CN113924363A - Method for extracting thick oil genome deoxyribonucleic acid, kit and application - Google Patents
Method for extracting thick oil genome deoxyribonucleic acid, kit and application Download PDFInfo
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Abstract
The invention provides a method for extracting thick oil genome deoxyribonucleic acid, a kit and application. The kit comprises: helicase, protease K, DNA extract containing triton X-100, decontamination solution, prion precipitation solution and adsorption column. The method mainly aims at high-density and high-viscosity oil products, and combines helicase, an extracting solution containing triton X-100 and a SDC solution. The kit can be applied to extraction of thick oil genome DNA.
Description
The invention belongs to the technical field of microbial geology, and relates to a method for extracting thick oil genome deoxyribonucleic acid (DNA), a kit and application.
International common degree API is more than or equal to 32
API 20-30: (
0.8654-0.9340), API 10-20 (API: (A)
0.9340-1) and DEG API ≦ 10(
) It is referred to as light crude oil, medium crude oil, heavy crude oil and extra heavy crude oil, respectively. Referring to the oil deposit classification of China oil and gas industry Standard (SY/T6169-1995), the viscosity is more than 50-10000 mPa.s, and the relative density is more than 0.9200 g.cm-3Crude oil at the temperature of 20 ℃ is common thick oil; viscosity > 10000-50000 and relative density greater than 0.9500 g-cm-3Crude oil at (20 ℃) is extra heavy oil; viscosity > 50000 and relative density greater than 0.9800g cm-3The crude oil at 20 ℃ is super heavy oil. The high viscosity and the high density are the most important characteristics of the thick oil and are main indexes for distinguishing the thick oil from the common thin oil. Because the thickened oil contains high content of non-hydrocarbon, asphaltene and other components, the collection of biomass in the pretreatment process and the interference of subsequent DNA extraction are influenced, and the DNA extraction difficulty is further increased, the selection of a more targeted pretreatment mode and a cell cracking mode is very important. At present, methods for extracting DNA from oil reservoir samples have been reported, but no heavy oil has been reported.
Disclosure of Invention
In order to overcome the defects of the prior art and further enhance the extraction effect of the DNA of the thick oil, the invention aims to provide a kit for extracting the DNA of a thick oil genome and a method for extracting the DNA of the thick oil genome by using the kit; the invention also aims to provide application of the kit in thick oil genome deoxyribonucleic acid extraction. The invention improves cell lysis and DNA extract components, and increases the effectiveness of heavy oil DNA extraction; simplifying and shortening the extraction steps and time of oscillation, thermal reaction and the like, and enhancing the extraction efficiency of DNA; meanwhile, the environmental protection property of the pretreatment process is improved, and a green reagent is used for replacing conventional organic reagents such as petroleum ether, isooctane and the like.
The purpose of the invention is realized by the following technical scheme:
in one aspect, the invention provides a method for extracting thick oil genome deoxyribonucleic acid, which comprises the following steps:
adding helicase, protease K, DNA extracting solution containing Triton X-100(Triton X-100) and decontamination solution into thick oil to perform cell lysis;
step two, centrifuging after standing to remove precipitates to obtain supernatant, adding a prion precipitation solution into the supernatant to remove organic macromolecular components, centrifuging and collecting the supernatant;
and step three, transferring the upper layer liquid collected by centrifugation to an adsorption column for adsorption, rinsing by using a rinsing liquid and eluting by using a DNA eluent, and collecting to obtain the thick oil genome deoxyribonucleic acid.
In the method, the adopted ingredients contain triton X-100 which can effectively dissolve cell membranes and improve the quality of polymerase chain reaction amplified DNA after DNA extraction; because the thick oil system is complex and is extracted as a total genome, the helicase is more beneficial to the breaking of fungal cells.
In the above method, preferably, the step one further includes a step of pretreating the thick oil, where the pretreatment is to add a pretreatment reagent into the thick oil to perform a treatment, so as to obtain pretreated thick oil.
