KR20170024388A - Markers for the differentiation of Polygonum multiflorum Thumberg, Cynanchum wilfordii Max. Hemsl, Cynanchum auriculatum Royle ex Wight - Google Patents
Markers for the differentiation of Polygonum multiflorum Thumberg, Cynanchum wilfordii Max. Hemsl, Cynanchum auriculatum Royle ex Wight Download PDFInfo
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Abstract
More particularly, the present invention relates to a marker specific for the discrimination of Hashuo, Bacillus subtilis and Bacillus subtilis using single nucleotide polymorphism, more specifically, a specific primer set derived from TrnL, TrnF and matK gene fragments present in chloroplasts The present invention relates to a method for distinguishing a hyssopoil, a bass gossypii and a bass gossypiper including information on the breed, mixing ratio and hybrids of the bass gass, the bass gass, and the bass gass.
Description
More particularly, the present invention relates to a marker specific for the discrimination of Hashuo, Bacillus subtilis and Bacillus subtilis using single nucleotide polymorphism, more specifically, a specific primer set derived from TrnL, TrnF and matK gene fragments present in chloroplasts The present invention relates to a method for distinguishing a hyssopoil, a bass gossypii and a bass gossypiper including information on the breed, mixing ratio and hybrids of the bass gass, the bass gass, and the bass gass.
Polygonum multiflorum Thumberg, Cynanchum wilfordii Max. Hemsley, and Cynanchum auriculatum Royle ex Wight are among the most mixed and distributed herbal medicines in Korea for various complicated reasons as follows.
First, the origin of the plant is defined differently according to the country. For example, the Chinese medicinal herb of the Cynanchum wilfordii Max. Hemsley, Cynanchum auriculatum Royle ex Wight, and C. bungei Dence are the origins of Baeksoo However, only Korean herbal medicines prescribes only the origins of Cynanchum wilfordii Max. Hemsley (Kim et al., Kor J. Med. Crop Sci. 13: 268- 272, 2005; Hwang et al., Kor J. Med. Crop Sci. 20: 136-139, 2012).
Second, different countries use different names. For example, in the case of sewage mistakes, Korea defines Polygonum multiflorum Thumberg as Cynanchum wilfordii Max. Hemsley, but North Korea classifies them as enemy Soso and White Soso, China and Japan have defined the enemy sewage as Polygonum multiflorum Thumberg, but Cynanchum wilfordii Max. Hemsley or White Sewer is not specified separately. For the origins of Polygonum multiflorum Thumberg, Korea, North Korea, China and Japan commonly define Polygonum multiflorum as Polygonaceae (Polygonaceae), and Baekhasuo is the most common species in Korea and North Korea Asclepiadaceae (Cynanchum wilfordii) is the origin of the plant. However, the name of the plant, which is also called the mockery of Korea, is called the big ridicule in Korea, and the North is called the roots of the newborn (Choi et al., Kor J. Ori. Med. 9: 81-89, 2003). Especially, in the distribution market in Korea, the sewage is divided into the enemy Soso (the enemy Soso) and the bag Soso (the Soso), and the same treatment is being done to deteriorate the soul and misuse.
Third, since dry root roots of these medicinal crops are mixed and distributed as herbal medicines, it is difficult for experts to easily distinguish them. In particular, it is very difficult to distinguish between the two types of bamboo shoots imported from China, and the shape and color of bamboo shoots are similar to those of bamboo shoots in Korea. Fourth, Baeksoo Owai Yooobo is confusing to farmers or researchers who grow herbs because the mature fruit or seeds of Metaplexis japonica Makino are morphologically very similar. In addition, according to farmers or experts who grow these herbs, it is known that there are interspecific hybrids between this leafhopper and white pickle.
The status of the development of technology to distinguish them is still insufficient, and more research is needed in the basic research stage. Especially, there is a desperate need for precise identification technology of those which are distributed in the form of dried roots used as raw materials for herbal medicines. It has been reported that the technique of dividing the external and internal forms of Hashuo and Bacillus by using an optical microscope has been reported, but it is difficult to apply to dry roots distributed in the market. It is necessary to develop a technology for discriminating them by using a nucleotide sequence of DNA which is a stable compound which is not chemically easily broken.
Much research has been done at the beginning by using RAPD and Sequence characterized amplified regions (SCARs) techniques for the identification of medicinal crops by molecular biology. Despite the advantages of RAPD technology being rapid, inexpensive, and extremely low in the amount of sample required for analysis, low reproducibility is a major disadvantage (Penner et al., PCR Methods Appl. 2: 341-345, 1993; Hosokawa et al., Planta Med. 66: 270-272, 2000; Agrawal et al., Plant Biotech. Rep. 1: 207-210, 2007; Shcher & Carles, Planta Med. 74: 603-623, 2008).
To overcome these problems, Moon et al. (Plant Biotech Rep. 4: 1-7, 2010, Korean Patent, Laid-open Patent No. 10-2009-0123113) applied the SCARs technology to discriminate between Hashuo, Markers have been developed and reported. This technique first clones the bands that are specific to Hashuo, Baishuo, and Baiji using the RAPD technology, and then determines primers that can specifically amplify these genes. These three herbal medicines We have developed a kit that can identify the mixed samples at one time by PCR.
Thus, a method using DNA markers that are not affected by external factors to accurately and quickly identify plant species has recently been actively studied for major crops. Unlike the morphological characteristics such as the shape and size of the plant, the method using the DNA marker can distinguish the species of the plant without being influenced by the external environment, and it is possible to accurately discriminate the number of the available markers
Accordingly, the present inventors have found that a part of TrnL and TrnF genes present in the chloroplast to distinguish between Hwasoo, Baechuo, and Bacillus; A fragment comprising intergenic DNA; And matK gene fragments were amplified by PCR and analyzed for their nucleotide sequences. Primers containing the SNP-appearing regions were developed, and markers were identified to identify the origin of each primer. In order to solve the problem of misuse and misuse of Bacillus subtilis, we developed a marker that can measure up to the blending ratio as well as the qualitative markers compared with existing discrimination markers. In order to identify interspecific hybrids between Bacillus subtilis and Bacillus subtilis, ARMS- The present invention has been accomplished by developing a discriminator capable of distinguishing each line in the ITS (internal transcribed sequence) I and II regions by applying the PCR technique.
It is a principal object of the present invention to provide a marker composition for the determination of diatomaceous earth, bamboo shoots and bamboo shoots comprising a specific set of primers.
Another object of the present invention is to provide a method for discriminating the variety, mixing ratio, and hybridization status of Hashuo, Bacillus auratus and Bacillus subtilis using the marker composition for distinguishing the Hacilus, Bacillus subtilis, and Bacillus subtilis.
It is another object of the present invention to provide a kit for distinguishing dysentery, white mushroom and mushroom containing the marker composition.
