CN115369114A - Improved method for extracting genome DNA from shrimp tissue - Google Patents

Improved method for extracting genome DNA from shrimp tissue Download PDF

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CN115369114A
CN115369114A CN202211147517.4A CN202211147517A CN115369114A CN 115369114 A CN115369114 A CN 115369114A CN 202211147517 A CN202211147517 A CN 202211147517A CN 115369114 A CN115369114 A CN 115369114A
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shrimp
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lysis solution
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侯家麟
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Qingdao Ouyi Bio Tech Co ltd
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    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads

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Abstract

The invention discloses an improved method for extracting genome DNA from shrimp tissues, which relates to the technical field of bioengineering, and has the technical scheme key points that the method comprises the following steps: s1, cracking: adding lysis solution and enzyme into shrimp tissue to obtain lysis solution containing nucleic acid; the cracking liquid comprises polyoxyethylene cetyl ether sodium sulfate, at least one surfactant and at least one moisture absorbent; the enzyme is proteinase K and RNase; s2, combining: adding a binding solution and magnetic beads into the lysis solution to obtain a binding body; the binding liquid comprises isopropanol, guanidine hydrochloride and nonionic detergent; s3, cleaning: cleaning the combination with ethanol; s4, elution: eluting the washed combined body with eluent to obtain shrimp tissue DNA; the eluent was TE buffer.