In the method, gamma-valerolactone is adopted as a pretreatment reagent, is different from isooctane, is a green and nontoxic chemical, can reduce the viscosity of oil products, and is easy for collecting microorganisms.
In the above method, preferably, the method specifically includes the steps of:
step one, adding 10-20 mu L of snailase into 500mg of thick oil per 300-inch thick oil, oscillating for 20-30min at 30-37 ℃ and 90-120rpm, then sequentially adding 10-20 mu L of protease K and 1-2mL of DNA extracting solution containing triton X-100, oscillating for 20-30min at 30-37 ℃ and 90-120rpm, finally adding 200 mu L of decontamination solution, and standing for 30-60min at 50-65 ℃;
or directly adding 10-20 μ L of helicase, 10-20 μ L of protease K, and 1-2mL of 200 μ L decontamination solution containing Triton X-100 and 100-;
step two, centrifuging for 2-3min at room temperature and 8000rpm (7870.72 Xg) to remove precipitates to obtain supernatant, transferring the supernatant into a new centrifuge tube, adding a prion precipitation solution with the same volume as the supernatant into the supernatant, centrifuging for 2-3min at 8000-10000rpm, transferring the supernatant into the new centrifuge tube, repeatedly adding the prion precipitation solution once, and centrifuging to collect the supernatant;
transferring the upper layer liquid collected by centrifugation to an adsorption column for adsorption, placing the adsorption column in a collecting pipe, centrifuging for 1-2min under the conditions of 10000-12000rpm (12298-17709.12 Xg) to remove the waste liquid, then adding precooled rinsing liquid into the adsorption column for rinsing, centrifuging for 1min under the conditions of 10000-12000rpm to remove the 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; and finally, suspending and dripping DNA eluent into the middle part of an adsorption membrane in the adsorption column, standing for 5-10min at room temperature, centrifuging for 2-3min under the conditions of 10000-12000rpm, and collecting the centrifuged supernatant, namely the thickened oil genome deoxyribonucleic acid.
In the above method, preferably, the step one further includes a step of pretreating the thick oil, where the pretreatment is to add 150-.
In the above method, preferably, the mass volume concentration of the helicase is 10-20mg/mL (aqueous solution of helicase).
In the above method, preferably, the pretreatment reagent is gamma valerolactone.
In the above process, preferably, the mass volume concentration of the protease K is 5 to 20mg/mL (aqueous solution of protease K).
In the above method, preferably, the DNA extract solution containing Triton X-100 comprises 50-200mmol of Tris-HCl, 50-200mmol of Triton X-100, and 50-200mmol of Na per liter3PO 4And 0.5 to 2mol of 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, Sodium Deoxycholate (SDC) solution is taken as decontamination solution, the sodium deoxycholate does not have a polar head end group, and polar groups are distributed in all parts of a molecular chain.
In the above method, preferably, the prion precipitation solution is a mixture of phenol, chloroform and isoamyl alcohol, and the volume ratio of the mixture of phenol, chloroform and isoamyl alcohol is (24-26): (23-25): (0.5-1.5).
In the above method, preferably, the rinsing liquid is chromatographically pure 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 membrane used in the adsorption column is a silica gel membrane.
In another aspect, the present invention also provides a kit for extracting dna from a thick oil genome, the kit comprising: helicase, protease K, DNA extract containing triton X-100, decontamination solution, prion precipitation solution and adsorption column.
In the kit, preferably, the kit further comprises a pretreatment reagent, and the pretreatment reagent is gamma-valerolactone.
In the kit, preferably, the kit further comprises a rinsing 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 another aspect, the invention also provides application of the kit in thick oil genome deoxyribonucleic acid extraction.