In order to solve the above problems, according to one embodiment of the present invention,
SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10;
SEQ ID NOS: 11 and 12, SEQ ID NOS: 13 and 14;
SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22; And
SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26
Wherein the primer set comprises at least one primer set selected from the group consisting of primers set forth in SEQ ID NO:
In this case, the primer sets of SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, and SEQ ID NOs: 9 and 10 are for discriminating the strains of Sasao, Bacillus aurora and Bacillus subtilis using single base polymorphism (SNP) 11 and 12, and SEQ ID NOs: 13 and 14 are preferably used in the HRM curve pattern analysis for the identification of the strains of Drosophila, Leucocephalus and Leucocephalus, and these primer sets can be used in known PCR techniques.
In addition, the primer set of SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, and SEQ ID NOs: 21 and 22 are for discriminating the mixing ratios of HASO, Can be used to obtain information on the mixing ratio.
In addition, the primer sets of SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26 are for hybrid identification of Bacillus subtilis and Bacillus subtilis. Particularly, the primer set is an ARMS-PCR technique including the SNP of nuclear ITS DNA Can be used to obtain information on whether or not the hybrid is hybridized.
Therefore, in another embodiment of the present invention, the primer set; And a reagent for carrying out an amplification reaction.
In this case, it will be apparent that a substance other than the primer set of the present invention can be appropriately controlled by those skilled in the art. For example, the reagent for carrying out the amplification reaction may include a DNA polymerase, dTNPs, .
As another embodiment, the present invention provides a method for distinguishing dewatering, blooming and blooming, including the following.
Isolating the genomic DNA from at least one sample selected from the group consisting of Hashuo, Baishuo, and Bifidobacterium;
Performing amplification reaction using the obtained DNA as a template using the above-described primer set to prepare an amplification product; And
Detecting the amplification product.
As described above, a primer set suitable for obtaining information on the variety, mixing ratio, and / or hybridization of Sasa, Pseudomonas aeruginosa and Bacillus subtilis among the above-described primer sets can be selected and carried out.
As described above, the present invention basically comprises:
SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10;
SEQ ID NOS: 11 and 12, SEQ ID NOS: 13 and 14;
SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22; And
SEQ ID NOS: 23 and 24, SEQ ID NOS: 25 and 26, and a variety of uses thereof.
The present invention can identify not only qualitative but also quantitative mixing ratios by providing a SNP discriminating primer for discriminating the origins of haploid, bovine anemia, and bistrous, and furthermore, using ARMS-PCR or HRM curve pattern analysis By establishing the discrimination markers and their conditions, various types of horn, used or mixed processed products can be grasped easily.
In addition, these crosses can be discriminated by using the discriminant markers produced using the SNPs present in the ITS Ⅰ and Ⅱ of the present invention for interspecific crosses between this and the other.
FIG. 1 is a schematic illustration of a specific primer of an aldehyde. FIG. 1 shows a region in which trnL-trnF genetic fragments of Drosophila, Bacillus subtilis, and Bacillus subtilis are compared.
FIG. 2 is a diagram showing the position of a primer for discriminating between a primate and a primate.
Fig. 3 shows the results of discrimination of strains collected from each collection area using a) aqua, bacillus, and cis-specific primers prepared from the nucleotide sequences of trnL-trnF or matK genes.
FIG. 4 is a schematic diagram of a primer design for HRM analysis using the trnL-trnF gene fragment.
5 is a schematic diagram of a primer design for HRM analysis using a matK gene fragment.
6 shows the result of PCR analysis using a primer for HRM curve analysis.
FIG. 7 shows the results of the HRM curve pattern after amplifying each gene fragment using the genomic DNA of Hashuo, Bacillus subtilis, and Bacillus subtilis using matK (left) and trnL-trnF (right) to be.
FIG. 8 is a result of discrimination by comparing and analyzing HRM curve patterns of Hsuo, Hwasooo, or Hwasooo after using a discriminant primer for HRM curve amplification to amplify a trnL-trnF gene fragment.
FIG. 9 is a graph showing the results of mixing ratio analysis by qPCR in a sample in which the genomic DNA of Bacillus subtilis (Cw) and Bacillus subtilis (Ca) are arbitrarily mixed.
FIG. 10 shows the positions of WIL and AUR specific primers using SNPs irradiated in the ITS I and II regions existing in the ribosomal DNA transcription units present in the nucleus, and the primers for ARMS-PCR Of the base sequence.
Fig. 11 shows the results of discrimination of strains using the primers (b) and (C) biopsy specimens prepared using the SNPs in the ITS region existing in the nucleus genome.
The terms used in the present invention are defined as follows.
"Genetic polymorphism" refers to a case where a genetic variation occurs in at least 1% of the population. The insertion, deletion, or substitution of a single nucleotide in DNA is called single nucleotide polymorphism (SNP).
The term "polymorphism " refers to a sequence in a sequence of genes that varies within a cluster. Polymorphisms consist of different "alleles ". The arrangement of this polymorphism can be confirmed by its position in the gene and the different amino acids or bases found therein. These amino acid variations are the result of two possible mutant bases, C and T, which are two different alleles. Since the genotype is composed of two different distinct alleles, any of the various possible variants can be observed in any individual (e. G., CC, CT or TT in this example). The individual polymorphisms are also known to those skilled in the art and are used in, for example, the Single Nucleotide Polymorphism Database (dbSNP) of the Nucleotide Sequence Variation of the nucleotide base mutations available on the NCBI website. ("Reference SNP", "refSNP", or "rs #").
"Single nucleotide polymorphism (SNP) refers to the diversity of DNA sequences that occur when a single nucleotide (A, T, C or G) in the genome is different between members of a species or between individual chromosomes For example, differences in single bases such as three DNA fragments of different individuals (eg, AAGT [A / A] AG, AAGT [A / G] AG, and AAGT [G / Within a population, SNPs are defined as minor allele frequencies (MAFs), which are the frequencies of allele frequencies (MAFs). (Deletion) or addition (insertion) of a polynucleotide sequence in the polynucleotide sequence of the polynucleotide sequence of the polynucleotide sequence of SEQ ID NO: It is possible to cause a change in the translation frame.
"Markers for the differentiation" is a substance that makes it possible to distinguish plants by information such as variety, blending ratio, and whether or not they are hybrid. In the present invention, the term "Markers for the differentiation" And organic biomolecules such as polypeptides or nucleic acids (e.g., mRNA), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, polysaccharides, etc.)
The term "functional equivalents" refers to, for example, one or more substitutions, deletions or additions from a reference sequence, a net effect that does not result in various functional dissimilarities between the reference and subject sequences, ≪ / RTI > and the nucleotide sequence of the mutated mutant sequence. Preferably, the nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, most preferably about 95% identity. For purposes of the present invention, sequences having substantially equivalent functions are treated as substantial equivalents.
"Nucleic acid" is meant to include any DNA or RNA, such as chromosomes, mitochondria, viruses and / or bacterial nucleic acids present in a tissue sample. Includes one or both strands of a double-stranded nucleic acid molecule and includes any fragment or portion of the intact nucleic acid molecule.
"Gene" means any nucleic acid sequence or portion thereof that has a functional role at the time of protein coding or transcription, or in the control of other gene expression. The gene may consist of only a portion of the nucleic acid encoding or expressing any nucleic acid or protein that encodes the functional protein. The nucleic acid sequence may comprise an exon, an intron, an initiation or termination region, a promoter sequence, another regulatory sequence, or a gene abnormality within a particular sequence adjacent to the gene.
