AU2021107435A4 - Method for extracting edible fish sperm DNA - Google Patents
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- 150000007523 nucleic acids Chemical class 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
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- 239000004472 Lysine Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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Abstract
The present disclosure provides a method for extracting edible fish sperm DNA. The
method comprises washing fish sperm as a raw material with a buffer solution; adding the
fish sperm as the raw material into the buffer solution, and homogenizing and centrifuging
to obtain a precipitate with an approximate skin color; dissolving the precipitate with a
concentrated salt solution to form a solution; adjusting pH of the solution to pH 9-11 with
an alkaline solution, and centrifuging to obtain a supernatant; adjusting pH of the
supernatant to pH 6-7 with an acid solution to obtain a supernatant containing DNA;
precipitating with 50~100% ethanol to obtain a fish sperm DNA precipitate; dissolving the
fish sperm DNA precipitate with sterile deionized water and purifying. The method for
extracting edible fish sperm DNA provided by the present disclosure avoids using toxic and
harmful reagents in other methods, and the obtained fish sperm DNA is safe and edible, has
high purity, and the method has a high recovery rate, can perform extraction and production
on a large scale, and is more suitable for use in animals and clinical research, thereby
providing enriched the application range of the fish sperm DNA.
Description
The present disclosure relates to a technical field of combining food, marine organisms and
medicine, and specifically relates to a method for extracting edible fish sperm DNA.
In a trend of increasingly serious population aging in China and in context of the limited
medical resources that cannot satisfy increasing medical needs, it is urgent to strengthen
pre-clinical intervention and home care, and precision medicine and precision nutrition are
also on the agenda. Nucleic acids constitute material basis of genetic information and are
important research contents of precision medicine and precision nutrition. According to
reports, dietary nucleic acids have physiological functions mainly comprising promoting
growth and development, regulating liver function, anti-oxidation; relieving physical fatigue,
and assisting in improving memory, which have a wide range of applications in cosmetics,
food, and health products.
Dietary nucleic acids contain a large proportion of RNA in most foods, while DNA is
generally rich in sperm-riched substances, such as fish sperm and pollen. The existing DNA
products are mainly fish sperm DNA and calf thymus DNA. The fish spermary, which is
used as waste during processing, is rich in DNA, and the fish sperm DNA has been proved
to have functions such as skin maintenance and anemia treatment. Thus, methord that
effectively extract DNA from fish spermary is very important.
However, during research on existing methods extracting fish sperm DNA, it is found
that the extraction process mostly involves use of toxic and harmful reagents, thereby
hindering its application in fields of food, health products, cosmetics and so on. WANG Jian
(Extracting Nucleic Acid from Marine Fish Sperm, Journal of Wuxi University of Light
Industry (Food and Biotechnology), Issue 2, 2003, pp. 71-74), TANG Xiaoli (Fast and
pollution-free extraction process of fish sperm DNA, Guangzhou Chemical Industry, 1996
Volume 24, Issue 4), LIU Runzhi (Research on Extraction of Deoxyribonucleic Acid (DNA)
from Carp Spermary, Journal of Natural Science of Hunan Normal University, Vol. 11,
Issue 3, 1988) recite that extraction rate of fish sperm DNA was 6-25%, but sodium dodecyl sulfate (SDS) was used in experiments to denature the protein. Patent CN110760509A uses filter membranes with different molecular weight cut-offs to obtain pure fish sperm DNA and protamine at the same time, but does not analyze purity and recovery rate of the obtained DNA.
To sum up, most of the existing methods use denaturants such as phenol,chloroform or
SDS, which do not correspond to requirements of green processing technology, energy
saving and emission reduction. A new and healthier method for extracting complete strip,
high purity, edible fish sperm DNA is desirable.
The purpose of the present disclosure is to provide a method for extracting edible fish sperm
DNA to overcome the shortcomings of the prior art.
In order to achieve the above purpose, the present disclosure adopts the specific technical
solutions as follows.
