KR100215084B1 - Classification of the korean ginseng by radp analysis - Google Patents

Abstract

The method for determining the production of Korean ginseng by RAPD analysis of the present invention isolates and purifies DNA of ginseng which is the subject of mountain determination; PCR amplification of ginseng DNA in a reaction solution containing purified ginseng DNA as a template DNA, Mg ²⁺ ion, Taq DNA plymerase, dNTP, and primers; the amplified ginseng DNA was ethidium bromide Electrophoresis on the included agarose gel; and selecting the RAPD label by comparing the pattern of DNA bands on the UV transilluminator to the agarose gel in which the electrophoresis-completed ginseng DNA is present; It is characterized by consisting of steps.

The present invention also includes specific primers that can be used in the RAPD assay.

Description

Method for Discriminating Korean Ginseng by RAPD Labeling and Primer Used in the Method

FIG. 1 is a photograph of a gel electrophoresed with DNA showing the effect of template DNA concentration on PCR (Polymerase Chain Reaction) amplification.

FIG. 2 is a photograph of a gel electrophoresed with DNA showing the effect of primer concentration on PCR amplification.

3 is a photograph taken of a gel electrophoresed with DNA showing the effect of dNTP concentration on the PCR amplification.

4 is a photograph of a gel electrophoresed with DNA showing the effect of Taq polymerase concentration on PCR amplification.

FIG. 5 is a photograph of a gel electrophoresed with DNA showing a RAPD band pattern according to DNA of each ginseng region.

FIG. 6 is a photograph of a gel electrophoresed with DNA after RAPD analysis with primer A02.

7 is a photograph of a gel electrophoresed with DNA after RAPD analysis with primer A04.

FIG. 8 is a photograph of a gel electrophoresed with DNA after RAPD analysis with primer Al0.

[Field of Invention]

The present invention relates to a method for determining the production of Korean ginseng (Pαnαx ginseng C. A. Meyer) by the labeling through RAPD analysis (Random Amplified Polymor Phic DNA analysis). More specifically, the present invention relates to a method for discriminating domestic and overseas Korean ginseng by RAPD label and a specific primer that can be used in the method.

[Background of invention]

Korean ginseng is distributed in the Far East of 34 ° to 48 ° north latitude, and is grown and grown in all parts of the Korean peninsula except Jeju Island, Manchuria in China and Yeonhae in Russia.

Korean ginseng is not only effective in preventing and treating various diseases, but also widely used as a tonic for those who do not have any diseases.In Korean medicine, mental nerve diseases, digestive diseases, respiratory diseases, circulatory diseases, and surgery Ginseng has been widely applied to diseases, eye diseases, pediatric diseases and gynecological diseases.

The production of Korean ginseng in Korea increased from about 10,000 tons in 1984 to about 14,800 tons in 1993 and increased by 50% over 10 years. The ratio of red ginseng and white ginseng was about 2: 8 in terms of use. Red ginseng raw ginseng is fully purchased by Korea Tobacco Ginseng Corporation. White ginseng and other processed raw ginseng have been distributed mainly in Ganghwa and Geumsan.

Recently, however, Chinese ginseng has been smuggled and illegally distributed in the domestic market, which is difficult to distinguish from Korean ginseng produced domestically. Therefore, for the protection of Korean ginseng and healthy distribution order, the determination of the exact method of discriminating Korean ginseng and Chinese ginseng is an important task.

Taxonomic studies of ginseng have been developed and used, such as morphological traits and isoenzyme analysis, but these methods are highly applicable to species classification of the same genus, but ginseng is phenotype. Because of this simplicity, these methods are not appropriate for the identification of varieties or breeds between varieties and breeding lines in relatively close species.

Recently, with the rapid development in the field of molecular biology, researches have been conducted to classify genus, species or intra-cultivar varieties of plants, and it is evaluated that the possibility is extremely high.

Differentiation of strains by DNA analysis at the molecular biology level can be understood by identifying a number of characteristics as well as the quantitative level of the strains, and has the advantage of excluding the effects of the environment.

Among them, RFLP (Restriction Fragment Length Polymorphism) is the number or length of DNA fragments produced when a series of DNA chains are treated with restriction enzymes. It is based on the fact that they appear differently and is used in various ways.

However, the mitochondrial DNA of Ginseng varieties, such as ginseng varieties, Pseudomonas spp., And Hwang Sook-jong, for the classification of ginseng varieties using the RFLP technique in recent years, 1990.The charactehzation of mitochondrial DNA of Korean ginseng.Korean J. Ginseng Sci. 14: 310-316) and chloroplast DNA (Lee et al., 1991, Purification and Characterization of chloroplast DNA in Korean ginseng.Recent progress in Molecular biology and Genetic engineering in Korea 1991, (2): 342). The structure was analyzed using restriction enzymes, and the differences were not found. Therefore, the RFLP method for the strain and growth system of ginseng was not found. Use has been found to be limited in cytoplasmic genes.