"Primer" refers to an oligonucleotide sequence that hybridizes to a complementary RNA or DNA-targeted polynucleotide and serves as a starting point for the stepwise synthesis of a polynucleotide from a mononucleotide by the action of, for example, the nucleotidyltransferase that occurs in the polymerase chain reaction .
"Protein" also includes fragments, analogs, and derivatives of proteins that retain essentially the same biological activity or function as the reference protein
"Label" or "label " means a compound or composition that facilitates the detection of a reagent, such as a reagent conjugated, conjugated, conjugated, or fused to a nucleic acid probe or antibody. The label may itself be detected (e. G., A radioactive isotope label or a fluorescent label), in the case of an enzyme label, to catalyze the chemical modification of the detectable substrate compound or composition.
"About" means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.
Throughout this specification, the words " comprising "and" comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.
Hereinafter, the present invention will be described in detail.
The object to be discriminated in the present invention includes the variety and related information of Polygonum multiflorum Thunberg, Cynanchum wilfordii Max. Hemsl. And Cynacum auriculatum Royle ex Wight.
Hashuo defines the root of Polygonum multiflorum Thunberg, a perennial herb of Polygonaceae, as dry (National Institute of Oriental Medicine, Department of Herbal Medicine, Graduate School of Oriental Medicine, Seoul, Younglim Publishing House, 1991, 583-584) (Cynanchum wilfordii Max. Hemsl.) Of Asclepiadaceae (Shin Min-gyo, Clinical Herbal Medicine, Seoul, Younglim Publishing House, 1986, 218-221). In China, however, it has been reported that the roots of Cynacum auriculatum Royle ex Wight and Cynanchum bungei Decne are used as white radish, while large radishes are used as other medicinal materials, such as oxygen (隆新民, 中國 药材學, Taipei, Keiji Bookstore, 1974, 515-517). At present, Korean traditional medicinal herb distribution market is divided into the red sea water (red sea water) and the white sea water (white sea water) and treats them equally. Especially, in case of white water, it is wrongly known as a red sea water imported from China. This is a completely different plant which is not listed in the standard of herbal medicine. It is reddish brown and has a starfish-shaped morphological characteristic. It is easily distinguishable from white cedar (white cedar), but in the case of cedar cedar, its shape and color are white And it is very difficult to distinguish it with the naked eye.
There is almost no effective method for distinguishing between the above-mentioned Hashuo, Bacillus subtilis, and Bacillus subtilis, and it is general that the external and internal forms are still distinguished by using a magnifying glass, a microscope or an optical microscope.
The present inventors have found that a part of the TrnL and TrnF genes present in the chloroplast to distinguish between the hyssop, A fragment comprising intergenic DNA; The matK gene fragment was amplified by PCR, and the nucleotide sequence was analyzed to prepare a primer containing the SNP region. In order to discriminate these strains by applying HRM technology, primers designed to center SNPs were developed and compared with HRM curve patterns using these primers, the conditions suitable for discrimination were established. Furthermore, in order to solve the problem of misuse and misuse of Lee Soo Woop and Bae Soo Oh, which have recently become a problem, we have developed a technology capable of measuring not only qualities but also mixing ratios as compared with existing discrimination markers. In order to confirm the interspecific hybridization between the bovine spongiform encephalopathy and Bacillus subtilis, the transcriptional unit of the ribosomal DNA present in the nucleus was amplified separately to identify the SNP, and then, in each of the internal transcribed sequence I and ITS II regions, We have developed discriminant markers that can distinguish phylogenies, and have identified these strains by applying the ARMS-PCR technique, and have also established a technique for discriminating transcripts of the interspecific hybrids in combination with the chloroplast discrimination markers.
Therefore, the present invention relates to a marker for discriminating a variety using a polymorphism of a chloroplast gene of a hapthouse, a papilloma, and a bacillus grandiflorum, and a use thereof, and more particularly, Therefore, it is possible to provide a variety of information of Hashuo, Baishuo, and Leebuchi, and it can be usefully used for prevention of misuse and miscellaneous distribution of goods.
<Marker>
The present invention relates to TrnL and TrnF genes present in chloroplasts in one aspect; The present invention relates to markers for distinguishing daphniaceous, mulberry, and mulberry leaves containing a specific primer set derived from matK gene, and uses thereof.
The base sequence constituting TrnL, TrnF, and matK can be obtained from a known one, for example, a well-known GeneBank DB, but is not limited thereto and includes functional equivalents thereof.
The selection and application of significant markers determines the reliability of the diagnostic results. Significant markers are those markers that are highly reliable with high validity and consistency in repeated measurements. The markers for distinguishing the Hashuo, Bacillus thuringiensis and Bacillus thuringiensis according to the present invention preferably have a constant sequence which is specifically included according to the origin (variety), blending ratio or hybrids of Drosophila, Bacillus subtilis and Bacillus subtilis Primers that have the same results in repeated experiments and are highly reliable markers with little or no chance of producing false results when compared to the control group. Therefore, the results obtained on the basis of the results obtained using the significant markers of the present invention can be reasonably reliable.
To confirm the single nucleotide polymorphisms in the chloroplast genes, the present inventors prepared primers using the nucleotide sequence information of known genes for the amplification of TrnL-TrnF gene fragment and matK gene fragment.
In one embodiment, the trnL-trnF gene fragment was prepared from the nucleotide sequences of GenBank accession number (JX028242.1) and matK gene fragment (GU373529.1), respectively.
Therefore, the present invention relates to TrnL and TrnF genes present in chloroplasts in one aspect; specific primer set derived from the matK gene.
More specifically,
SEQ ID NOS: 11 and 12, SEQ ID NOS: 13 and 14;
SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22; And
SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26, and a primer set selected from the group consisting of primers set forth in SEQ ID NOs: 25 and 26. The term " primer set "
The distinction of the above-mentioned Hashuo, Bacillus oryzae and Bacillus anthracis contains all information on the variety, mixing ratio and whether or not hybrid.
Preferably, as one embodiment (I) of the present invention,
A primer set selected from the group consisting of primers set forth in SEQ ID NOS: 5 and 6, SEQ ID NOS: 7 and 8, SEQ ID NOS: 9 and 10, and a primer set selected from the group consisting of SEQ ID NOs: .
Preferably, as another embodiment (II) of the present invention,
A marker composition for HRM curve pattern analysis for the identification of a variety of strains of Drosophila, Leucocephalus and Leucovorus, comprising at least one primer set selected from the group consisting of primers set forth in SEQ ID NOs: 11 and 12 and SEQ ID NOs: 13 and 14 .
Preferably, as yet another embodiment (III) of the present invention,
A primer set selected from the group consisting of primers set forth in SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, and SEQ ID NOs: 21 and 22, The present invention relates to a marker composition characterized in that it is for discriminating a blending ratio of an ash.
Preferably, as another embodiment (IV) of the present invention,
A primer set selected from the group consisting of primers set forth in SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26, and a primer set selected from the group consisting of SEQ ID NOs: .
