CN103898090B - The Capsicum chinense leaves genomic DNA extracting method of improvement - Google Patents
The Capsicum chinense leaves genomic DNA extracting method of improvement Download PDFInfo
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Abstract
The invention discloses the Capsicum chinense leaves genomic DNA extracting method of a kind of improvement, for in Capsicum chinense blade rich in the feature of phenols, saccharide and albumen, orthogonal design is utilized to optimize the concentration of several important component in Capsicum chinense extracting genome DNA, filter out a kind of extracting method obtaining high quality DNA molecule, solve that the DNA molecular impurity that conventional method extracts is more, productivity is relatively low, brown stain degradable, easy, agents useful for same toxicity relatively big, affect a difficult problem for next step molecular biology research.Adopt the DNA purity that the method for the present invention obtains higher.DNA integrity is better, nothing ruptures, without signs of degradation.
Description
Technical field
The present invention relates to the Capsicum chinense leaves genomic DNA extracting method of a kind of improvement, particularly relate to orthogonal design and optimize the concentration of several important component in Capsicum chinense extracting genome DNA, it is intended to filter out a kind of extracting method obtaining high quality DNA molecule.
Background technology
Hainan Huang sweetbell redpepper (CapsicumchinenseJacquin) is Solanaceae (Solanaceae) Capsicum (Capsicum) herbaceos perennial, originate from South America Amazon, also known as yellow Fructus Capsici, Yellow Emperor, a legendary ruler green pepper etc., Plant Taxonomy belongs to Chinese Capsicum (Capsicumchinense), it it is the rare capsicum variety in distinctive place, Hainan, long cultivation and edible history is had in Hainan, it is distributed mainly on the southeast, Hainan Island, coastal area, southwest, happiness high temperature, high light, growth potential is strong, branch and leaf are luxuriant, mature fruit yellow or golden yellow, shape is like lantern, containing abundant vitamin C, carotene, mineral, aminoacid and trace element.Additionally, Huang sweetbell redpepper contains abundant Capsaicinoids in Hainan, has extremely strong pungent, its fresh fruit peppery degree 170,000 about SHU, simultaneously possibly together with other aromatic substance, being one of processing Fructus Capsici sauce and the optimum feed stock extracting capsaicin, yellow sweetbell redpepper Fructus Capsici sauce has become the travelling products that Hainan is well-known, the deep welcome by domestic and international consumer, the pharmacologically active effect of capsaicin is extensively paid attention to by countries in the world, energy gastric acid secretion inhibiting, stimulates alkaline viscid secretion, contributes to prevention and treatment gastric ulcer；The metabolism of energy acceleration energy and lipid, reduces body fat accumulation；The metabolism about cell can be stoped, reduce the incidence rate of cancer cell.In a word, Hainan Huang sweetbell redpepper has high economic worth, nutritive value and medical value, is widely used, is the Specialty vegetable crop of Hainan most potentiality to be exploited on diet and medical product.
Extracting genome DNA is the basis of molecular biology research.Conventional DNA extraction method mainly has cetyltriethylammonium bromide (CTAB) method, sodium lauryl sulphate (SDS) method and Low pH extraction with high salts method.SDS method is simple to operate, but products therefrom is impure more；CTAB can fracturing cell walls and cell membrane, and complex can be formed with nucleic acid, and then from the plant tissue rich in polyphenol and polysaccharide, isolate high-quality genomic DNA；It is relatively low that high salt pH method extracts DNA productivity, and can remain more small-molecule substance.Material composition and the content difference of various composition contained by different materials are relatively big, therefore, extract for different plant genome DNAs and should take diverse ways.
