KR19980038936A - Method for Discrimination of Korean Ginseng by RAPD Marker and Primer Used in the Method - Google Patents

Abstract

According to the present invention, the method for determining the production of Korean ginseng by RAPD analysis is performed by separating and purifying the DNA of ginseng which is the subject of mountain determination and using purified ginseng DNA as template DNA, M ²⁺ ion, T _aq DNA polymerase, dNTP. , Amplifying ginseng DNA by PCR in a reaction solution containing a primer, electrophoresing the amplified ginseng DNA on an agarose gel containing ethidium bromide, and UV light the agarose gel in which the electrophoresis is completed. Comparing the aspect of the DNA band on the transilluminator is characterized by consisting of the step of selecting the RAPD markers.

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

Description

Method for Discrimination of Korean Ginseng by RAPD Marker and Primer Used in the Method

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

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

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

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

Figure 5 is a photograph of a gel electrophoresis of the DNA showing the RAPD band (band) pattern according to the DNA for each ginseng region.

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.

8 is a photograph of a gel electrophoresed with DNA after RAPD analysis with primer A10.

[Purpose of invention]

[Field of Invention]

The present invention relates to a method for discriminating the mountain of Korean ginseng (Panax ginseng C. A. Meyer) by the labeling through RAPD analysis (Random Amplified Polymorphic DNA analysis). More specifically, the present invention relates to a method for determining domestic and foreign Korean ginseng by RAPD markers and to specific primers 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. 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, increasing by 50% over 10 years. The ratio of red ginseng and white ginseng was about 2: 8. 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.

Although taxonomic studies of ginseng have been developed and used, such as morphological traits and isoenzyme analysis, 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 strain, and has the advantage of excluding the effects of the environment.

Among them, RFLP (Restriction Fragment Length Polymorphism) is the number and length of DNA fragments produced when a series of DNA chains are treated with restriction enzymes, depending on the genetic composition of the individual, that is, the difference in nucleotide sequence. 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 characterizahon of mitochondrial DNA of Korean ginseng.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. In Molecular Biology and Genetic Engineering in Korea 1991, (2): 342), the structure was analyzed using restriction enzymes. The use of the RFLP method 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 the high cost, the test procedure is complicated and the use of radioisotopes is required.

Applicants of the present invention has been invented a method for determining the origin of Korean ginseng using a RAPD marker 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 markers, which is easy to trace polymorphism, takes a small amount of DNA, and has a fast analysis process.

Still another object of the present invention is 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 in the RAPD analysis.

[Summary of invention]

Origin detection method of Korean by RAPD markers of the present invention by the DNA of ginseng to be subjected to the producer determines the isolated and purified DNA as a template DNA and the purified ginseng Mg ²⁺ ions, Taq DNA polymerase, dNTP, primer In the reaction solution containing amplified ginseng DNA by PCR, the amplified ginseng DNA is electrophoresed on agarose gel with a buffer containing ethidium bromide, and the electrophoresis is completed ginseng DNA The agarose gel is characterized by consisting of the step of selecting the RAPD markers by comparing the aspect of the DNA band on the UV transilluminator (transilluminator).

The 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, and this aspect of the band is called the RAPD marker.

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 marker selection step It will be described in detail below.

[A. DNA Separation 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 phenolized compounds, so phenol extraction should be repeated one or two more times during the extraction process. Pure DNA can be obtained by reducing 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. DNA Amplification 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, can be performed with only a small amount of DNA, and the test process is fast and safe.

In performing 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 at appropriate cycles (about 50 cycles) 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.

Preassayed Primer Codes, Sequences, and Properties Code number Sequence (5'-3 ') Molecular Weight PCR amplification capacity A01 CAGGCCCTTC 2955 P A02 TGCCGAGCTG 3035 E A03 AGTCAGCCAC 2988 P A04 AATCGGGCTG 3059 E A05 AGGGGTCTTG 3090 P A06 GGTCCCTGAC 2995 - A07 GAAACGGGTG 3108 E A08 GTGACGTAGG 3099 P A09 GGGTAACGCC 3044 E A10 GTGATCGCAG 3059 E A11 CAATCGCCGT 2979 E A12 TCGGCGATAG 3059 E A13 CAGCACCCAC 2933 E A14 TCTGTGCTGG 3041 P A15 TTCCGAACCC 2939 P A16 AGCCAGCGAA 3037 P A17 GACCGCTTGT 3910 P A18 AGGTGACCGT 3059 P A19 CAAACGTCGG 3028 P A20 GTTGCGATCC 3010 E

∴-: no amplification,

P: poor amplification,

G: amplification is good,

E: Excellent amplification.