In addition, RFLP requires a large amount of high-purity DNA and takes a lot of time and effort. In addition to being costly, the testing process is complicated and there are difficulties in using radioisotopes.

Applicants of the present invention has been invented a method for determining the production of Korean ginseng using RAPD label that is technically simple and easy to trace polymorphism in view of the above problems.

[Purpose of invention]

An object of the present invention is to provide a method for determining the origin of Korean ginseng by RAPD labeling, which is easy to trace polymorphism, takes a small amount of DNA, and has a fast analysis process.

It is another object of the present invention to provide conditions for RAPD analysis suitable for determining the origin of Korean ginseng.

Another object of the present invention is to provide a specific primer used for the RAPD assay.

[Summary of invention]

The production method of Korean ginseng using RAPD label of the present invention is to isolate and purify DNA of ginseng which is the subject of mountain determination; Using the purified ginseng DNA as a template DNA, ginseng DNA was amplified by PCR in a reaction solution containing Mg ²⁺ ion, Taq DNA polymerase, dNTP, and primers; the amplified ginseng DNA was ethidium bromide on agarose gel. electrophoresis with a buffer containing (ethidium bromide); And the agarose gel in which the ginseng DNA is completed electrophoresis is compared to the aspect of the DNA band on the UV transilluminator (transilluminator) to select the RAPD label; characterized in that it comprises a step.

The present invention also includes specific primers that can be used in the RAPD assay.

Detailed Description of the Invention

In the RAPD analysis of the present invention, conditions are slightly different depending on crops using PCR, but in the case of ginseng, double-stranded DNA is made of two single-stranded DNA at high temperature (near 90 ° C) and then random primer and target at low temperature (near 50 ° C). Annealing the DNA, repeating the process of making double-stranded DNA again at medium temperature (near 72 ° C), exponentially amplifying the number of specific DNA fragments, electrophoresing the amplified DNA on a gel, and staining with ethidium bromide It is based on what appears to be a visible band on the UV transluminator, a pattern of which is called the RAPD label.

Even if there is a slight difference in genes of individual strains of Korean ginseng, DNA is amplified according to specific primer and complementarity at the time of amplification by PCR, and the number and location of bands generated during electrophoresis are different. The band due to phoresis does not appear. Therefore, it is possible to discriminate Korean ginseng by region having a slight difference in genes among individuals.

Advantages of the RAPD analysis of the present invention is that even if only one base of the primer is changed, the expression pattern of the band is completely different, so that the polymorphism assay is sufficient by using oligonucleotides of about 10 nucleotides (10-mer) as primers for DNA amplification. Is that you can.

(A) DNA isolation and purification of ginseng, (A) DNA amplification by PCR, (C) electrophoresis, and (D) RAPD label selection step It will be described in detail below.

A. DNA Isolation and Purification of Ginseng

The success of RAPD analysis depends on the acquisition of high-purity DNA. In case of PCR using low purity DNA, the band pattern is not clear, there are many impurities, and there is also a problem in reproducibility.

The shorter the time required for the separation of ginseng DNA, the longer the amount of DNA obtained and higher purity. When a large amount of time is spent in the grinding process of the sample, which is the initial stage of DNA separation, the extraction solution is oxidized to dark brown to brown, and it is difficult to secure DNA with a small amount of finally obtained DNA and high purity.

The method of separating total DNA from ginseng can be carried out by conventional methods known in the art. It should be noted that the roots of ginseng generally contain a lot of starch and phenolic compounds, so phenol extraction should be repeated one or two more times during the extraction process. Pure DNA can be obtained by measuring the grinding and extraction time of ginseng samples and carefully repeating the phenol-chloroform extraction process.

Finally, the total DNA obtained is electrophoresed on agarose gel to appear as a single band, which is quantified using UV / VIS spectroscopy.

B. Amplification of DNA by PCR

PCR is a method of exponentially amplifying the number of specific DNA fragments by repeating a single strand of double-stranded DNA at high temperature, then annealing the primer and target DNA at low temperature and double-stranded DNA at medium temperature.

PCR is an effective method for screening large populations because it is highly sensitive enough to amplify even a single DNA fragment and can be performed with a small amount of DNA.

In PCR, template DNA, Mg ²⁺ ions, Taq polymerase, dNTP, primers, etc. are essential components of the reaction solution. Amplification can be achieved.