At this time, the polynucleotide or its complementary polynucleotide constituting the sequence number may be composed of 10 or more, preferably 10 to 100, more preferably 10 to 50 contiguous bases.
In particular, primers represented by SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10; And the polynucleotide according to the primers represented by SEQ ID NOs: 11 and 12, and SEQ ID NOs: 13 and 14, or a complementary polynucleotide thereof, is a polymorphic sequence. A polymorphic sequence refers to a sequence comprising a polymorphic site representing a single base polymorphism in the nucleotide sequence. A polymorphic site is a site in a polymorphic sequence where a single base polymorphism occurs.
The single nucleotide polymorphism (SNP) may be included in the coding sequence of the gene, in the non-coding region of the gene or in intergenic regions between the genes. SNPs in the coding sequence of a gene do not necessarily cause changes in the amino acid sequence of the target protein due to the degeneracy of the genetic code. SNPs that form the same polypeptide sequence are called synonymous (also called silent mutations) and SNPs that form other polypeptide sequences are said to be non-synonymous. Non-consensual SNPs can be missense or nonsense, and mismatch changes produce other amino acids while nonsense changes form non-mature termination codons. SNPs that are not in the protein-coding region can induce gene silencing, transcription factor binding, or non-coding RNA sequences.
The high-resolution melting (HRM) technique is applied to the primers represented by SEQ ID NOs: 11 and 12, and SEQ ID NOs: 13 and 14 to design the SNP to be centrally located to identify these lines.
Identification of genotypes of SNPs can be performed by sequencing analysis, sequencing analysis using an automated sequencer, pyrosequencing, microarray hybridization, restriction fragment length polymorphism (PCR), single strand conformation polymorphism, PCR-SSO (specific sequence oligonucleotide), ASO (allele specific oligonucleotide) hybridization method using PCR-SSO method and dot hybridization method, TaqMan-PCR method, MALDI-TOF / MS method, rolling circle amplification ), High resolution melting (HRM), primer extension, Southern blot hybridization, dot hybridization and the like.
Recently, studies on the development of a barcode using a technique using a single nucleotide polymorphism (SNP) of a specific gene have been actively conducted (He et al., J. Med. Plants Res. 5: 3653-3659, 2011; Herbology 27: 37-42, 2012; Moon et al., Kor. J. Herbology 28: 75-84). In addition, a number of studies have been carried out to develop PCR primers capable of identifying specific species after identifying SNPs, and to identify species with and without specific amplification bands by PCR. However, most single base differences are difficult to discriminate by PCR In order to compensate for the problem, an amplification refractory mutation system (ARMS) -PCR technique may be applied in which two mismatch primers are generated by modifying the second or third nucleotide adjacent to the SNP to perform PCR.
Further, in the present invention, a high resolution melting curve analysis method can be further performed as a quicker and more sensitive technique (Jaakola et al., Food Chem. 123: 492-500, 2010; Kalivas et al. Mol. Biol., Rep. 41: 5147-5155, 2014, Kim et al., Genet. Resour. Crop Evol. 60: 413-425, 2013; Mader et al., Anal. Biochem. 409: 153-155,2011).
The HRM technique is a highly sensitive primer designed to center a single SNP, amplifying a target gene fragment of about 200 bp in size, and then gradually increasing the temperature to make the double strand completely single stranded It is a technique to distinguish a strain or a variety by the difference of a melting curve curve which is obtained by measuring the degree of color development which occurs when a fluorescent dye reagent (for example, EVA green) inserted in a double helix turns into a single strand in a reaction process.
In one embodiment of the present invention, it was confirmed that the species can be distinguished by showing different HRM curve patterns for the three species of matsutake, papilloma, and bivalvia in both matK and trnL-trnF fragments. In particular, in the case of the trnL-trnF fragment, the Hashuo showed a totally different pattern from the bovine spongiform encephalopathy and bovine spongiform encephalopathy.
As described above, the analysis of HRM curve analysis pattern of the present invention makes it easy to distinguish three kinds of medicinal materials of Hashuo, Baishuo, and Leejyeobo, which are mixed in various forms such as dried roots or powdered state.
In addition, the marker of the present invention can measure not only the qualities but also the mixing ratios in comparison with existing discrimination markers in order to solve the problem of misuse and misuse of the two-headed male and female male.
Preferably, the amount of DNA is determined by quantitative PCR (qPCR) using primers set forth in SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, A sample in which the genomic DNA of the bovine calf (Ca) is arbitrarily mixed can be analyzed.
In one embodiment, the genomic DNA was mixed at the blending ratio described on the x-axis, and the DNA fragment was amplified with each specific primer to express the relative amplification value on the y-axis. The intact actin gene and the trnL-trnF gene were used as a control After the amplification, the relative DNA content was compared with the corrected value. As a result, it was confirmed that the ratio was intentionally mixed.
In addition, the marker of the present invention can confirm whether there is interspecific hybridization between this leafhopper and Bacillus subtilis.
A discrimination marker capable of discriminating the respective strains in the ITS (internal transcribed sequence) I region after confirming the SNP by amplifying the transcription unit of the ribosomal DNA present in the nucleus, preferably SEQ ID NOs: 23 and 24, The primers set forth in SEQ ID NOs: 25 and 26 can be developed to identify these strains by applying the ARMS-PCR technique, and to identify the transcripts and transcripts of interspecific hybrids in combination with the chloroplast identification markers described above.
"ITS sequence" means a region on gDNA comprising part or all of the ITS sequence (ITS I & ITS II) and including all or part of the sequence of rDNA (18S, 5.8S & 28S) Depending on the context, an amplification product obtained from the region on the gDNA, a transcript, a cDNA obtained from the transcript, or an amplification product of the cDNA.
The results of the SNP polymorphism can be statistically processed using a statistical analysis method commonly used in the art. For example, Student's t-test, Chi- continuous variables, categorical variables, odds ratios, and 95% confidence intervals, obtained through linear regression analysis, linear regression line analysis, and multiple logistic regression analysis, % Confidence interval, and so on.
The primer set of the present invention can be used to hybridize the probe so that the base of the polymorphic site can be specifically identified by a known method. Here, hybridization is usually carried out under stringent conditions, for example, a salt concentration of 1 M or less and a temperature of 25 ° C or higher.
In the present invention, a probe means a hybridization probe, and means an oligonucleotide capable of binding sequence-specifically to a complementary strand of a nucleic acid. The hybridization conditions show a significant difference in the intensity of hybridization between alleles and should be sufficiently stringent to hybridize to only one of the alleles. Preferably, the probe of the present invention aligns with the polymorphic site of the polymorphic sequence. This can lead to good hybridization differences between different allelic forms.
The probe may be used in a kit or a prediction method such as a microarray for detecting alleles and obtaining information on the distinction of Hwaseo, Baekgoo, and bovine corpus callosum.
Important probes can be labeled for detection and can be labeled, for example, as radioactive isotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. It is well known in the art to appropriately label such a probe, and can be carried out by a conventional method.
