and method for extracting genomic dna using the same |
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