In primers used for RAPD analysis for determination of the acidity of Korean ginseng of the present invention, it is preferable to use primers indicating E, that is, primers A02, A04, A07, A09, A10, A11, A12, A13 and A20. However, in addition to the 9 kinds of primers, those skilled in the art can replace the primers, that is, primers having high complementarity because the base sequence is similar to the 9 kinds of primers will be included in the present invention.

(b) concentration of template DNA

The appropriate amount of template DNA for the PCR reaction (when the total reaction solution is 25 μl) is 25ng to 250ng, preferably 50ng to 200ng. As shown in Fig. 1, the long bands of DNA sequences are not clear at low concentrations, and at high concentrations, the expression intensity of short bands of DNA sequences tends to be weak or disappear. In the template DNA concentration range of 25 ng to 200 ng, a relatively clear and repeatable RAPD band is identified.

(c) concentration of primer

The proper concentration of the primer for amplifying ginseng DNA by PCR is 1pM to 2pM. As the primer concentration is lower than 1 pM, the long band of the DNA sequence is not clear. As the primer concentration is higher than 2 pM, the band of the short DNA sequence is not clear. As shown in FIG. 2, all bands are uniformly expressed in the range of 1 pM to 2 pM.

(d) dNTP concentration

The dNTP titer for optimal PCR amplification is 100 μM to 200 μM, as shown in FIG. 3. Above 200μM, polymerase action causes the formation of unspecific bands and minimizes the possibility of producing unreproducible bands from primers attached or misattached primers, even though PCR is not fully complementary. In order to achieve this, it is advantageous to lower the concentration of dNTP as much as possible.

(e) Taq polymerase concentration

The optimal concentration of Taq polymerase, a heat-resistant enzyme, is related to the amount of DNA contained therein rather than the total amount of the reaction solution. The optimal concentration of Taq polymerase is 1.0 to 2.5 units per 100 μl of the reaction solution. It may depend on the nature and primer type.

As shown in FIG. 4, in the 1.0 unit and the 2.0 unit, a large band is expressed with high intensity, whereas in the 4.0 unit, the expression intensity of the small band is weak.

However, Taq polymerase is a kind of enzyme, and its activity may vary depending on the manufacturing method, storage conditions, etc., and there is a shortage of simply mentioning the concentration because the activity varies depending on the manufacturer.

(f) the concentration of Mg ²⁺ ions

If the Mg ²⁺ ion in the reaction solution is less than 1mM, the number of RAPD bands may become small or no bands may appear.

(g) RAPD by site of ginseng

In performing RAPD of ginseng, the difference of tissues used for DNA separation does not affect the DNA band pattern after PCR.

As shown in Figure 5 in the case of ginseng is the same plant, even if the DNA is separated from any tissue irrespective of the sampling site of the same result. That is, when RAPD was performed using DNA from the root, stem and fruit tissues of ginseng, the DNA band pattern is similar.

C. Electrophoresis

Electrophoresis is performed using a buffer containing ethidium bromide to load the amplified DNA on an agarose gel and stain the DNA. The DNA is located near the shorter one according to the length of the sequence, and far from the longer one. The length of the sequence is calculated by Kb (kilobase) by electrophoresis of the assay DNA (denoted by M in the figure) treated with the restriction enzymes Hind III and Pst I with the DNA of the lambda-phage so as to express the number of bases.

After electrophoresis is completed, the DNA band pattern is observed and photographed on a UV transluminator.

D. Selection of RAPD Markers of Korean Ginseng Collected in Korea

In an embodiment of the present invention, the analysis of 167 bands of which the diversity of fat collection species was recognized among 169 RAPD bands obtained by PCR with a total of 9 primers resulted in 31 specificities in which common bands appeared in 25 reported fat collection species. After tying up the bands, RAPD markers were selected to discriminate each lipoma from one to several bands for domestic and 8 U.S. collections (Table 2).

As can be seen from Fig. 6 to Fig. 8, five regional collection species of Jinggyu, Fuchon, Chang Pai, Shiju and Nagano can be distinguished from other collection species by the presence or absence of expression of only one band. .