High-quality DNA is by analysis of genetic diversity, genetic linkage maps builds and the premise of molecular marker assisted selection, is the guarantee that experimental result reproducibility and reliability is good.Template quality will directly affect pcr amplification reaction.Aldehydes matter and polysaccharide are to affect 2 key factors that plant genome DNA extracts, and often result in DNA molecular degraded, have a strong impact on DNA purity and yield.Capsicum chinense blade is rich in materials such as saccharide, protein and phenols, and aldehydes matter is susceptible to oxidation, is combined formation yellow or pitchy precipitation with DNA, it is difficult to dissolving, DNA purity is relatively low, it is difficult to meet Capsicum chinense molecular biology research demand.Effective Polysaccharide removing, protein and aldehydes matter etc., be the key extracting high quality DNA molecule.Effectively remove vegetable polysaccharides at present, the DNA extraction method of aldehydes matter mainly has two kinds: one is CsCl density－gradient centrifuga－tion method；One is CTAB method, and the former requires higher by instrument and equipment, time-consuming, and cost is high；The latter operates relatively simple, it is adaptable to most plants DNA extraction, applies increasingly extensive.Accordingly, it would be desirable to adopt the CTAB method of improvement to optimize the extraction of Capsicum chinense genomic DNA, extract Capsicum chinense genomic DNA molecule quick, economical, safe efficiently.
Polyvinylpyrrolidone (PVP), as cleaning agent, has adsorption, can adsorb polysaccharide and be removed.Dithiothreitol, DTT (DTT) is a kind of reducing agent, Wheat Protein can effectively prevent phenols to be oxidized to quinone, it is to avoid the generation of DNA brown stain, but the penetrating odor of DTT is little, and toxicity is also much lower than mercaptoethanol.The concentration of several important component (CTAB, PVP and DTT) in Capsicum chinense extracting genome DNA is optimized by the CTAB method of improvement, contribute to filtering out the extracting method that can obtain high quality DNA molecule, lay the foundation for carrying out Capsicum chinense molecular biology research further.
Summary of the invention
The present invention seeks to in Capsicum chinense blade rich in phenols, the feature of saccharide and albumen, orthogonal design is utilized to optimize the concentration of several important component in Capsicum chinense extracting genome DNA, filter out a kind of extracting method obtaining high quality DNA molecule, the DNA molecular impurity solving conventional method extraction is more, productivity is relatively low, degradable, easy brown stain, agents useful for same toxicity is bigger, as document (to, Song little Li, the pretty .DNA-AFLP of Shi Qian analyzes with the optimization of Fructus Capsici genome DNA extracting method. Changjiang University's journal, 2009, 6 (2): 48-51), affect a difficult problem for next step molecular biology research.
Technical scheme is as follows:
The Capsicum chinense leaves genomic DNA extracting method of improvement, comprises the following steps:
(1) preparation CTAB-3 extracting solution, after high temperature sterilize, 65 DEG C of preheatings；CTAB-3 extracting solution is: 0.1molL-1TrispH7.5,0.05molL-1EDTApH8.0,0.7molL-1NaCl, 2%CTAB, 0.5%PVP, 0.2%DDT；Wherein, percentage ratio is mass percent concentration；
(2) liquid nitrogen is utilized to load in 2.0mL centrifuge tube by Capsicum chinense blade grind into powder tender for children, sample reaches 1/4 place and is advisable, and often pipe adds the CTAB-3 extracting solution 700 μ L of preheating and is placed in 65 DEG C of constant water bath box 60min and is shaken gently for once every 10min；
(3) addition phenol, chloroform, isoamyl alcohol mixed liquor 700 μ L fully mix 15min, slow in one's movements, 4 DEG C of centrifugal 6min of 12000rpm；Phenol/chloroform/isoamyl alcohol volume ratio is 25: 24: 1；
(4) Aspirate supernatant proceeds in 1.5mL centrifuge tube, adds isopyknic chloroform, isoamyl alcohol mixed liquor fully mixes 15min, slow in one's movements, 4 DEG C of centrifugal 6min of 12000rpm；Chloroform, isoamyl alcohol volume ratio are 24: 1；
(5) Aspirate supernatant proceeds in 1.5mL centrifuge tube, adds the dehydrated alcohol of 2 times of volume pre-coolings, mixes gently, puts-20 DEG C of refrigerator 30min precipitations；
(6) choose DNA flocculent deposit with sterilizing toothpick, put in 1.5mL centrifuge tube, adopt 70% ethanol rinse twice, then dry；
(7) adding 100 μ L sterilizing ultra-pure waters, 4 DEG C of refrigerators dissolve.