The invention has the beneficial effects that:
the method for extracting the thick oil genome DNA has more advantages for extracting the thick oil, firstly, the method can directly extract the thick oil as is, and also can select gamma-valerolactone as a pretreatment reagent according to the condition of the oil product, and the pretreatment reagent is a green reagent and is more environment-friendly than pretreatment by using isooctane, petroleum ether and other reagents. Secondly, the method has pertinence to the thick oil sample, and has better cell cracking and decontamination effects on microorganisms in high-density and high-viscosity oil products by jointly applying the helicase, the extracting solution containing the triton X-100 and the SDC solution. Finally, the invention simplifies the extraction steps, shortens the extraction time, can quickly complete the whole extraction process within 1 and a half hour, has high extraction concentration and extremely small dimer production amount, and can be used for subsequent qPCR detection, high-throughput sequencing, metagenome and other analysis. The kit simplifies the operation method, can conveniently apply the extraction of the genome DNA of the thick oil, and achieves the purpose of efficiently extracting the total deoxyribonucleic acid in the thick oil. Compared with the conventional genomic DNA extraction method (ZL 20170116846.5) aiming at common crude oil by the applicant, the method is shorter in time (saving more than 100min on year-on-year basis), better in effect, more environment-friendly and economical and the like.
FIG. 1 is an electropherogram of the extracted genomic DNA PCR product amplification 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 is an electropherogram of the amplified PCR products from genomic DNA extracted in examples 2 and 3 of the present invention (wherein: 5 to 6 are parallel samples of the same oil sample as in example 2; 7 to 8 are parallel samples of the same oil sample as in example 3; and M is a molecular weight Marker).
FIG. 3 is a comparison of the electropherograms of the PCR products of the genomic DNA extracted 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) and the electropherograms of the PCR products of the genomic DNA extracted 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; M is a molecular weight Marker).
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.
The concentration and purity of the DNA extract (A260/A280) were measured using a nucleic acid prion detector, and the concentration was measured in combination with the amount of bits per mouth (Qubit).
In the examples, all the heavy oil samples involved were collected from the Liaohe oil field, L2YHC23, density 0.994g/cm3The viscosity was 5860 mPas, the gum + asphaltene content was 46.8%, the heavy crude oil was classified according to the Chinese Standard SY/T6169-1995 as the common heavy oil and according to the International Normal degree API classification.
The raw materials and reagents used in the examples were all commercially available unless otherwise specified.
Example 1
The embodiment provides a kit for extracting thick oil genome deoxyribonucleic acid, which comprises: the pretreatment reagent, glass beads, helicase, protease K, DNA extracting solution containing triton X-100, decontamination solution, prion precipitation solution, rinsing solution, DNA eluent, an adsorption column, a collecting pipe, a centrifugal pipe and other components and materials, wherein the content and the material of each component are as follows:
pretreatment reagents: gamma valerolactone;
glass beads: 20 monodisperse glass beads with the particle size of 1.5 mm;
snailase: the mass volume concentration is 20 mg/mL;
protease K: the mass volume concentration is 20 mg/mL;
extract (ph 8.0): each liter of the extract contains 100mmol of Tris-HCl, 100mmol of Triton-X and 100mmol of Na3PO 4And 1.5mol NaCl;
decontamination solution: a SDC solution with the mass volume concentration of 100 mg/mL;
protein precipitation liquid: the volume ratio is 25: 24: 1 in a mixed solvent of phenol/chloroform/isoamyl alcohol;
rinsing liquid: chromatographically pure grade 70% ethanol by volume;
DNA eluent: sterile ultrapure water;
adsorption column: contains a silica gel adsorption film;
collecting and centrifuging tubes: an EP tube.
The extraction method in the embodiment can be conveniently applied by adopting the kit so as to achieve the aim of efficiently extracting the total deoxyribonucleic acid in the thick oil.