It was not possible to distinguish it with only one band, but when two bands amplified by one primer were used as discriminating markers, it was confirmed that 17 species were collected in Korea and cultivated in Kaesong. The differences between the collected species are distinguished. If the plantation is in Korea, it can be identified as A02-1.25 (1) and A02-0.34 (1) in the RAPD band (in primer-Kb order), and A04-1.25 (0) and A04-0.53 (for Kaesong). 1) can be identified.

Among ginseng collected from foreign countries, Gian and Liaoning can distinguish from other collected ginseng in one primer only by identifying three or seven or more bands.

Among the foreign collected ginseng, two primers are used for Jinan and Liaoning, and two bands for Jinan and three bands for Liaoning can be distinguished from other collected ginseng.

Therefore, the DNA band pattern shown by RAPD analysis performed by 1 or 2 primers can be selected as a single RAPD marker, and at most several bands can be selected as RAPD markers.

The present invention will be further illustrated by the following examples, which are merely illustrative of the present invention and are not intended to limit or limit the scope of the present invention.

EXAMPLE

Example 1 DNA Separation and Purification of Ginseng

In this example, a total of 25 points (Table 3) such as ginseng collected from major tobacco ginseng cultures in Korea and Korea and Korea ginseng grown by Korea Ginseng and Tobacco Research Institute and ginseng collected directly from Japan, etc. are disclosed. Gene DNA was extracted from the root of each ginseng and used as a sample.

The centrifuges used in this example were VS-21SMT and VS-15000CF models of Vision Science.

The ginseng was first washed and then dried to remove water, and then collected in about 2 g and placed in a mortar, and then ground in a short time using a pestle while adding liquid nitrogen to use a pestle. The ground samples were mixed by shaking in a 50 ml falcon tube containing a 6 ml urea extraction buffer (see Table 4) to prevent oxidation, and then mixed with 3 ml of phenol and 3 ml of chloroform solution. After standing for 15 minutes at 4 ℃ was centrifuged at 8,000rpm for 10 minutes.

After centrifugation, the supernatant was taken and placed in a 15 ml Falcon tube, a solution of 4.4 moles of ammonium acetate (pH 5.2) (105 ml of unionized water was added to a 500 ml beaker with stirring and 50 ml of gracial acetic acid was added, followed by ammonium hydride). Add 1 ml of 45% of the lockside (molecular weight 35.05) dropping and add 6 ml of isopropanol to bring the total volume to 13 ml. The solution was mixed evenly and centrifuged again at 10,000 rpm.

Composition of Urea Extraction Buffer Component density Volume Element - 168 g NaCl 5.0mol 25 ml Tris-hcl 1.0 mol (pH 8.0) 20 ml EDTA 0.5 mol (pH 8.0) 16 ml Sarcosine 20.0% 20 ml water - 190 ml sum 400 ml

: Tris-Hcl, EDTA, NaCl and water are used after autoclaving.

After centrifugation, the supernatant was discarded and the DNA mass that had settled at the bottom of the tube was washed twice with 70% ethanol and then dried in vacuo or at room temperature. After drying, the DNA pellet was re-dissolved in 200 μl of TE buffer (100 μl of 1.0 mole Tris-Hcl (pH 8.0), 20 μl of 0.5 mole EDTA (pH 8.0) and 9,880 μl of water). A phenol-coroform solution of the same volume as RNase was added and mixed, followed by centrifugation at 15,000 rpm for 10 minutes.

After centrifugation, the supernatant was collected and the same amount of isopropanol was added to precipitate DNA, followed by centrifugation at 15,000 rpm for 3 minutes. After centrifugation, the supernatant was discarded and the precipitated DNA was suspended in 200 µl of 70% ethanol.

The finished DNA was electrophoresed on an agarose gel and quantified by UV / VIS spectroscopy to use only 1.8 purity.

Example 2 Amplification of DNA by PCR

The PCR model used in the test of the present invention was Thermal Cycler 480 from Perkin Elmer Cetus.

The reaction vessel of PCR was a 0.5 ml eppen rope tube, and the reaction solution was added to the tube and the surface of the solution was coated with about 10 μl of mineral oil to prevent evaporation of the solution during the PCR reaction.

Double-stranded DNA was first isolated from PCR at 94 ° C. for 5 minutes, and then amplified DNA was treated by 1 minute at 94 ° C., 1 minute at 35 ° C., and 2 minutes at 72 ° C., respectively. After 50 cycles, DNA amplification was completed and finally stabilized at 72 ° C. for 10 minutes and then maintained at 4 ° C.