The method for extracting edible fish sperm DNA includes the following steps:
(1) washing a fish sperm as a raw material with a buffer solution ;
(2) adding the fish sperm as the raw material processed in (1) into the buffer solution,
and homogenizing and centrifuging to obtain a precipitate with an approximate skin color;
(3) dissolving the precipitate obtained in (2) with a concentrated salt solution to form a
solution;
(4) adjusting pH of the solution in (3) to pH 9-11 with an alkaline solution, and
centrifuging to obtain a supernatant;
(5) adjusting pH of the supernatant in (4) to pH 6-7 with an acid solution to obtain a
supernatant containing DNA;
(6) precipitating with 50~100% ethanol to obtain a fish sperm DNA precipitate;
(7) dissolving the fish sperm DNA precipitate in (6) with sterile deionized water and
purifying.
Further, the fish can be selected as walleye pollack which is relatively easy to obtain in life.
Further, the buffer solution used in the step (1) is an SSC solution, specifically a mixture of
0.05-1 M sodium chloride and 0.01-0.1 M sodium citrate (14:5, c/c).
Further, the specific procedure of the step (2) is: adding a 2 times the weight of the pre-cooled SSC solution compared to the raw material and homogenizing for 1.5 min; centrifuging at 4000 rpm for 15 min at 4C and discarding the supernatant; transfering the precipitate to 2 times the weight of the pre-cooled SSC solution compared to the raw material and homogenizing for 1 min; and centrifuging at 4000 rpm for 15 min at 4C, discarding the supernatant, with two replications. The specific operation of the centrifugation has been verified by many experiments, and it can bring about a better centrifugal effect.
Further, the concentrated salt solution in the step (3) is specifically selected as a 10-20 wt%
sodium chloride solution; the specific operation is adding a 10 times the weight of 10-20
wt% sodium chloride solution compared to the raw material, stirring to fully dissolving a
fish sperm desoxyribose nucleoprotein(DNP), and standing at 4C for 48 h.
Further, in the step (4), the alkaline solution is prepared by sodium carbonate, sodium
bicarbonate or a mixture of both; specifically, by adding the alkaline solution, adjusting pH of the
DNP solution according to amino acid composition within a general adjustment range of pH 9 -11.
Taking extraction of walleye pollack sperm as an example, the pH of the DNP solution is adjusted
to pH 9.7; the precipitate obtained by centrifugation at 10,000 rpm at 4C for 10 min is discarded;
alkaline solution is added to adjust the pH of the DNP solution to pH 10.8; the precipitate
obtained by centrifugation at 10,000 rpm at 4C for 10 min is discarded.
Further, in the step (5), the acidic solution is a mixture of one or more of hydrochloric acid,
acetic acid, and citric acid, the pH of the DNP solution is adjusted to pH 6.5; and the
precipitate obtained by centrifugation at 10,000 rpm at 4C for 10 min is discarded.
The specific operation of the step (6) includes: adding 2 times volume of pre-cooled
-100% ethanol,centrifuging at 10,000 rpm at 4C for 10 min, and discarding the
supernatant, with two replications; washing the precipitate with pre-cooled 50-100% ethanol
twice, and air-drying to remove ethanol.
Further, in the step (7), purification is performed by an ultrafiltration membrane having a
molecular weight cut-off of 5000.
Advantages and beneficial effects of the present disclosure:
The present disclosure uses sodium chloride, edible alkaline solution, hydrochloric acid and
other solutions instead of phenol, chloroform, sodium dodecyl sulfate (SDS) to obtain fish sperm
DNA, providing a safe method for extracting edible fish sperm DNA.
The method for extracting fish sperm DNA provided by the present disclosure avoids using phenol, chloroform, SDS and other reagents in other methods, and the obtained fish sperm DNA is safe and edible, has high purity. The method has a higher recovery rate, and can perform extraction and production on a large scale and is more suitable for animal and clinical research, providing a enriched application range of fish sperm DNA, especially is suitable for the extraction of marine fish-walleye pollack sperm.
Fig. 1 is a schematic flow chart of the method for extracting edible walleye pollack
sperm DNA in Examples.
Fig. 2 is a graph showing the results of analysis of the amino acid composition and
content of walleye pollack used in Examples.
Fig. 3 is a absorbance-change curve of DNA obtained by the extraction method in
Examples at different wavelengths .
Fig. 4 is a graph showing results of agarose gel electrophoresis for DNA obtained by the
extraction method in Examples.
The present disclosure will be further explained by way of Examples below, but the
present disclosure is not limited to the scope of the described Examples. In the following
Examples, experimental methods without specific conditions are selected according to
conventional methods and conditions, or according to product specification.