In the present invention, the appropriate length of the primer may vary depending on the purpose of use, but it is generally composed of 15 to 30 bases.
The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. The primers can be hybridized to a DNA sequence containing a polymorphic site to amplify a DNA fragment containing the polymorphic site. The primer of the present invention can be used in a kit such as a microarray for predicting the survival prognosis of lung cancer and a prediction method by detecting alleles.
The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with analogs, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
In another embodiment, the present invention relates to a microarray for discriminating daphnia, leucorrhoea, and leucocephalus including the marker composition.
The microarray may be composed of a conventional microarray except that it comprises the polynucleotide, primer or probe of the present invention.
The hybridization of nucleic acids on a microarray and the detection of hybridization results are well known in the art. The detection may be performed, for example, by labeling the nucleic acid sample with a labeling substance capable of generating a detectable signal including a fluorescent substance, such as Cy3 and Cy5, and then hybridizing on the microarray, The hybridization result can be detected.
The microarray method can simultaneously study the RNA expression of a plurality of genes, thereby enabling a more comprehensive insight into the molecular basis for the identification of Hashuo, Bacillus, and Bacillus.
In a similar aspect, the present invention also relates to a kit for distinguishing daphniae, leucocephalus, and leucocephalus including the marker composition as another example.
The kit of the present invention can be used for distinguishing the polleny regions (SNP) and the like of a specific sequence by using the above-described primer set, which is a marker for discriminating between Hwaseo, Baechuo, have.
The kit for the identification of the Hashuo, Bacillus subtilis and Bacillus anthracis of the present invention may contain not only the primers of the present invention but also one or more other component compositions, solutions or devices suitable for the analysis method.
In one embodiment, the kit of the present invention may be a kit containing the necessary elements necessary for performing PCR. The PCR kit may contain test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq polymerases and reverse transcriptase enzymes, as well as specific polynucleotides, primers or probes specific for the SNPs. , DNase, RNAse inhibitors, DEPC-water and sterile water, and the like.
<How to identify and provide information>
In another aspect, the present invention relates to a method for distinguishing between a primer marker and a primer marker, and more particularly, to a method for identifying a hybrid primer marker, It is possible to provide a variety of information of Baeksoo Oh and Lee Yeol Woobo so that they can be usefully used for prevention of misuse and miscellaneous distribution of goods.
In one embodiment, the method of the present invention comprises
Isolating the genomic DNA from at least one sample selected from the group consisting of Hashuo, Baishuo, and Bifidobacterium;
Amplifying the amplified product by performing amplification reaction using the obtained DNA as a template and using the primer set of
Detecting the amplification product;
, And a method for distinguishing between the two species
The method of discrimination is particularly concerned with examining the characteristics of a particular expression pattern according to each of the Hashuo, Bacillus, and Bacillus grandiflorum, and the methods disclosed herein are useful for obtaining appropriate and useful data and information for the identification of the plant Efficient, and cost-effective means. ≪ RTI ID = 0.0 >
Genomic DNA isolation is not particularly limited as long as it is a method used for isolating genomic DNA from a plant. Preferably, the plant sample is pulverized and / or disrupted to produce a powder, and then a kit for plant DNA extraction is used Genomic DNA can be isolated.
The method of amplifying the target sequence is not particularly limited as long as it is a method of amplifying the target sequence in the method of discriminating the species of the plant. For example, a polymerase chain reaction (PCR), a ligase chain reaction, nucleic acid sequence-based amplification, a transcription-based amplification system, , Amplification with strand displacement amplification or Qβ replicase, or any other suitable method for amplifying nucleic acid molecules known in the art
Among these methods, the polymerase chain reaction (PCR) is a method of amplifying a target sequence from a primer set that specifically binds to a target sequence using a polymerase. Such PCR methods are well known in the art and commercially available kits can be used
In addition, the amplified target sequence may be labeled with a detectable labeling substance. The labeling substance is not particularly limited as long as it is ordinarily used as a primer-labeled substance. For example, fluorescent, phosphorescent or radioactive materials can be used, and ethidium bromide or Hoechst 33258 dyes can preferably be used. When the radioactive isotope such as 32 P or 35 S is added to the reaction solution, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive.
Next, the step of detecting the amplification product will be described
The method of detecting the amplification product is not particularly limited as long as it is a method of detecting an amplified product, but it is preferably performed by capillary electrophoresis, DNA chip, gel electrophoresis, radioactive measurement, fluorescence measurement or phosphorescence measurement have.
In addition, according to one embodiment of the present invention, the method for distinguishing between Hosuo, Bacillus subtilis, and Bacillus subtilis may further comprise determining the amplified product of the Hacsus, Bacillus subtilis, and Bacillus subtilis, the mixing ratio, .
As described above, the present invention includes all uses using discrimination markers of Hashuo, Baishuo, and Bifida.
< Example >
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.
Unless otherwise indicated, nucleic acids are recorded in a 5 'to 3' orientation from left to right. The numerical ranges recited in the specification include numerals defining the ranges and include each integer or any non-integral fraction within a defined range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein.
material
(Gyeongnam), Chungbuk Agricultural Research and Extension Service (Ochang), National Horticultural Research Institute (Gospel), Kyungnam Agricultural Research and Extension Service (Gyeongnam), Gyeongbuk Agricultural Research and Extension Service ) (See Table 1).
Example 1: Genome-specific genome DNA detach
Genomic DNA was isolated according to the experimental method provided by the company using GeneAll's ExgeneTM Plant SV Kit using powder obtained by rapidly freezing a certain amount of roots, seeds, leaves, etc. in liquid nitrogen in each collected line.
In order to investigate the purity of the purified DNA, a predetermined amount of fractions were electrophoresed on 1% agarose gel and confirmed by using a micro-spectrometer (BioPrince, SD-2000, Gangwon, South Korea) , The degree of contamination of RNA and proteins was checked by examining the absorbance of A 260 / A 280 in the range of 1.8-2.2 and the range of A 234 / A 260 within the range of 0.5-0.8. Respectively.
Example 2: Amplification and sequencing of chloroplast genes
To amplify the TrnL-TrnF gene fragment and the matK gene fragment in order to confirm the single nucleotide polymorphism in the chloroplast gene, a primer was prepared as summarized in Table 2. The trnL-trnF gene fragment was prepared from the nucleotide sequences of accession number (JX028242.1) and matK gene fragment (GU373529.1) in GenBank.
No
.
()
(
bp
)
(SEQ ID NO: 1)
(SEQ ID NO: 2)
(SEQ ID NO: 3)
(SEQ ID NO: 4)
The amplification of the gene was minimized by using a buffer solution for PCR reaction and Solg TM Pfu DNA polymerase provided by SolGent (Seoul, South Korea), which could be caused by polymerization reaction.
50 ng of pure DNA and 10 pmoles of primers were mixed with each other and denatured at 94 ° C for 5 minutes. The DNA was denatured at 94 ° C for 30 seconds, at 60 ° C for 10 seconds, at 72 ° C for 30
Each amplified gene fragment was cloned using the T-Blunt ™ PCR Cloning Kit (Solgent) and sequenced by SolGent Company (Seoul, South Korea). However, since the mutation may occur during the cloning process, the amplified bands were eluted from the agarose gel without cloning, and direct sequencing was performed simultaneously after the restriction of net cleavage. In addition, the final confirmed single nucleotide polymorphism (SNP) was tested by performing at least 5 repetitions.