Description

Improved method for extracting genome DNA from shrimp tissue
Technical Field
The invention relates to the field of bioengineering, in particular to an improved method for extracting genome DNA from shrimp tissues.
Background
There are numerous methods in the prior art for isolating nucleic acids from non-identical samples. However, the same method is not equally effective for all samples. For example, using one method, nucleic acids can be efficiently isolated from a particular sample, such as tissue, plasma, serum, or urine. However, this method may not be effective in isolating nucleic acids from other samples, such as shrimp tissue samples, particularly shrimp tissue samples containing high cell counts, protein and/or fat content, such as the muscle and head tissues of marine and freshwater shrimps such as prawns, metapenaeus, prawns, shrimp, crawfish, and the like, pose particular challenges.
At present, the applicability of a nucleic acid separation method based on a rotating column to processing shrimp meat samples is very limited, and especially the muscle and head tissue samples of marine and freshwater shrimps such as prawns, metapenaeus ensis, shrimps, small shrimps, crayfish and the like. When nucleic acid is isolated from various shrimp meat samples using established nucleic acid isolation methods, the easy clogging of silicon columns is a major problem due to the specific composition of these samples. Especially high cell numbers, large amounts of proteins and/or lipids may lead to such clogging effects. The silica column is clogged with substances such as proteins and fats, resulting in low purification efficiency, and thus little or no nucleic acid is separated. Furthermore, the isolated nucleic acids are often contaminated with impurities/inhibitory substances and nucleases, resulting in low nucleic acid yields, and this impairs the downstream performance of the isolated nucleic acids, particularly in amplification reactions such as RT-PCR. This makes it difficult to detect a target nucleic acid among the separated nucleic acids. In addition, the clogging effect results in that only a small amount of sample can be processed at a time, and generally, in order to reduce clogging, the sample loading amount must be limited not to exceed 10mg.
The above-described clogging is particularly problematic when using 96-lane silicon columns or automated systems to separate nucleic acids from large numbers of samples. Thus, especially when processing large numbers of samples and/or using an automated system, the corresponding nucleic acid samples may be lost, which would result in having to re-provide the samples.
The low purification efficiency and limitation of the loading volume are significant disadvantages when isolating specific target nucleic acids that may be contained in a shrimp meat tissue sample. For example, shrimp head tissue nucleic acid, may be present in small amounts in shrimp meat tissue samples. The low purification efficiency and limited loading results in that conventional nucleic acid isolation methods often do not allow isolation of sufficient amounts of target tissue nucleic acid for subsequent standard detection assays, such as polymerase chain reaction. The detection of the nucleic acid in different parts of the shrimp meat tissue is one of the main reasons for separating the nucleic acid from various shrimp meat tissues.
The prior patent application document with the reference application publication number of WO2011157683A discloses a nucleic acid extraction method based on a silicon column, wherein the main components of a cracking mixed solution are based on different damp salts (such as guanidine hydrochloride or guanidine thiocyanate) and surfactants, the silicon column is eliminated in the step of combining nucleic acid and a stationary phase, magnetic beads more suitable for high-flux extraction equipment and automatic equipment are used instead, and ethanol with different concentrations is suitable for subsequent washing solutions, so that the method is not ideal for inhibiting DNase I activity expression in the process of extracting genomic DNA.
The prior patent application documents with the reference application publication numbers CN201810404724.0, CN201910971430.0 and CN202111251984.7 mention that the use of magnetic beads rather than silica columns is beneficial for high-throughput automated extraction of feasible tissues, but the components of the lysis mixture are only or possibly only suitable for extraction of certain mammalian tissues and blood, and are not suitable for shrimp meat tissues containing high cell number and large amount of protein and/or lipid, especially shrimp head tissues.
Thus, there is a need for a reliable, efficient method that is also amenable to automation and that can isolate nucleic acids from shrimp tissue.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an improved method for extracting genomic DNA from shrimp tissues, which solves the problems of low nucleic acid yield and nucleic acid degradation in the extraction of nucleic acid from shrimp tissues.
In order to achieve the purpose, the invention provides the following technical scheme: an improved method for extracting genomic DNA from shrimp tissues, comprising the following steps:
s1, cracking: adding lysis solution and enzyme into shrimp tissue to obtain lysis solution containing nucleic acid; the cracking liquid comprises polyoxyethylene cetyl ether sodium sulfate, at least one surfactant and at least one moisture absorbent; the enzyme is proteinase K and RNase;
s2, combining: adding a binding solution and magnetic beads into the lysis solution to obtain a binding body; the binding solution comprises isopropanol, guanidine hydrochloride and a non-ionic detergent;
s3, cleaning: cleaning the combination with ethanol;