The numerical values disclosed in the Examples of the present disclosure are
approximate values, rather than definite values. Where error or experimental conditions are
allowable, all values within the error range can be included and are not limited to the
specific values disclosed in Examples of the present disclosure.
Example 1:
In this Example, amino acid analysis of the fish sperm is used to determine pH of the
alkaline solution adjustment solution. For the amino acid analysis, referring to the method
described in GB 5009.124-2016 "Determination of Amino Acids in Foods in National Food
Safety Standard", the method includes:
(1) thawing the fish sperm, homogenizing for 1 min, with 3 times repetition;
(2) taking an appropriate amount of the fish sperm, homogenizing to determine the
protein concentration in the walleye pollack sperm, specifically including:
taking 1 mL of the fish sperm to homogenize and adding 3 mL of PBS buffer to dilute,
and recording as "M";
taking 200 L of the homogenate of "M4" and adding 200 L of PBS buffer to dilute, repeating the operation to dilute the homogenate of the walleye pollack sperm to 4 times, 8
times, 16 times, 32 times and 64 times, and recording as "M4 ", "M8", "M16", "M32", "M64", respectively;
adding 20 L of the homogenates of fish sperm having different diluted concentration to
sample wells of a 96-well plate, adding 200 L of BCA working solution, and standing at
37°C for 30 min; and
measuring A562 and calculating the protein concentration of fish sperm, thereby
determining that the mass of the homogenate of fish sperm to be weighed is 0.3501 g.
(3) adding 0.3501 g of the homogenate of the fish sperm to an ampoule, adding 15 mL
of 6 M HCl (containing 5% mercaptoethanol), filling with nitrogen, and sealing the ampoule
by melting with an alcohol lamp;
(4) putting the ampoule into a 110 °C electric heating air-blowing thermostat to
hydrolyze for 22 h, taking out and cooling, and diluting the hydrolysate to 50 mL with 0.02
M HCl;
(5) transferring 1 mL of hydrolysate into an ampoule, placing the ampoule in a water
bath at 60°C, and blowing dry with a compressed air machine.
(6) adding 3-5 drops of ultrapure water, blowing dry and removing the acid, repeating
the operation 3 times; and
(7) redissolving with 2 mL of 0.02 M HCl, filtering through a 0.22 m water-based
filter membrane into a liquid phase bottle as a solution to be tested, and finally analyzing
with Hitachi L-8900 automatic amino acid analyzer.
The results of amino acid analysis of the fish sperm are shown in Figure 2 and Table 1.
The results show that the fish sperm has large proportion of basic amino acids, wherein the
proportions of lysine and arginine proportions of are 11.32% and 10.76% respectively. The
isoelectric point of arginine is pH 10.8, the isoelectric point of lysine is pH 9.7, and the
approximate range is pH 9-11. According to the property of "the solubility of protein or amino acid at the isoelectric point being the smallest", the pH is finally obtained. Thus, the pH adjustment range when the alkaline solution is used to remove the fish sperm is pH 9-11.
Table 1. Amino acid composition and content analysis for walleye pollack sperm ESTD Pk# RT Name Height Area /1 Conc/ng conc/nmol 1 2.227 Tau 52480 607191 0.418 52.375192 4 4.893 Asp 417182 5227255 3.088 411.039249 5 5.520 Thr 346813 4473658 2.487 296.169766 6 6.140 Ser 331914 4301340 2.348 246.741467 7 6.940 Glu 491867 7676249 4.257 626.214510 9 9.880 Gly 414108 7299470 4.121 309.350475 10 10.840 Ala 286059 7154775 4.115 366.618624 11 12.220 Cys 7382 88257 0.046 11.139554 12 12.900 Val 369330 4778155 2.621 306.900998 13 14.200 Met 47639 950699 0.516 76.989824 15 16.507 Ile 98751 2709809 1.590 208.545397 16 17.513 Leu 250830 5667473 3.541 464.598778 17 18.053 Tyr 117826 1854998 1.099 199.139009 18 18.987 Phe 96014 1883380 1.138 187.974000 21 20.973 Lys 634343 7557830 3.992 583.577225 23 22.413 NH3 379707 7798645 7.585 129.165361 24 23.333 His 88332 1398991 0.792 122.893651 25 27.340 Arg 209344 531575 3.191 555.800731 Totals 4639321 76743902 46.943
Example 2
The Example aims to select appropriate centrifugal conditions.