The CTAB method of improvement is extracted Capsicum chinense genomic DNA and is had feature: (1) DNA is precipitated as floccule, utilizes the cotton-shaped DNA of the toothpick picking after sterilizing to replace centrifugation, and the DNA purity so obtained is higher.(2) the DNA integrity extracted is better, without fracture, without signs of degradation.(3) the method can remove protein effectively, it is thus achieved that DNA purity higher.(4) in extracting solution, add the PVP being simultaneously introduced 0.5-1% of DTT, can effective Polysaccharide removing, it is suppressed that aldehydes matter is oxidized into brown, it is ensured that the DNA molecular color and luster of extraction is white.(5) utilize DTT substituted sulfhydryl ethanol, can effectively suppress aldehydes matter to aoxidize on the one hand, additionally, DTT has antioxidative effect, and the effect to mercaptoethanol is similar, but the penetrating odor of DTT is much smaller, toxicity is also much lower than mercaptoethanol, it is possible to be substantially reduced the pollution to environment.
Accompanying drawing explanation
Fig. 1: Capsicum chinense leaves genomic DNA agarose gel electrophoresis result.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
(1) L is adopted423Orthogonal design experiment arrangement processes, preparation DNA extraction liquid, extract Capsicum chinense leaves genomic DNA molecule (2) respectively and utilize the DNA molecular of agarose gel electrophoresis technology Detection and Extraction, detect DNA with or without whether degraded and impurity thoroughly remove according to band situation.(3) utilize the DNA of nucleic acid-protein instrument Detection and Extraction at 260nm and the 280nm light absorption value located, judge the DNA purity extracted according to OD260/OD280 value.
Instrument and equipment needed for the technical program: High speed refrigerated centrifuge, thermostatic water bath, electrophresis apparatus, Horizontal electrophoresis tank, nucleic acid-protein analyser, gel imaging system.
Reagent needed for the technical program: the saturated phenol of EDTA, NaCl, Tris, CTAB, PVP, DDT, Tris, chloroform, isoamyl alcohol, dehydrated alcohol, agarose, Methanamide, bromophenol blue, diformazan cyanophenyl.
The detailed step adopting the technical program extraction Capsicum chinense leaves genomic DNA is as follows:
(1) preparation extract with CTAB liquid, after high temperature sterilize, 65 DEG C of preheatings；
(2) liquid nitrogen is utilized to load in 2.0mL centrifuge tube by Capsicum chinense blade grind into powder tender for children, sample reaches 1/4 place and is advisable, and often pipe adds the extract with CTAB liquid 700 μ L of preheating and is placed in 65 DEG C of constant water bath box 60min (being shaken gently for once every 10min)；
(3) add phenol/chloroform/isoamyl alcohol 700 μ L and fully mix 15min, slow in one's movements, 4 DEG C of centrifugal 6min of 12000rpm；
(4) Aspirate supernatant proceeds in 1.5mL centrifuge tube, adds isopyknic chloroform/isoamyl alcohol and fully mixes 15min, slow in one's movements, 4 DEG C of centrifugal 6min of 12000rpm；
(5) Aspirate supernatant proceeds in 1.5mL centrifuge tube, adds the dehydrated alcohol of 2 times of volume pre-coolings, mixes gently, puts-20 DEG C of refrigerator 30min precipitations；
(6) choose DNA flocculent deposit with the toothpick of sterilizing, put in 1.5mL centrifuge tube, adopt 70% ethanol rinse twice, then dry；
(7) adding 100 μ L sterilizing ultra-pure waters, 4 DEG C of refrigerators dissolve；
(8) draw 5 μ LDNA solution, add 2.5 μ L indicators, carry out agarose gel electrophoresis, differentiate DNA is with or without whether degraded and impurity thoroughly remove according to band situation；
(9) utilize the DNA of nucleic acid-protein instrument Detection and Extraction at 260nm and the 280nm light absorption value located, judge the DNA purity extracted according to OD260/OD280 value.
Wherein, (1) DNA extraction liquid adopts L423Orthogonal design is prepared respectively, including 3 factors (CTAB concentration, PVP concentration and DDT concentration), 2 level (1%CTAB, 2%CTAB；0.5%PVP, 1%PVP；0.1%DDT, 0.2%DDT), extract with CTAB liquid (CTAB-1, CTAB-2, CTAB-3 and CTAB-4) 4 kinds different, different extracting solution (table 1) composed as follows need to be prepared altogether.