s4, elution: eluting the washed combined body with eluent to obtain shrimp tissue DNA; the eluent is TE buffer solution.
By adopting the technical scheme, because the lysis solution and the enzyme are added into the shrimp tissues, the protein combined with nucleic acid can be enzymolyzed, and RNA can be degraded; by adding polyoxyethylene fatty alcohol ether sodium sulfate into the lysate, the activity of DNase I in a sample can be effectively inhibited, the degradation of genome DNA in the extraction process is prevented, the degradation of histone combined with a DNA double helix structure is promoted, and the purity and yield of the DNA are improved.
Further, the polyoxyethylene cetyl ether sodium sulfate is polyoxyethylene fatty alcohol ether sodium sulfate.
Further, the lysis solution comprises polyoxyethylene fatty alcohol ether sodium sulfate, 0.1% -5% of sodium dodecyl sulfate and 1.0-2.0M guanidine thiocyanate.
Further, the lysis solution also comprises any mixture of ethylenediamine tetraacetic acid, tris (hydroxymethyl) aminomethane, tritonX-100 and Tween 20;
further, the concentration of the polyoxyethylene fatty alcohol ether sodium sulfate is 1% -10%.
Further, the concentration of the polyoxyethylene fatty alcohol ether sodium sulfate is 5.5%.
Further, the shrimp tissue includes, but is not limited to, shrimp head tissue and shrimp meat tissue.
Further, the shrimp includes, but is not limited to, marine shrimp such as prawn, metapenaeus ensis, shrimp, crayfish, and freshwater shrimp.
In conclusion, the invention has the following beneficial effects:
because the invention adopts the method that the lysis solution and the enzyme are added into the shrimp tissues, the protein combined with the nucleic acid can be enzymolyzed and the RNA can be degraded; by adding the polyoxyethylene fatty alcohol ether sodium sulfate into the lysate, the activity of DNase I in a sample can be effectively inhibited, the genomic DNA is prevented from being degraded in the extraction process, the degradation of histone combined with a DNA double-helix structure is promoted, and the purity and the yield of the DNA are further improved. By the method, the sample loading amount of the shrimp tissue can be increased to 20mg, the purity of the obtained shrimp tissue nucleic acid is improved, and the yield of the shrimp tissue nucleic acid is increased from less than 10ng/uL to more than 50 ng/uL.
Drawings
FIG. 1 is an agarose gel electrophoresis image of genomic DNA extracted from shrimp tissues, showing example 1, example 2 and comparative example 1 in the order from left to right;
FIG. 2 is a diagram of agarose gel electrophoresis of examples 3-5 of the present invention and comparative example 2;
FIG. 3 is a photograph of agarose gel electrophoresis of examples 6-11 of the present invention;
FIG. 4 is a photograph of agarose gel electrophoresis of examples 12 to 16 of the present invention and comparative example 3.
Detailed Description
The present invention will be described in further detail with reference to examples. The reagents used in the present invention are commercially available.
Example 1
Adding a lysis solution consisting of polyoxyethylene fatty alcohol ether sodium sulfate, ethylene diamine tetraacetic acid, tris (hydroxymethyl) aminomethane and sodium dodecyl sulfate into 20mg of shrimp tissues for lysis, and adding 20ul of protease K and RNase to obtain a lysis solution. Then, a combined solution of isopropanol, guanidine hydrochloride and the non-ionic detergents Tween 20, triton X-100 was added to the lysis solution, and the resulting combined mixture comprised 2.31M guanidine thiocyanate, 13% isopropanol, 10mM Tris and 3.5% sodium polyoxyethylene fatty alcohol ether sulfate. Subsequently, the nucleic acid was eluted by a magnetic bead method with 75% ethanol twice and TE buffer.
Example 2
Adding lysis solution composed of sodium dodecyl sulfate, ethylene diamine tetraacetic acid and tris (hydroxymethyl) aminomethane into 20mg of shrimp tissue for lysis, and adding protease K and RNase 20ul to obtain lysis solution. Then, a binding solution consisting of isopropanol, guanidine thiocyanate and non-ionic detergents Tween 20, triton X-100 was added to the lysis solution, and the resulting binding mixture comprised of 2.3M total concentration of the damp salts GTC and GuHCL,13% isopropanol and 10mM Tris. Subsequently, the nucleic acid was eluted by a magnetic bead method with 75% ethanol twice and TE buffer.
Comparative example 1
Adding a lysis solution consisting of guanidine thiocyanate, isopropanol, nonionic detergents Tween 20 and Triton X-100 into 20mg of shrimp tissues for lysis, and adding 20ul of proteinase K and RNase to obtain a lysis solution. Then, a binding solution of guanidine thiocyanate and ethanol was added to the lysis solution, and the resulting binding mixture comprised a mixture of 2M guanidine thiocyanate, 33% ethanol and Triton X-100 in total concentration in a wet salt. Subsequently, the nucleic acid was eluted by a magnetic bead method with 75% ethanol twice and TE buffer.
Mu.l of each of the nucleic acids isolated in examples 1-2 and comparative example 1 above was used for the subsequent nanodrop nucleic acid quantification and agarose electrophoresis, wherein PBS was used as a negative control. The higher the concentration value of the nanodrop nucleic acid obtained in the experiment is, the higher the concentration of the DNA in the separated nucleic acid is, the brighter main DNA band of the agarose electrophoresis is the genome DNA, the more obvious the main band is, the more complete the genome DNA separated from the nucleic acid is, and the phenomenon of tailing and degradation does not occur, so that the genome DNA is not degraded in the extraction process. The results are shown in FIG. 1 and Table 1.
TABLE 1 results of nanodrop measurements of shrimp tissue-extracted genomic DNA of examples 1-2 and comparative example 1