The centrifugation speed and time are depending on the conditions of the test precipitation. if
the centrifugation speed is too low, the precipitate will float in the solution and cannot be
removed. If the centrifugation speed is too high, it may cause degradation of fish sperm DNA and
affect subsequent experiments. The centrifugation speed involved in the Example is 4000-10,000
rpm, and the time is 10-15 min. The centrifugation conditions for obtaining DNP solution were at
4°C, at 4000 rpm for 15 min; the centrifugation conditions in the step of adjusting the pH of DNP
solution to remove the fish sperm were at 4C at 10,000 rpm for 10 min; the centrifugation
conditions in the ultrafiltration purification step were at 4C at 7000 rpm for 20 min. Under these
conditions, the obtained DNA bands are complete and in high purity.
Example 3
The Example determines the pH range for removing an excess of the alkaline solution.
According to the results shown in Figure 2, the pH of the DNP solution is adjusted to about
3.2 due to the high content of glutamic acid in the protamine, such that the protamine is removed
and at the same time the excess of alkaline solution is neutralized; but the effect of removing the
protein is not significant and under the condition of pH 3.2, DNA may be degraded, thereby the
pH of the DNP solution is adjusted back to the pH before adding the alkaline solution, ranging
from pH 6.0 to pH 7.0 to ensure that the excess of the alkaline solution is neutralized.
Example 4:
In the Example, a spermary tissue of the walleye pollack was used as a raw material, and the
extraction was performed according to the schematic diagram of the method for extracting edible
walleye pollack sperm DNA in FIG. 1. The method includes:
(1) washing the spermary tissue of the walleye pollack by using a SSC solution (a mixed
solution of 0.14 M sodium chloride and 0.05 M sodium citrate), bloting with a filter paper,
weighing, putting into a masher;
(2) adding a 2 times the weight of the pre-cooled SSC solution compared to the raw
material to the walleye pollack spermary obtained in (1), homogenizing for 1.5 min; centrifuging
at 4C at 4000 rpm for 15 min, discarding a supernatant; transferring a precipitate to a 2 times
the weight of the pre-cooled SSC solution compared to the raw material, homogenizing for 1
min; centrifuging at 4000 rpm for 15 min at 4C, discarding a supernatant, and repeating the
operation twice to obtain a precipitate with a approximate skin color;
(3) adding a 10 times the weight of 10% sodium chloride solution compared to the raw
material to the precipitate described in (2), stirring to fully dissolve the precipitate described in
(2), and standing at 4C for 48 hours;
(4) adding a saturated sodium carbonate solution to the solution obtained in (3), adjusting pH
of the DNP solution to pH 9.7, centrifuging at 4C, at 10,000 rpm for 10 min, discarding the
precipitate; adding a saturated sodium carbonate solution, adjusting pH of the DNP solution pH
10.8; centrifuging at 4C, at 10,000 rpm for 10 min, discarding the precipitate, and taking a
supernatant;
(5) adding hydrochloric acid to the supernatant in the step (5), adjusting pH of the DNP
solution to pH 6.5; centrifuging at 10,000 rpm at 4C for 10 min, discarding a precipitate, and
taking a supernatant; wherein the pH involved in steps (4) and (5) is determined according to the amino acid composition and content of the fish sperm of the used walleye pollack; and Figure 2 is a graph of the amino acid composition and content analysis of the walleye pollack used in the present disclosure.
(6) adding the supernatant obtained in (5) to 2 times volume of pre-cooled 75% ethanol,
centrifuging at 10,000 rpm at 4C for 10 min, discarding a supernatant, and repeating the
operation twice; washing the precipitate with absolute ethanol twice, air-drying to remove
ethanol, and dissolving the DNA in sterile deionized water to obtain a DNA solution; and
(7) transferring the solution in (6) to an ultrafiltration tube having a molecular weight cut-off
of 5000, and centrifuging at 7000 rpm for 20 minutes to purify the DNA.
The method for evaluating the content and purity of the DNA extracted in the Examples of
the present disclosure is as follows.