The implementation also provides a method for extracting the thick oil genome deoxyribonucleic acid, which comprises the following steps:
step one, taking 500mg of thick oil, adding 200 and 300 mu L of gamma-valerolactone, uniformly mixing by vortex, and removing subnatant to obtain pretreated thick oil;
step two, taking the pretreated thick oil into a centrifuge tube, adding 10-20 mu L of helicase, oscillating for 30min at 37 ℃ and 120rpm, then sequentially adding 10-20 mu L of protease 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 decontamination solution, standing for 1h at 65 ℃, centrifuging for 3min at room temperature and 6000-12000rpm to remove precipitates to obtain supernatant, transferring the supernatant into a new centrifuge tube, adding the prion precipitation solution with the same volume as the supernatant into the supernatant, centrifuging for 3min at 6000-12000rpm, transferring the supernatant into the new centrifuge tube, repeatedly adding the prion precipitation solution once, centrifuging and collecting the supernatant;
step three, transferring 1000 mu L of supernatant fluid collected by centrifugation into an adsorption column for adsorption, placing the adsorption column into a collecting pipe, centrifuging at 12000rpm of 6000-; standing the adsorption column at room temperature for 10min to air-dry the residual rinsing liquid in the adsorption film in the adsorption column; and finally suspending and dripping 110 mu L of DNA eluent into the middle part of the adsorption membrane in the adsorption column, standing for 8min at room temperature, centrifuging for 2min under the conditions of 10000-12000rpm, and collecting the centrifuged supernatant, namely the thickened oil genome deoxyribonucleic acid (DNA).
The purity of the thickened oil genome DNA extracted in the embodiment is verified by PCR amplification, and the specific steps are as follows:
the forward Primer (Primer1) was 515F: 5 '-GTGYCAGCMGCCGCGGTAA-3',
reverse Primer (Primer2) 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' (shown in SEQ ID NO: 1);
5'-GTGCCAGCCGCCGCGGTAA-3' (shown in SEQ ID NO: 2);
5'-GTGTCAGCAGCCGCGGTAA-3' (shown in SEQ ID NO: 3);
5'-GTGTCAGCCGCCGCGGTAA-3' (shown in SEQ ID NO: 4).
The base sequence of the reverse primer may be selected from one of the following sequences:
5'-GGACTACACGGGTATCTAAT-3' (shown in SEQ ID NO: 5);
5'-GGACTACCCGGGTATCTAAT-3' (shown in SEQ ID NO: 6);
5'-GGACTACGCGGGTATCTAAT-3' (shown in SEQ ID NO: 7);
5'-GGACTACAGGGGTATCTAAT-3' (shown in SEQ ID NO: 8);
5'-GGACTACCGGGGTATCTAAT-3' (shown in SEQ ID NO: 9);
5'-GGACTACGGGGGTATCTAAT-3' (shown in SEQ ID NO: 10).
And (3) carrying out electrophoresis on the amplification product on a 1% agarose gel at a voltage of 100V for 45min, and shooting by using an ultraviolet gel imaging system. L2YHC23-1 and L2YHC23-2 were a set of replicates, and L2YHC23p was the mean and standard deviation of the oil samples (Table 1). The DNA extracted using this example 1 was relatively pure, with an A260/A280 ratio of about 1.6 and a relatively high concentration (Table 1). Electrophoresis No. 1 and 2 (FIG. 1) show that the DNA amplification product has single and clear band, so that the effect of extracting DNA by using the method is better.
TABLE 1 concentration and purity of thickened oil genomic DNA extracted in EXAMPLE 1
Comparative example 1
500mg of thickened oil is taken, the pretreatment mode is the same as that of the embodiment 1, 200-. L2YHC23-3 and L2YHC23-4 were a set of replicates, and L2YHC23p was the mean and standard deviation of the oil samples (Table 2). The purity and concentration of DNA extracted from heavy oil by the Qiagen kit method (Table 2) are lower than those of the method (example 1), and the difference seems to be small, but the subsequent sequencing shows that the subsequent experiment can be carried out only by the method.