The different DNA extraction buffer solution system composition of table 1
(3) phenol: chloroform: isoamyl alcohol=25: 24: 1；
(4) chloroform: isoamyl alcohol=24: 1；
(8) indicator formula: 98mL Methanamide, 2mL0.5molL-1EDTApH8.0,0.1g bromophenol blue, 0.1g diformazan cyanophenyl.Agarose gel concentration is 1%.
The theoretical foundation of high-quality Capsicum chinense leaves genomic DNA detection:
1) agarose gel electrophoresis: in conventional Capsicum chinense leaves genomic DNA extraction process, aldehydes matter not only may result in DNA degradation, and is easily combined formation yellow with DNA to the precipitate being difficult to dissolve of pitchy, hardly results in highly purified DNA molecular.If the DNA molecular extracted is precipitated as, white, band be clear, neat and consistent, brightness are higher, without signs of degradation, it was shown that the DNA mass of extraction is higher.Test result indicate that, DNA molecular concentration and quality that CTAB-3 extracts are the highest.
2) UV absorbance detection: utilize the DNA of nucleic acid-protein instrument Detection and Extraction at 260nm and the 280nm light absorption value located, judge the DNA purity extracted according to OD260/OD280 value.The OD260/OD280 of high-purity DNA sample should be 1.8～2.0, when OD260/OD280 is more than 2.0, it was shown that has RNA to pollute, during less than 1.8, it was shown that there is protein contamination in sample.Test result indicate that, the DNA molecular OD260/OD280 that CTAB-3 method is extracted is 1.8～2.0, it was shown that the DNA molecular purity of extraction is higher.
Embodiment 2
One, the preparation of CTAB-3DNA Extraction buffer: the concentration of each composition is 0.1molL-1TrispH7.5,0.05molL-1EDTApH8.0,0.7molL-1NaCl, 2%CTAB, 0.5%PVP, 0.2%DDT.
Two, CTAB-3DNA Extraction buffer is utilized to extract the concrete steps of Capsicum chinense leaves genomic DNA:
(1) 65 DEG C of preheating CTAB-3DNA Extraction buffer；
(2) liquid nitrogen is utilized to load in 2.0mL centrifuge tube by Capsicum chinense blade grind into powder tender for children, sample reaches 1/4 place and is advisable, and often pipe adds the CTAB-3 extracting solution 700 μ L of preheating and is placed in 65 DEG C of constant water bath box 60min (being shaken gently for once every 10min)；
(3) add phenol/chloroform/isoamyl alcohol (25: 24: 1) 700 μ L and fully mix 15min, slow in one's movements, 4 DEG C of centrifugal 6min of 12000rpm；
(4) Aspirate supernatant proceeds in 1.5mL centrifuge tube, adds isopyknic chloroform/isoamyl alcohol (24: 1) and fully mixes 15min, slow in one's movements, 4 DEG C of centrifugal 6min of 12000rpm；
(5) Aspirate supernatant proceeds in 1.5mL centrifuge tube, adds the dehydrated alcohol of 2 times of volume pre-coolings, mixes gently, puts-20 DEG C of refrigerator 30min precipitations；
(6) choose DNA flocculent deposit with sterilizing toothpick, put in 1.5mL centrifuge tube, adopt 70% ethanol rinse twice, then dry；
(7) adding 100 μ L sterilizing ultra-pure waters, 4 DEG C of refrigerators dissolve.
Three, DNA mass and purity detecting:
In order to verify quality and the purity of the Capsicum chinense leaves genomic DNA utilizing CTAB-3DNA Extraction buffer to extract, this example adopt following two analyze method.
1) agarose gel electrophoresis detection method.Take 5 μ LDNA solution, after 2.5 μ L indicator mix homogeneously, carry out 1% agarose gel electrophoresis, after dyeing, observe under uviol lamp, present a clear neat master tape, without hangover, then illustrate that the DNA integrity extracted is better, without fracture, without signs of degradation.5 and No. 6 samples in accompanying drawing 1.
2) UV absorbance detection method, utilizes the DNAOD260/OD280 that CTAB-3DNA Extraction buffer extracts between 1.8～2.0, illustrates that the DNA extracted has typical DNA standard absorption spectrum.