In the present invention, to determine the appropriate conditions of RAPD using the DNA of ginseng, it is confirmed how the difference in the amount of addition of the major factors involved in PCR affects the PCR product and presents the appropriate concentration of each element.

(a) Primer preliminary assay

Primers suitable for PCR of ginseng should be selected. That is, the most important factor in the present invention is to properly select a primer that can be annealed to single-stranded DNA of ginseng to synthesize DNA when PCR is performed. Even if the same species of ginseng was PCR under the same conditions, the RAPD band pattern appeared variously depending on the primer type, and the number of bands and the clarity were remarkably different. Therefore, preliminary tests should be performed to select suitable primers.

PCR cycles are tested for each primer in an appropriate cycle (between 50 and about) to select primers with distinct band patterns in RAPD.

In the embodiment of the present invention, the primers of high availability are selected by preliminarily assaying the 10-mer primers by expressing thick, distinct and clear bands in RAPD. The base sequence, molecular weight and RAPD amplification capacity of each primer used in the preliminary assay of the examples are mentioned in Table 1.

TABLE 1

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Claims (10)

Separating and purifying the DNA of ginseng which is the subject of mountain determination; Amplifying the ginseng DNA by PCR (Polymerase Chain Reaction) in a reaction solution containing Mg ²⁺ ions, Taq DNA polymerase, dNTP and a primer using the purified ginseng DNA as a template DNA; Electrophoresis of the amplified ginseng DNA on an agarose gel containing ethidium bromide; And Agarose gel in which electrophoresis-completed ginseng DNA is present is used to select RAPD (Random Amplified Ploymorphic DNA) label by comparing the pattern of DNA bands on UV (ultraviolet) transilluminator; Mountain ginseng determination method of Korean ginseng by RAPD label, characterized in that consisting of steps. The method of claim 1, wherein the concentration of the template DNA is 25ng to 250ng, preferably 50ng to 200ng when the total reaction solution is 25μl. The method of claim 1, wherein the concentration of the primer is 1pM to 2pM. The method of claim 1, wherein the concentration of dNTP is 100μM to 200μM. The method according to claim 1, wherein the Taq polymerase concentration is 1.0 to 2.5 units per 100 μl of the reaction solution. The method of claim 1, wherein the primer is selected from the group consisting of primers A02, A04, A07, A09, A10, A11, A12, A13 and A20 having the following sequence: Separating and purifying the DNA of domestic Korean ginseng and Chinese ginseng; PCR of the ginseng DNA in a reaction solution containing Taq DNA polymerase, dNTP, and primer A02 (5'-TGCCGAGCTG-3 ') using the purified ginseng DNA as a template DNA was carried out by PCR (polymerase chain reaction). Amplified by; Electrophoresis of the amplified ginseng DNA on an agarose gel containing ethidium bromide; And the agarose gel in which the electrophoresis-completed ginseng DNA is present to select the RAPD (Random Amplified Polymorphic DNA) label by comparing the DNA band pattern on the UV (ultraviolet) transilluminator; Identification method of domestic Korean ginseng and Chinese ginseng by RAPD analysis, characterized in that consisting of steps. Separating and purifying the DNA of domestic Korean ginseng and Gaeseong ginseng; PCR of the ginseng DNA in a reaction solution containing Taq DNA polymerase, dNTP, and primer A04 (5'-AATCGGGCTG-3 ') using the purified ginseng DNA as a template DNA (Polymerase Chain Reaction) Amplified by; Electrophoresis of the amplified ginseng DNA on an agarose gel containing ethidium bromide; And agarose gel containing the electrophoresis of ginseng DNA is present to compare the pattern of the DNA band on the UV (ultraviolet) transilluminator to select RAPD (Random Amplified Polymorphic DNA) label; Identification method of Korean ginseng and Gaeseong ginseng produced by RAPD label, characterized in that consisting of steps. Separating and purifying the DNA of domestic Korean ginseng and Japanese ginseng; PCR of the ginseng DNA in a reaction solution containing Taq DNA polymerase, dNTP, and primer A10 (5'-GTGATCGCAG-3 ') using the purified ginseng DNA as a template DNA (Polymerase Chain Reaction) Amplified by; Electrophoresis of the amplified ginseng DNA on an agarose gel containing ethidium bromide; And agarose gel containing the electrophoresis of ginseng DNA is present to compare the pattern of the DNA band on the UV (vltraviolet) transilluminator to select the RAPD label; Identification method of domestic Korean ginseng and Japanese ginseng by RAPD label, characterized in that consisting of steps. A primer having the following sequence to be used in the method for determining the origin of Korean ginseng by RAPD (Random Amplified Polymorphic DNA) labeling;