Example 3: Sewage , Baewooo Oh , Bovine To determine the origin Marker Development
It is classified as Cynanchum wilfordii, Cynanchum auriculatum in the case of Baishuo, and Cynanchum wilfordii in the case of Baishuo and Yibeobu asphyxia. It is called and distinguished by other species.
As a result of comparison of their nucleotide sequences, as expected, there was a great difference in the number of Hashuo compared to that of Bacillus auris and Bacillus thuringiensis. ), Deletion, and so on.
Based on these results, we designed each species specific primer of red mulberry, white mulberry and white mulberry.
FIG. 1 is a schematic representation of a specific HSA-specific primer. In the region where the trnL-trnF genomic fragment of Drosophila, Bacillus subtilis, and Bacillus subtilis is compared, the thymine base (T) and the adenine base (A) A reverse primer was prepared using a primer (trnLF-R) used to amplify the full-length trnL-trnF gene fragment described in Table 2,
Thus, in the case of the red seaweed, a gene fragment having a size of 829 bp was amplified.
As shown in FIG. 2, the primers used to discriminate between the primers were trnLF-F described in Table 2 as the forward primers, and the nucleotide sequences of the primers reverse primer (WILtrnLF-R, AURtrnLF-R) were designed in such a manner that a base containing single nucleotide polymorphism was located at the 3'-end of the nucleotide sequence.
Therefore, each gene amplified fragment was 828 bp. Table 3 summarizes the primer information for the discrimination of Hashuo, Baishuo, and Yiwu.
No
.
(° C)
(
bp
)
( Cynanchum
wilfordii Max.
Hemsley)
(SEQ ID NO: 5)
828
(SEQ ID NO: 6)
( Cynanchum
auriculatum
Royle ex
Wight)
(SEQ ID NO: 7)
(SEQ ID NO: 8)
( Polygonum
multiflorum
Thumberg)
(SEQ ID NO: 9)
829
(SEQ ID NO: 10)
Using the primers shown in Table 3, the PCR conditions for amplifying Hwasoo, Baechuo, and Bacillus thuringiensis were the same as those of the trnL-trnF gene described in Example 1, except that the annealing temperature was set to a value of Tm .
As a result, as summarized in FIG. 3, Hashuo and Gyeongnam Herbal Medicine Agricultural Cooperative, which were sold in Chungbuk Agricultural Research and Extension Service (Ochang), National Horticultural Research Institute (Voice), Gyeongnam Agricultural Research and Extension Service (Ochang), National Institute of Horticulture (Voice), Gyeongbuk Agricultural Research and Extension Center (Bonghwa), Gyeongbuk Agricultural Research and Extension Service (Bonghwa), Gyeongbuk Agricultural Research and Extension Service Were amplified from the single-stranded DNA fragments that could be identified as the species-specific primers.
Example
4 :
HRM
(
High
resolution
melting
)
curve
Through pattern analysis
Sewage
,
Baewooo Oh
,
Lee Byung-Woo
System identification
The primers for analyzing HRM curves were designed based on the single nucleotide polymorphism patterns found by comparing the nucleotide sequences of trnL-trnF and matK genes present in the chloroplast genome.
FIG. 4 shows the positions of the primers prepared in order to amplify a short fragment of 236 bp from three species and to compare and analyze the patterns of the three kinds of HRM curves.
(HRM trnLF-F) was prepared by comparing the nucleotide sequences of the three trnL-trnF genes, namely, Hsu, Hsu, and Lee, The nucleotide sequence of this bacteriophage is 100% identical to that of Bacillus anthracis. The homology of this bacterium is low, so 10 bases at the 5 'end and 5 bases at the 3' end are identical, but the inner six bases are inserted, The three bases disagree. The reverse primer used was trnLF-R as described in Table 2.
FIG. 5 is a graph showing the positions of the primers prepared in the most homologous regions by comparing and analyzing the nucleotide sequences of three species to investigate HRM curve analysis patterns using matK gene fragments.
The forward primer (HRM matK-F) was a 27-mer, and only the second base was prepared at a different position, and the matK-R primer described in Table 2 was used as the reverse primer. Details of the above primers are summarized in Table 4. Therefore, the size of the matK gene fragment amplified by this primer combination is expected to be 174 bp, and when the homology between the nucleotide sequences in these fragments is compared, the nucleotide sequence of 21 mutant strains is different from that of Bacillus subtilis and Bacillus subtilis , And the two bases differed only in the two bases.
(° C)
(
bp
)
trnLF-F
(SEQ ID NO: 11)
236
trnLF-R
(SEQ ID NO: 12)
matK-F
(SEQ ID NO: 13)
(SEQ ID NO: 14)
Before analyzing the HRM curve pattern using the primers summarized in Table 4, in order to investigate whether these primers accurately amplify the target gene fragments, the primer combinations of the respective primers Was used to amplify the genomic DNA isolated from Hashuo, Bacillus subtilis, and Bacillus grandiflorum as a template by PCR and confirmed by electrophoresis on 2.0% agarose gel.
As a result, as shown in Fig. 6, it was confirmed that a single-band DNA fragment was amplified at the predicted position. In addition, these bands were purified from agarose gel and analyzed by sequencing. After confirming that they were 100% identical to the trnL-trnF or matK genes identified in each species, HRM curve pattern analysis was performed.
10 ng of each of the genomic DNAs isolated from Hashuo, Bacillus thuringiensis, and Bacillus thunbergii were added to each well, and 5 pmol of each of the primers described in Table 4 was added. 10 μl of SsoFast ™ EvaGreen Supermix BIO-RAD and 172-5200 premixture were added, After adjusting to 20 μl, HRM curve analysis was performed using Mx3005P QPCR Systems (Agilent Technologies, Santa Clara, USA).
The conditions were as follows: the enzyme was activated at 98 ° C for 2 minutes, denatured the DNA at 98 ° C for 5 seconds, maintained at 57 ° C for 20 seconds, annealed and extended for 30 times, After termination, the temperature was lowered to 40 ° C, and the melting curve pattern was analyzed by the input program while increasing the temperature to 95 ° C again.
As a result, as shown in Fig. 7, it was found that the discrimination of the species was possible by showing different HRM curve patterns for the three kinds of the matK and trnL-trnF fragments.
In the case of matK, only two monoclonal polymorphisms were observed in the bovine spongiform encephalopathy. However, in the case of the trnL-trnF fragment, the Hashuo showed a completely different pattern from the bovine spongiform encephalopathy and bovine spongiform encephalopathy.