Example 3: RAPD titration conditions of ginseng

In the present example, the test ginseng KG101 was used, and the PCR reaction solution of each test was 50 μg of template DNA, 1 pM of primer, dATP, dCTP, dGTP and dTTP, 1.5 mM of Mg ²⁺ ion, and 1.5 units of Taq DNA polymerase. 2.5 μl of the 10 × reaction buffer and the rest were sterilized in distilled water to give 25 μl of the total reaction solution.

A. Template DNA Concentration

In order to check the appropriate DNA amount in 25μl of total reaction solution in PCR amplification, the concentration of template DNA isolated from KG101 ginseng root was varied by 10, 25, 50, 100, 200, 250, 500, 1000, 2500ng The solution was prepared and subjected to PCR and the DNA band pattern was compared on the agarose gel. In addition to the template DNA addition, other factors were added based on the reaction solution concentration described above, and primer A08 was used.

As shown in FIG. 1, when the primer A08 was used, seven bands distinguishable between 0.5 and 2.0 Kb were expressed. When a serial number was given from a large band (low Kb), the band 2 appeared as a high intensity band only in the 10 to 50 ng treatment and the expression intensity of the other treatment was weak. In case of band 4, the expression intensity tended to be gradually weakened when treated with more than 50ng. In band 7, the expression intensity was significantly weakened at 10-20ng.

B. Concentration of Primer

In order to determine the proper concentration of the primer, PCR reaction solution was prepared at a primer A20 concentration of 0.2, 0.5, 1, 2, 4, 8 pM, respectively. Other elements than the primer were added according to the standard concentration of the reaction solution described above, and the prepared reaction solution was subjected to PCR and electrophoresed on agarose gel to examine the DNA band pattern.

In the expression patterns of the six bands according to the primer concentration, the long bands 1 to 5 were clear in the 0.2, 0.5 and 2 pM treatments, and the short band 6 was clearly expressed in the 4 and 8 pM (see FIG. 2). ).

C. dNTP concentration

In the dNTP titration test for optimal PCR amplification, 12.5, 25, 50, 100, 200, and 400 μM of dNTPs were prepared for each PCR reaction solution. Elements other than dNTP were used by adjusting the reaction solution standard concentration described above and primer A20 was used.

When PCR was performed with the A20 primer, a total of eight bands were expressed between 0.5 and 6.5 Kb. Band 2 was expressed only in the 100 and 200 μM treatments, and the expression intensity of the band was more significant at the 100 μM treatment. Bands 3 and 4 were expressed in 50,100 and 200 μM treatments, and clearer bands were expressed in 100 and 200 μM treatments. Band 6 was expressed at the highest intensity in 50 and 100 μM treatments (see FIG. 3).

D. Taq Polymerase Concentration

In order to determine the proper amount of Taq polymerase that can perform the PCR amplification reaction of total DNA of ginseng most effectively, the PCR reaction solution was prepared by varying the Taq polymerase concentration by 0.2, 0.5, 1, 2, and 4 units.

Primer A08 was used and other additives other than Taq polymerase concentration were adjusted to the reaction solution baseline described above.

One band was expressed at 0.2 units of Taq polymerase concentration and six bands at 0.5 units. In 1.0 units and 2.0 units, the large bands were expressed at high intensity, while in 4.0 units, the intensity of the small bands was weak or disappeared (see FIG. 4).

E. Electrophoresis

After completion of the PCR reaction, 20 μl of the reaction solution for each treatment was mixed and loaded on a pre-prepared 1.5% agarose gel, followed by electrophoresis at 120 V for about 2 hours and 30 minutes, and ethidium bromide (0.5 μg / ml) as an electrophoresis buffer. 0.5 × TBE buffer (45 mM, Tris-borate, 1 mM EDTA) was used. After electrophoresis was completed, the DNA band pattern was observed and photographed on a UV transluminator.

Example 4 RAPD by Region of Ginseng

Tissue samples were taken from the pedoma species and tested. Tissues used in this test were DNA isolated from these tissues as root, stem and fruit, and then PCR was performed using primer A02, followed by electrophoresis on agarose gel to compare DNA band patterns. Three replicates were tested for each tissue.