Capsicum chinense leaves genomic DNA quality and purity that two kinds of detection methods all show to utilize CTAB-3DNA Extraction buffer to extract are all better.
As it is shown in figure 1, adopt the method for the present invention to obtain Capsicum chinense leaves genomic DNA agarose gel electrophoresis result, DNA is precipitated as white, agarose gel electrophoresis result presents a clear neat master tape, without hangover, illustrate that the DNA integrity extracted is better, without fracture, without signs of degradation.M representation DNA molecular weight standard；1 and 2 DNA moleculars extracted for CTAB-1 method, are precipitated as brown, almost all degrade, without clearly neat master tape；3 and 4 is the DNA moleculars that CTAB-2 method is extracted, be precipitated as that white, band be clear, neat and consistent, without signs of degradation, but brightness to be not so good as CTAB-3 bright；5 and 6 is the DNA moleculars that CTAB-3 method is extracted, be precipitated as that white, band be clear, neat and consistent, brightness are higher, without hangover, without signs of degradation；7 and 8 DNA moleculars extracted for CTAB-4 method, are precipitated as brown, there is hangover, have degraded in various degree.
It should be appreciated that for those of ordinary skills, it is possible to improved according to the above description or converted, and all these are improved and convert the protection domain that all should belong to claims of the present invention.
Claims (1)
1. the Capsicum chinense leaves genomic DNA extracting method of an improvement, it is characterised in that comprise the following steps:
(1) preparation CTAB-3 extracting solution, after high temperature sterilize, 65 DEG C of preheatings；CTAB-3 extracting solution is: 0.1molL-1TrispH7.5,0.05molL-1EDTApH8.0,0.7molL-1NaCl, 2%CTAB, 0.5%PVP, 0.2%DTT；Wherein, percentage ratio is mass percent concentration；
(2) liquid nitrogen is utilized to load in 2.0mL centrifuge tube by Capsicum chinense blade grind into powder tender for children, sample reaches 1/4 place and is advisable, often the CTAB-3 extracting solution 700 μ L of pipe addition preheating is placed in 60min in 65 DEG C of constant water bath box, is shaken gently for once every 10min；
(3) addition phenol, chloroform, isoamyl alcohol mixed liquor 700 μ L fully mix 15min, slow in one's movements, 4 DEG C of centrifugal 6min of 12000rpm；Phenol/chloroform/isoamyl alcohol volume ratio is 25: 24: 1；
(4) Aspirate supernatant proceeds in 1.5mL centrifuge tube, adds isopyknic chloroform, isoamyl alcohol mixed liquor fully mixes 15min, slow in one's movements, 4 DEG C of centrifugal 6min of 12000rpm；Chloroform, isoamyl alcohol volume ratio are 24: 1；
(5) Aspirate supernatant proceeds in 1.5mL centrifuge tube, adds the dehydrated alcohol of 2 times of volume pre-coolings, mixes gently, puts-20 DEG C of refrigerator 30min precipitations；
(6) choose DNA flocculent deposit with sterilizing toothpick, put in 1.5mL centrifuge tube, adopt 70% ethanol rinse twice, then dry；
(7) adding 100 μ L sterilizing ultra-pure waters, 4 DEG C of refrigerators dissolve.
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| CN103173444A (en) * | 2013-03-27 | 2013-06-26 | 广东省农业科学院蔬菜研究所 | Functional marker for accurately distinguishing chilli from pimento as well as detection primer group and detection method thereof |
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| CN103173444A (en) * | 2013-03-27 | 2013-06-26 | 广东省农业科学院蔬菜研究所 | Functional marker for accurately distinguishing chilli from pimento as well as detection primer group and detection method thereof |
Non-Patent Citations (3)
| Title |
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| DNA-AFLP分析用辣椒基因组DNA提取方法的优化;向珣等;《长江大学学报（自然科学版）》;20090630;第6卷(第2期);第48-64页 * |
| 辣椒叶片基因组DNA提取方法的研究;周春阳等;《辣椒杂志》;20111231(第3期);第36页左栏第4-右栏第1段，第36页右栏第4段，第37页右栏第1、4段，表1-2 * |
| 辣椒基因组DNA两种提取方法的比较;桑维维等;《宿州学院学报》;20120831;第27卷(第8期);第21-24页 * |
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