(1) analysis of recovery rate
The recovery rate of DNA extracted in Examples of the present disclosure can reach 1-5%,
and the specific operation and calculation method are as follows.
In the present disclosure, the DNA recovery rate refers to percentage of dry weight of the
extracted fish sperm DNA to dry weight of the fish sperm. The method for calculating the dry
weight of the fish sperm is measured by measuring a moisture content of the fish sperm, referring
to GB 5009.3-2016 "Determination of Moisture in Foods in National Food Safety Standards".
The fish sperm in the present disclosure is suitably determined using a two-step drying method in
a direct drying method. The specific method is as follows:
weighing 2-5 g of clean fish sperm (with the accuracy to 0.0001 g), recording as "ml", and
air-drying for 15-20 h, crushing and mixing the air-dried sample;
taking a flat weighing bottle made of clean glass and placing in a drying oven at 101-105°C
for 1 h, covering, taking out and putting in a desiccator, and cooling for 0.5 h, and weighing;
repeating the drying operation until mass difference between two drying processes as before
and after is < 2 mg, and the last weighed value is recorded as "m5";
weighing the air-dried fish sperm and recording as "m2", putting into a dry weighing bottle
to make sure that the sample thickness is <5 mm, weighing and recording as "m3;
capping and putting in a drying oven at 101-105°C, and placing the cap on the side of the
bottle obliquely, drying for 2-4 hours, after capping, taking out and cooling in a desiccator for 0.5
h, and weighing; capping and putting in a drying oven at 101-105°C, and placing the cap on the side of the bottle obliquely, drying for 1 hour, after capping, taking out and cooling in a desiccator for 0.5 h, and weighing; repeating the drying operation until mass difference between two drying processes as before and after is < 2 mg, and the last weighed value is recorded as "m4";
Calculation formula of the moisture content:
m1 -m2 +m2 xm3 -m4 m3 - m5 X 100% m1 Upon calculating as described above, the recovery rate of the fish sperm DNA in the method
of the present disclosure is 1-5%. In the research on extracting deoxyribonucleic acid (DNA)
from a spermary in the prior art, the DNA extraction rate is about 8-10%, but during the
extraction process, the addition of sodium dodecyl sulfate (SDS) may contaminate the obtained
fish sperm DNA, which is also a technical problem to be solved by the present disclosure; the
recovery rate of the fish sperm DNA in the present Examples and the yields of the fish sperm
DNA-Na and calf thymus DNA described in the patent CN106031709B and Xu Zhipeng et al
(Study on the Process of Large-scale Preparation of Calf Thymus DNA, Pharmaceutical
Biotechnology, 2013 Vol. 20, Issue 5, pp. 435-438) are similar, while the fish sperm DNA
prepared by the latter two methods are non-edible. That is, the present disclosure can be used for
subsequent large-scale preparation and activity studies of the fish sperm DNA.
(2) Purity analysis
2 L of the DNA solution obtained in step (6) is taken and placed in Nano-100 for detection
to obtain an absorbance change curve of the DNA obtained by the method according to the
Examples of the present disclosure at different wavelengths in Fig. 3, and the purity of the
obtained DNA is calculated. The calculation formula is as follows.
(3) Purity = A260/A280
As shown in Figure 3, A260/A280 of the extracted walleye pollack sperm DNA is 1.80,
which can confirm its high purity, proving that this Example can obtain a high-purity DNA with
low contamination of protein.
7.5 pL of the DNA solution obtained in step (6) is taken and mixed evenly with 1.5 L of 6x
loading buffer. 6 L of the sample is taken, and electrophoresis is performed on a 1% agarose gel
for 1 h at a voltage of 120 V. The detection result of the agarose gel electrophoresis is shown in
Fig. 4, the first lane is the fish sperm DNA extracted in this Example and the band is complete,
and the molecular size is between 10kb and 15kb, which can meet requirements of subsequent
activity analysis.
The foregoing descriptions are only preferred examples of the present disclosure, and do not
constitute a limitation to the scope of the claims. Other substantially equivalent alternatives that
can be envisaged by those skilled in the art are all within the protection scope of the present
disclosure.