Using these conditions, Hashuo and Gyeongnam Herbal Medicine Agricultural Cooperative (Gyeongnam), which were sold respectively by Chungbuk Agricultural Research and Extension Service (Ochang), National Horticultural Research Institute (Voice), Gyeongnam Agricultural Research and Extension Service He has been sold from the Institute of Technology (Ochang), Gyeongnam Agricultural Research and Extension Service (Gyeongbuk), Gyeongbuk Agricultural Research and Extension Service (Bonghwa), Chungbuk Agricultural Research and Extension Service (Ochang), National Horticultural Research Institute (Voice) As shown in FIG. 8, the HRM curve analysis patterns of the cows showed that the three species could be identified exactly in agreement with each other, and these three kinds of medicines were mixed in various forms such as dried roots or powder states It can be easily distinguished.
Example 5: Baisho Owa Lee Yoo To determine the mixing ratio of the mixed samples PCR Establish amplification conditions
As described in Example 3, in the trnL-trnF gene fragment, primers were prepared using single nucleotide polymorphisms in which two nucleotides differ from nucleotide sequences of polynucleotide polynucleotides and polynucleotides. Markers were developed.
However, qualitative analysis to discriminate only the coexistence is necessary. However, quantitative discrimination technology capable of discriminating the exact amount of incorporation when the problem occurs in the actual distribution market is considered to be important. In Table 3, a primer containing a single nucleotide polymorphism (TrnLF-F-2) amplified to a suitable size to quantify the amount of DNA by quantitative PCR (qPCR) using reverse primer WILTRnLF-R and AURtrnLF-R as reverse primers. Primers were prepared to amplify trnL-trnF gene fragments of similar size to the intrinsic actin gene.
Then, the genomic DNAs isolated from Bacillus thuringiensis and Bacillus thuringiensis were quantified by UV spectrophotometer and then mixed with each other at random ratios. Then, Respectively.
Experimental conditions were as follows: 10 ng of genomic DNA and 5 pmoles of each primer were mixed and then the total reaction solution was adjusted to 20 μl by mixing iTaq ™ Universal SYBR Green Supermix (BIO-RAD, Hercules, USA) After the denaturation, the program was run to perform 30 cycles of 30 seconds at 95 ° C, 30 seconds at 58 ° C, and 1 minute at 72 ° C.
FIG. 9 shows the results of comparing the amplified values obtained after completion of the reaction with the amplified values of the intrinsic genes (actin) and trnL-trnF gene fragments used as a control, and comparing the relative DNA contents. That is, a mixture of the genomic DNAs of Cp and Ca was mixed with each genomic DNA at the mixing ratios described in the x axis, And the relative amplification value was expressed on the y-axis. The intrinsic actin and trnL-trnF genes were amplified with the control and the values obtained were corrected. The experiment was performed with three iterations to calculate the SE value. As a result, it was confirmed that the mixing tendency almost coincided with the mixing ratio intentionally mixed.
This suggests that it is possible to estimate the relative mixing ratio when two herbal medicines are mixed.
No
.
(° C)
(
bp
)
(SEQ ID NO: 15)
(SEQ ID NO: 16)
(control)
(SEQ ID NO: 17)
(SEQ ID NO: 18)
( Cynanchum
wilfordii Max.
Hemsley)
(SEQ ID NO: 19)
(SEQ ID NO: 20)
( Cynanchum
auriculatum
Royle ex
Wight)
(SEQ ID NO: 21)
(SEQ ID NO: 22)
Example 6: Baisho Owa Lee Byung-Woo That can distinguish between species Nucleus DNA discrimination markers And ARMS - PCR Applied technology development
According to the opinions of the farmers and experts who cultivate them, the morphological characteristics of the leaves, stems, fire structures and roots of the ground are mixed together. It is said that the plants can be easily found. Such hybrids may not be distinguishable from the discrimination markers present in the chloroplast.
Therefore, the inventors of the present invention have found that the genes present in the chloroplast are inherited by the mother and the genes present in the nucleus are inherited by crossing, intergenic transcribed spacer (SNP) regions of the genome, and to develop discriminant markers that can distinguish the polymorphisms. In other words, if there is a hybrid of the bovine anemone and the bovine oval, they are to be distinguished.
First, in order to obtain the nucleotide sequence of the ITS gene fragment, the nucleotide sequence of the gene encoding 18S rRNA and 28S rRNA present in the transcription unit of the ribosomal DNA was searched in GenBank, and Cynanchum wilfordii isolate cy1, Accession No. AY548207. ITSI, 5.8S rDNA, ITSII, and 28S rDNA were amplified using a primer prepared using a primer (Cynanchum auriculatum, accession No. AB109974.1) and a single band After purification of the DNA, the base sequence was analyzed.
The results were compared with each other to confirm some single nucleotide polymorphisms. Based on this, a primer for amplification refractory mutation system (ARMS) -PCR in Table 6 was prepared.
In other words, we confirmed three single nucleotide polymorphisms in the ITSI region and the ITS II region, and the forward primer positioned the first single nucleotide polymorphism of ITSI at the 3'-end and the original WIL ITS-F (AUR ITS-F) was used as a primer for the mutation of the third base T to A at the 5'-end in the 3'-terminal base (C) Respectively.
The reverse primer was designed to have a second single nucleotide polymorphism in the ITS II region, that is, a primer G, a primer T, and a primer T. The reverse primer (AUR ITS-R) And the third nucleotide A at the terminus was used to induce mutation to increase the discrimination efficiency. However, Baiksoo's reverse primer (WIL ITS-R) used the original sequence without modification (see Fig. 10).
Table 6 summarizes the information of the primers prepared to discriminate between the primate and the primate.
No
.
(° C)
(
bp
)
( Cynanchum
wilfordii Max.
Hemsley)
(SEQ ID NO: 23)
(SEQ ID NO: 24)
( Cynanchum
auriculatum
Royle ex
Wight)
(SEQ ID NO: 25)
(SEQ ID NO: 26)
PCR was carried out by adjusting the annealing temperature according to the value of Tm using the method described in Example 2 using purely separated genomic DNAs to distinguish between the primers and the primers. Respectively.
The results are shown in Fig.
The control was amplified with a primer prepared using the nucleotide sequence information obtained from GenBank. It was confirmed that a fragment of the transcription unit of 18S-ITS I-5.8S-ITSII-28S of 762 bp was amplified in all samples, (Table 6) prepared for differentiation of each species, and it was confirmed that the bands were amplified uniquely for each species.
In other words, we obtained the expected results at 5 points purchased from Ochang, Ahn, Bonghwa, and Sancheong. Also, as expected, Ophiopods purchased from Ochang, Voice, and Sancheong were only used when species specific primers were used Amplification of a single band of the corresponding size was confirmed. Therefore, using these primer combinations, it is possible to easily discriminate whether or not a mixture of Bacillus thuringiensis and Bacillus thuringiensis can be easily identified. In addition, cross-breeding experiments with the chloroplast primer combinations can easily discriminate the hybrid of Bacillus thuringiensis and Bacillus thuringiensis will be.
From these results, it can be seen that it is possible to accurately discriminate those which are used as raw materials of herbal medicines and which are distributed in the form of dry roots, using the markers for discrimination of Hashuo, Baishuo, and bamboo shoots of the present invention.