Figure BDA0003852338150000051
Figure BDA0003852338150000061
FIG. 1 shows that the extracted main band genomic DNA of example 1 is intact and has a higher yield, and the concentration of the extracted genomic DNA of example 1 is higher according to the data in Table 1; FIG. 1 shows that the yield of the extracted genomic DNA of example 2 is low, and the concentration of the extracted genomic DNA of example 2 is low according to the data in Table 1; FIG. 1 shows that the major band of the extracted genomic DNA of comparative example 1 was almost completely degraded, however, according to the data in Table 1, the concentration of DNA obtained in comparative example 1 was higher than that obtained in example 2, and the results of nanodrop alone did not indicate the actual concentration of genomic DNA, which was due to DNA degradation. As shown above, the polyoxyethylene fatty alcohol ether sodium sulfate is added into the lysis solution, and although the subsequent operation is the same as or similar to other schemes, the yield of the shrimp meat genome DNA can be effectively improved, and the DNA degradation can be prevented.
Comparative example 2
The difference from example 1 was that the lysis solution was 1.81M guanidine thiocyanate, 5% sodium dodecyl sulfate and 6.5% Tween 20.
Example 3
The difference from example 1 is that the lysis solution was 1.81M guanidinium thiocyanate, 2% sodium polyoxyethylene fatty alcohol ether sulfate and 6.5% Tween 20.
Example 4
The difference from example 1 is that the lysis solution is 1.81M guanidine thiocyanate, 4% sodium polyoxyethylene fatty alcohol ether sulfate, 0.3% sodium dodecyl sulfate.
Example 5
The difference from example 1 is that the lysis solution is 1.81M guanidine thiocyanate, 8% sodium polyoxyethylene fatty alcohol ether sulfate, 0.3% sodium dodecyl sulfate.
The results of the nanodorp detection and the agarose gel electrophoresis patterns of comparative example 2, examples 3 to 5 are shown in Table 2 and FIG. 2, respectively.
TABLE 2 results of nanodrop measurements of genomic DNA extracted from tissues of comparative example 2 and examples 3 to 5 of shrimps
Figure BDA0003852338150000071
As is clear from FIG. 2 and Table 2, in comparative example 2, even though the results of the nanodorp assay showed that the DNA concentration was high, the nucleic acid was degraded; in examples 3 to 5, although the final concentrations of sodium polyoxyethylene fatty alcohol ether sulfate were different, higher concentrations of DNA were obtained, and as can be seen from FIG. 2, as the concentration of sodium polyoxyethylene fatty alcohol ether sulfate increased, the integrity of genomic DNA did not change significantly and was relatively intact, and when the final concentration of sodium polyoxyethylene fatty alcohol ether sulfate was around 2.5%, the concentration of genomic DNA obtained was higher and the main band was brighter.
Example 6
The difference from example 1 is that the lysis solution was 4% sodium polyoxyethylene fatty alcohol ether sulfate, 1.81M guanidine thiocyanate, 5% sodium dodecyl sulfate, and 20mM magnesium chloride.
Example 7
The difference from example 1 is that the lysis solution is 4% sodium polyoxyethylene fatty alcohol ether sulfate, 2.5M guanidine hydrochloride, 5% sodium dodecyl sulfate, and 20mM magnesium chloride.
Example 8
The difference from example 1 is that the lysis solution was 4% sodium polyoxyethylene fatty alcohol ether sulfate, 1.81M guanidine thiocyanate, 5% cetyltrimethylammonium bromide, and 20mM magnesium chloride.
Example 9
The difference from example 1 is that the lysis solution was 4% sodium polyoxyethylene fatty alcohol ether sulfate, 2.5M guanidine hydrochloride, 5% hexadecyl trimethyl ammonium bromide, 20mM magnesium chloride.
Example 10
The difference from example 1 is that the lysis solution was 1.81M guanidinium thiocyanate, 5% cetyltrimethylammonium bromide, 20mM magnesium chloride.
Example 11
The difference from example 1 is that the lysis solution was 2.5M guanidine hydrochloride, 5% cetyltrimethylammonium bromide, 20mM magnesium chloride.
The agarose gel electrophoresis patterns of examples 6-11 are shown in FIG. 3. As can be seen from FIG. 3, the nucleic acid can be isolated from the shrimp tissue samples by using different combinations of lysates according to the method of the present invention, but the degradation of genomic DNA is affected differently by using different combinations of lysates, and the genomic DNA can be effectively prevented from being degraded even if different combinations of surfactants and salt particles are used in the lysate to which the polyoxyethylene fatty alcohol ether sodium sulfate is added, and the nucleic acid with higher concentration can be obtained.
Different shrimp tissue samples were treated according to the method of example 1, and their genomic DNAs were extracted therefrom, i.e., examples 12 to 16, and subjected to nucleic acid spectrophotometric measurement and agarose gel electrophoresis, and the results are shown in Table 3 and FIG. 4. Wherein, the method of the comparative example 1 is used for treating the prawns in the comparative example 3.
TABLE 3 Nanodrop measurements of genomic DNA extracted from prawn, metapenaeus ensis, prawns, shrimps, crayfish
Figure BDA0003852338150000091
As can be seen from Table 3, using the method of the present invention, it is possible to treat various shrimp tissue samples and extract genomic DNA therefrom, and from FIG. 4, it is clear that the nucleic acid electrophoresis bands prepared in examples 12 to 16 are clear, indicating that nucleic acids of higher concentration and integrity can be prepared, and that nucleic acid degradation of the above-mentioned various shrimp tissue samples can be effectively prevented.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.