Three bands were expressed between 0.5 and 1.1 Kb, and these three bands were expressed regardless of tissues, and the intensity of band expression was slightly higher in DNA extracted from the roots, but there was no difference (see FIG. 5).

Example 5 Preliminary Assay

Twenty random primers (Operon, Almeda, CA, USA) were used, and PCR was performed on the total DNA of nine Korean ginseng strains collected in Korea under the same conditions.

The PCR reaction solution was used as the reference solution, and the number of PCR was fixed at 50 cycles, and each primer was tested to select primers having distinct band patterns from RAPD.

In the present invention, the base sequence, molecular weight and RAPD amplification ability of each primer preliminarily assayed 20 10-mer primers are mentioned in Table 1.

Example 5 Selection of RAPD Markers of Korean Ginseng

To identify Korean, Japanese and Chinese cultivated regions of Korean ginseng, PCR was carried out using 9 primers selected from the roots of 25 domestically collected ginseng extracts. Was compared.

Among the nine primers in series, the band patterns expressed as a result of PCR of DNA extracted from 25 kinds of ginseng at home and abroad using A02 primers are shown in Table 5 and FIG. 6. Only Korean ginseng showed bands at 1.25Kb and 0.34Kb, which could be distinguished from other cultured ginseng.

PCR using the A04 primer resulted in 16 bands being expressed between 1.85 Kb and 0.15 Kb. The expression patterns of the bands are shown in Table 5 and FIG. The band was expressed at 0.53 Kb and the band was not expressed at 1.25 Kb, so it was distinguished from other cultures.

As a result of PCR using the A10 primer, 21 bands were expressed between 2.10 Kb and 0.28 Kb, and the bands are shown in Table 6 and FIG. 8.

All of the ginseng that was able to select RAPD markers could be distinguished with only one primer, and it was thought that the use of these primers, which required two or more RAPD bands for the collection of ginseng, could be practically used. Only Japanese ginseng expressed the 1.70Kb band, which could be distinguished from other cultures.

Claims (10)

Isolate and purify the DNA of ginseng that is the subject of mountain determination, and use the purified ginseng DNA as a template DNA to PCR the ginseng DNA in a reaction solution containing Mg ²⁺ ion, Taq DNA polymerase, dNTP, and primer. The amplified ginseng DNA was electrophoresed on an agarose gel containing ethidium bromide, and the agarose gel containing the electrophoresis-completed ginseng DNA was compared to the DNA bands on a UV transluminator. Mountain ginseng determination method of Korean ginseng by RAPD markers, characterized in that the selection step. The method according to claim 1, wherein the concentration of 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 of 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, Al1, A12, A13 and A20. Isolate and purify the DNA of Korean ginseng and Chinese ginseng from Korea and use the purified ginseng DNA as template DNA to amplify the ginseng DNA by PCR in a reaction solution containing Taq DNA polymerase, dNTP, and primer A02. Ginseng DNA was electrophoresed on agarose gel containing ethidium bromide, and agarose gel containing electrophoresis of ginseng DNA was selected to compare the pattern of DNA band on UV transluminator to select RAPD marker. Identification method of domestic Korean ginseng and Chinese ginseng by RAPD analysis. Isolate and purify the DNA of Korean ginseng and Gaeseong ginseng from Korea, and amplify the ginseng DNA by PCR in a reaction solution containing Taq DNA polymerase, dNTP, and primer A04 using the purified ginseng DNA as template DNA. Ginseng DNA was electrophoresed on agarose gel containing ethidium bromide, and agarose gel containing electrophoresis of ginseng DNA was selected to compare the pattern of DNA band on UV transluminator to select RAPD marker. Identification method of Korean ginseng and Gaeseong ginseng produced by the RAPD markers. Isolate and purify the DNA of Korean ginseng and Japanese ginseng from Korea and use the purified ginseng DNA as template DNA to amplify the ginseng DNA by PCR in a reaction solution containing Taq DNA polymerase, dNTP, and primer A10. Ginseng DNA was electrophoresed on an agarose gel containing ethidium bromide, and agarose gels containing electrophoretic ginseng DNA were compared on the UV transluminator to select RAPD markers. Identification method of domestic Korean ginseng and Japanese ginseng by RAPD marker, characterized in that the configuration. The primer according to any one of claims 1 to 10, wherein the primer is replaceable with primers used in the identification method of Korean ginseng and has a similar nucleotide sequence and has high complementarity.