TABLE 2 concentration and purity of genomic DNA from thickened oil extracted in comparative example 1
Example 2
The only difference between this example and example 1 is the absence of step one, i.e. without the use of a pretreatment agent (gamma valerolactone), the thick oil is used directly for extraction, and the rest of the operations are completely identical. L2YHC23-5 and L2YHC23-6 were a set of replicates and L2YHC23p was the mean and standard deviation of the oil samples (Table 3). The extraction results and the verification results show that the DNA concentration extracted by applying the method is higher (Table 3), and the electrophoresis bands of No. 5 and No. 6 (figure 2) are single and clear, which shows that the DNA extraction effect of the thickened oil is better.
TABLE 3 concentration and purity of the genomic DNA of thickened oil extracted in EXAMPLE 2
Example 3
Compared with the embodiment 1, the embodiment omits the step one, and directly uses the thick oil for extraction, and the specific process is as follows: taking 500mg of thick oil in a centrifuge tube, adding 10-20 mu L of snailase, then sequentially adding 10-20 mu L of protease K and 1-2mL of DNA extracting solution containing triton X-100, finally adding 100-200 mu L of decontamination solution, standing at 65 ℃ for 1h, centrifuging at room temperature and 8000rpm for 3min to remove precipitates to obtain supernatant, transferring the supernatant into a new centrifuge tube, adding a prion precipitation solution with the same volume as the supernatant into the supernatant, centrifuging at 8000rpm for 3min, transferring the supernatant into a new centrifuge tube, repeatedly adding the prion precipitation solution once, centrifuging and collecting the supernatant; the subsequent operation was completely identical to example 1.
L2YHC23-7 and L2YHC23-8 were a set of replicates and L2YHC23p was the mean and standard deviation of the oil samples (Table 4). The extraction results and the verification results show that the DNA extracted by the method has higher concentration (Table 4), and the No. 7 and No. 8 electrophoresis bands (figure 2) are clearest and brighter, which indicates that the extraction effect of the heavy oil DNA is better.
TABLE 4 concentration and purity of the genomic DNA of thickened oil extracted in EXAMPLE 3
Example 4
In this example, the same thickened oil (different storage conditions after collection) was used, and the amount used was completely the same as the experimental operation, compared to example 3. L2YHC23-9 and L2YHC23-10 were a set of replicates and L2YHC23p was the mean and standard deviation of the oil samples (Table 5). The extraction results and the verification results show that the DNA extracted by the method has higher concentration and purity (Table 5), and the electrophoresis bands (a in figure 3) of No. 9 and No. 10 are clearer and brighter, which indicates that the extraction effect of the DNA of the thickened oil is better.
TABLE 5 concentration and purity of thickened oil genomic DNA extracted in EXAMPLE 4
Comparative example 2
In comparison with example 4, the same thick oil was used in this comparative example 2, and the extraction method was carried out according to the method of patent ZL201710116846.5 of the present invention. L2YHC23-11 and L2YHC23-12 were a set of parallel samples, and L2YHC23p was the mean value and standard deviation (Table 6) of the oil samples, and the extraction results and the verification results showed that the concentration and purity of DNA extracted by the method were lower than those of the method of the present invention (example 4) (Table 6), and that electrophoresis bands No. 11 and No. 12 (b in FIG. 3) were not as bright as the method of the present invention (a in FIG. 3), indicating that the extraction effect of the present invention was better. In addition, the time for extracting DNA is shorter than that of the prior invention, the DNA can be quickly extracted within 90min, and at least 100min can be saved compared with the prior invention; meanwhile, organic reagents such as isooctane and the like can not be used in the pretreatment process, so that the method is more environment-friendly than the method.
TABLE 6 concentration and purity of genomic DNA of thickened oil extracted in comparative example 2
The genomic DNA extracted by the method can meet the analysis requirements of subsequent qPCR detection, high-throughput sequencing, metagenome and the like.
Claims (15)
- A method for extracting thick oil genome deoxyribonucleic acid comprises the following steps:adding helicase, protease K, DNA extracting solution containing triton X-100 and decontamination solution into thick oil to perform cell lysis;step two, centrifuging after standing to remove precipitates to obtain supernatant, adding a prion precipitation solution into the supernatant to remove organic macromolecular components, centrifuging and collecting the supernatant;and step three, transferring the upper layer liquid collected by centrifugation to an adsorption column for adsorption, rinsing by using a rinsing liquid and eluting by using a DNA eluent, and collecting to obtain the thick oil genome deoxyribonucleic acid.