Claims (8)

1. An improved method for extracting genomic DNA from shrimp tissue, comprising the steps of:
s1, cracking: adding lysis solution and enzyme into shrimp tissue to obtain lysis solution containing nucleic acid; the cracking liquid comprises polyoxyethylene cetyl ether sodium sulfate, at least one surfactant and at least one moisture absorbent; the enzyme is proteinase K and RNase;
s2, combining: adding a binding solution and magnetic beads into the lysis solution to obtain a binding body; the binding solution comprises isopropanol, guanidine hydrochloride and a non-ionic detergent;
s3, cleaning: cleaning the combination with ethanol;
s4, elution: eluting the washed combined body with eluent to obtain shrimp tissue DNA; the eluent is TE buffer solution.
2. The improved method for extracting genomic DNA from shrimp tissue as claimed in claim 1, wherein said sodium polyoxyethylene cetyl ether sulfate is sodium polyoxyethylene fatty alcohol ether sulfate.
3. The improved method of claim 1, wherein the lysis solution comprises sodium polyoxyethylene alcohol ether sulfate, 0.1% -5% sodium dodecyl sulfate, and 1.0-2.0M guanidinium thiocyanate. .
4. The improved method of claim 3, wherein the lysis solution further comprises any mixture of EDTA, tris, triton X-100, tween 20.
5. The improved method for extracting genomic DNA from shrimp tissue as claimed in claim 2, wherein the concentration of sodium polyoxyethylene fatty alcohol ether sulfate is 1% -10%.
6. The improved method for extracting genomic DNA from shrimp tissue as claimed in claim 2, wherein the concentration of the sodium polyoxyethylene fatty alcohol ether sulfate is 5.5%.
7. The improved method of claim 1, wherein the shrimp tissue includes but is not limited to shrimp head tissue and shrimp meat tissue.
8. The improved method of extracting genomic DNA from shrimp tissue as claimed in any one of claims 1 to 7 wherein said shrimp includes but is not limited to marine shrimp such as prawn, metapenaeus shrimp, crayfish, and freshwater shrimp.
CN202211147517.4A 2022-09-19 2022-09-19 Improved method for extracting genome DNA from shrimp tissue Pending CN115369114A (en)

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CN105602941A (en) * 2016-03-20 2016-05-25 北京农学院 Fungal hypha total DNA (deoxyribonucleic acid) extracting solution and method for extracting fungal hypha total DNA
CN106498044A (en) * 2016-10-18 2017-03-15 淮海工学院 A kind of quantitative detecting method of dusky white prawn mitochondrial genome copy number
CN109486959A (en) * 2018-10-23 2019-03-19 浙江海洋大学 The Variations of liver mtDNA copy number in Sepiella maindroni aging course
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Inventor after: Hou Jialin

Inventor after: Zhou Lisha

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