Claims (10)
1. A method for extracting edible fish sperm DNA, comprising the following steps:
(1) washing a fish sperm as a raw material with a buffer solution ;
(2) adding the fish sperm as the raw material processed in (1) into the buffer solution,
and homogenizing and centrifuging to obtain a precipitate with an approximate skin color;
(3) dissolving the precipitate obtained in (2) with a concentrated salt solution to form a
solution;
(4) adjusting pH of the solution in (3) to pH 9-11 with an alkaline solution, and
centrifuging to obtain a supernatant;
(5) adjusting pH of the supernatant in (4) to pH 6-7 with an acid solution to obtain a
supernatant containing DNA;
(6) precipitating with 50-100% ethanol to obtain a fish sperm DNA precipitate;
(7) dissolving the fish sperm DNA precipitate in (6) with sterile deionized water and
purifying.
2. The method for extracting edible fish sperm DNA according to claim 1, wherein the
buffer solution used in the step (1) is an SSC solution, which is specifically a mixed solution
of 0.05-1 M sodium chloride and 0.01-0.1 M sodium citrate with a concentration ratio of
14:5.
3. The method for extracting edible fish sperm DNA according to claim 1, wherein the
specific procedure of the step (2) is: adding a 2 times the weight of the pre-cooled SSC
solution compared to the raw material and homogenizing for 1.5 min; centrifuging at 4000
rpm for 15 min at 4C and discarding the supernatant; transfering the precipitate to 2 times
the weight of the pre-cooled SSC solution compared to the raw material and homogenizing
for 1 min; and centrifuging at 4000 rpm for 15 min at 4C, discarding the supernatant, with
two replications.
4. The method for extracting edible fish sperm DNA according to claim 1, wherein the
concentrated salt solution in the step (3) is specifically selected as a 10-20 wt% sodium
chloride solution; the specific operation is adding a 10 times the weight of 10-20 wt%
sodium chloride solution compared to the raw material, stirring to fully dissolving a fish
sperm DNP(desoxyribose nucleoprotein), and standing at 4C for 48 h.
5. The method for extracting edible fish sperm DNA according to claim 1, wherein the
alkaline solution in the step (4) is prepared by sodium carbonate, sodium bicarbonate or a
mixture of the sodium carbonate and sodium bicarbonate; specifically, by adding the
alkaline solution, adjusting pH of the DNP solution according to amino acid composition
within a general adjustment range of pH 9-11.
6. The method for extracting edible fish sperm DNA according to claim 5, wherein the
pH of the DNP solution is adjusted to pH 9.7; the precipitate obtained by centrifugation at
,000 rpm at 4C for 10 min is discarded; an alkaline solution is added to adjust the pH of
the DNP solution to pH 10.8; and the precipitate obtained by centrifugation at 10,000 rpm at
4°C for 10 min is discarded.
7. The method for extracting edible fish sperm DNA according to claim 1, wherein the
acidic solution in the step (5) is one or more of hydrochloric acid, acetic acid, and citric acid;
the pH of the DNP solution is adjusted to pH 6.5; and the precipitate obtained by
centrifugation at 10,000 rpm at 4C for 10 min is discarded.
8. The method for extracting edible fish sperm DNA according to claim 1, wherein the
specific operation of the step (6) comprises adding 2 times volume of pre-cooled 50-100%
ethanol, centrifuging at 10,000 rpm at 4C for 10 min, and discarding the supernatant, with
two replications; washing the precipitate with pre-cooled 50-100% ethanol twice, and
air-drying to remove ethanol.
9. The method for extracting edible fish sperm DNA according to claim 1, wherein in
the step (7), purification is performed by an ultrafiltration membrane having a molecular
weight cut-off of 5000.
10. The method for extracting edible fish sperm DNA according to claim 1, wherein the
fish is selected as a marine fish, specifically walleye pollack.
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CN1112124A (en) * | 1994-05-16 | 1995-11-22 | 刘润芝 | Process for extracting deoxyribonucleic acid (DNA) |
CN106031709B (en) * | 2015-03-20 | 2019-01-01 | 上海辉文生物技术股份有限公司 | Milt DNA-NA, nucleoprotamine extract and preparation method thereof |
CN110760509A (en) * | 2019-07-03 | 2020-02-07 | 深圳瑞达生物股份有限公司 | Method for extracting milt component of globefish testis |
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