- The method as claimed in claim 1, wherein the step one further comprises the step of pretreating the thick oil, and the pretreatment is carried out by adding a pretreatment agent into the thick oil to obtain pretreated thick oil.
- The method according to claim 1, wherein the method comprises in particular the steps of:step one, adding 10-20 mu L of snailase into 500mg of thick oil per 300-inch thick oil, oscillating for 20-30min at 30-37 ℃ and 90-120rpm, then sequentially adding 10-20 mu L of protease K and 1-2mL of DNA extracting solution containing triton X-100, oscillating for 20-30min at 30-37 ℃ and 90-120rpm, finally adding 200 mu L of decontamination solution, and standing for 30-60min at 50-65 ℃;or directly adding 10-20 μ L of helicase, 10-20 μ L of protease K, 1-2mL of DNA extract containing triton X-100 and 100 μ L of decontamination solution into 500mg of thick oil per 300-;step two, centrifuging for 2-3min at room temperature and 8000-;transferring the upper layer liquid collected by centrifugation to an adsorption column for adsorption, putting the adsorption column into a collecting pipe, centrifuging for 1-2min under the conditions of 10000-plus 12000rpm to remove the waste liquid, then adding precooled rinsing liquid into the adsorption column for rinsing, centrifuging for 1-2min under the conditions of 10000-plus 12000rpm to remove the 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; and finally suspending and dripping the DNA eluent in the middle part of the adsorption membrane in the adsorption column, standing for 5-10min at room temperature, centrifuging for 2-3min under the conditions of 10000-12000rpm, and collecting the centrifuged supernatant, namely the thickened oil genome deoxyribonucleic acid.
- The method as claimed in claim 3, wherein the step one further comprises a step of pretreating the thick oil, wherein the pretreatment comprises adding 150-250 μ L of pretreatment reagent into every 300-500mg of thick oil, and removing the underflow after vortex mixing to obtain the pretreated thick oil.
- A method as claimed in any one of claims 1 to 4, wherein the snailase is at a mass volume concentration of 10-20 mg/mL.
- The method of claim 2 or 4, wherein the pre-treatment agent is gamma valerolactone.
- The process according to any one of claims 1 to 4, wherein the mass volume concentration of the protease K is from 5 to 20 mg/mL.
- The method according to any one of claims 1-4, 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 liter3PO 4And 0.5 to 2mol of NaCl.
- The method of any one of claims 1-4, wherein the decontamination solution is a sodium deoxycholate solution at a mass volume concentration of 50-100 mg/mL.
- The method as claimed in any one of claims 1 to 4, wherein the prion precipitation solution is a mixture of phenol, chloroform and isoamyl alcohol, and the volume ratio of the mixture of phenol, chloroform and isoamyl alcohol is (24-26): (23-25): (0.5-1.5).
- The method of any one of claims 1-4, wherein the rinse solution is chromatographically pure ethanol at a concentration of 70% by volume;preferably, the DNA eluent is sterile ultrapure water;preferably, the adsorption membrane adopted in the adsorption column is a silica gel membrane.
- A kit for extracting deoxyribonucleic acid of a thick oil genome, wherein the kit comprises: helicase, protease K, DNA extract containing triton X-100, decontamination solution, prion precipitation solution and adsorption column.
- The kit of claim 12, wherein the kit further comprises a pretreatment reagent, wherein the pretreatment reagent is gamma valerolactone;preferably, the kit further comprises a rinsing solution and a DNA eluent.
- The kit according to claim 12, wherein the kit further comprises glass beads, the glass beads being monodisperse glass beads having a particle size of 1.5-2.0 mm.
- Use of the kit according to any one of claims 12 to 14 for the extraction of deoxyribonucleic acid from thick oil genomes.
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