The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
<110> Industry Academic Cooperation Foundation of Gyeongnam National University <120> Markers for the differentiation of Polygonum multiflorum Thumberg, Cynanchum wilfordii Max. Hemsl, Cynanchum auriculatum Royle ex Wight <130> pn1507-207 <160> 26 <170> Kopatentin 2.0 <210> 1 <211> 35 <212> DNA <213> matK-F <400> 1 ctatatccac ttatctttca ggagtatagt tatgc 35 <210> 2 <211> 33 <212> DNA <213> matK-R <400> 2 aaagttctag cacaagaaag tcgaagtata tac 33 <210> 3 <211> 32 <212> DNA <213> trnLF-F-1 <400> 3 cgtacggact taattggatt gagccttagt aa 32 <210> 4 <211> 22 <212> DNA <213> trnLF-R <400> 4 ggccgagatt ttcagtcctc tg 22 <210> 5 <211> 32 <212> DNA <213> trnLF-F-1 <400> 5 cgtacggact taattggatt gagccttagt aa 32 <210> 6 <211> 26 <212> DNA <213> WILtrnLF-R <400> 6 tcaattaaaa gggcgaaaga agattc 26 <210> 7 <211> 32 <212> DNA <213> trnLF-F-1 <400> 7 cgtacggact taattggatt gagccttagt aa 32 <210> 8 <211> 26 <212> DNA <213> AURtrnLF-R <400> 8 tcaattaaaa gggcgaaaga aggtta 26 <210> 9 <211> 31 <212> DNA <213> FAL trnLF-F <400> 9 aaactccaaa aaggatgaag aataaatcta g 31 <210> 10 <211> 22 <212> DNA <213> trnLF-R <400> 10 ggccgagatt ttcagtcctc tg 22 <210> 11 <211> 23 <212> DNA <213> HRM trnLF-F <400> 11 tttgagcaag aaatccccat ttg 23 <210> 12 <211> 22 <212> DNA <213> HRM trnLF-R <400> 12 ggccgagatt ttcagtcctc tg 22 <210> 13 <211> 27 <212> DNA <213> HRM matK-F <400> 13 cgtattagga catcccatta gtaagcc 27 <210> 14 <211> 33 <212> DNA <213> matK-R <400> 14 aaagttctag cacaagaaag tcgaagtata tac 33 <210> 15 <211> 20 <212> DNA <213> Actin F <400> 15 ggctggattt gctggtgatg 20 <210> 16 <211> 20 <212> DNA <213> Actin R <400> 16 ccgcctgaat agcaacatac 20 <210> 17 <211> 23 <212> DNA <213> trnLF-F-2 <400> 17 tttgagcaag aaatccccat ttg 23 <210> 18 <211> 22 <212> DNA <213> trnLF-R <400> 18 ggccgagatt ttcagtcctc tg 22 <210> 19 <211> 23 <212> DNA <213> trnLF-F-2 <400> 19 tttgagcaag aaatccccat ttg 23 <210> 20 <211> 26 <212> DNA <213> WILtrnLF-R <400> 20 tcaattaaaa gggcgaaaga agattc 26 <210> 21 <211> 23 <212> DNA <213> trnLF-F-2 <400> 21 tttgagcaag aaatccccat ttg 23 <210> 22 <211> 26 <212> DNA <213> AURtrnLF-R <400> 22 tcaattaaaa gggcgaaaga aggtta 26 <210> 23 <211> 18 <212> DNA <213> WIL ITS-F <400> 23 cccttgctcg gtcggtca 18 <210> 24 <211> 18 <212> DNA <213> WIL ITS-R <400> 24 tgccggcaac tcgcgtac 18 <210> 25 <211> 18 <212> DNA <213> AUR ITS-F <400> 25 cccttgctcg gtcggacc 18 <210> 26 <211> 18 <212> DNA <213> AUR ITS-R <400> 26 tgccggcaac tcgcgaaa 18
Claims (16)
SEQ ID NOS: 11 and 12, SEQ ID NOS: 13 and 14;
SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22; And
SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26
Wherein the primer set comprises at least one primer set selected from the group consisting of primers set forth in SEQ ID NO:
Wherein the primer set of SEQ ID NOs: 5 and 6, SEQ ID NOs: 7 and 8, and SEQ ID NOs: 9 and 10 are for discriminating the strains of Sasao, Bacillus anthracis and Bacillus anthracis using single nucleotide polymorphism (SNP).
Wherein the primer set of SEQ ID NOs: 11 and 12, and SEQ ID NOs: 13 and 14 are used for HRM curve pattern analysis for the identification of the strains of Hassuo, Bacillus subtilis and Bacillus subtilis.
Wherein the primer set is used in a PCR technique.
Wherein the primer set of SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, and SEQ ID NOs: 21 and 22 are for determining the mixing ratio of the hyssop,
Wherein said primer set is used in a qPCR technology.
Wherein the primer sets of SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26 are for hybrid identification of the primrose and the second primrose.
Wherein the primer set is for ARMS-PCR comprising a SNP of nuclear ITS DNA.
Wherein the reagent for performing the amplification reaction comprises a DNA polymerase, dTNPs, and a buffer.
Amplifying the amplified product by performing amplification reaction using the obtained DNA as a template and using the primer set of claim 1; And
Detecting the amplification product;
Wherein the method comprises the steps of:
Wherein the primers set forth in SEQ ID NOS: 5 and 6, SEQ ID NOS: 7 and 8, and SEQ ID NOS: 9 and 10 are used in a PCR method for discriminating the strains of Hashuo, Bacillus subtilis and Bacillus subtilis.
Wherein the primers set forth in SEQ ID NOS: 11 and 12, and SEQ ID NOS: 13 and 14 are used for HRM curve pattern analysis.
The primer sets of SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, and SEQ ID NOs: 21 and 22 are characterized in that they are used in the qPCR method for discriminating the mixing ratio of the hyssop, How to.
Wherein the primer set of SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26 is used in the ARMS-PCR method for hybridization of Bacillus subtilis and Bacillus subtilis.
SEQ ID NOS: 11 and 12, SEQ ID NOS: 13 and 14;
SEQ ID NOs: 15 and 16, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22; And
SEQ ID NOs: 23 and 24, SEQ ID NOs: 25 and 26
A primer set selected from the group consisting of primers set forth in SEQ ID NO:
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| KR1020150119573A KR20170024388A (en) | 2015-08-25 | 2015-08-25 | Markers for the differentiation of Polygonum multiflorum Thumberg, Cynanchum wilfordii Max. Hemsl, Cynanchum auriculatum Royle ex Wight |
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| KR1020150119573A KR20170024388A (en) | 2015-08-25 | 2015-08-25 | Markers for the differentiation of Polygonum multiflorum Thumberg, Cynanchum wilfordii Max. Hemsl, Cynanchum auriculatum Royle ex Wight |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101866163B1 (en) | 2018-03-20 | 2018-06-08 | 재단법인 전남생물산업진흥원 | InDel markers for discrimination of Cynanchum wilfordii Mas. Hemsley. and Cynanchum auriculatum Royle ex Wight and method for use thereof |
| KR102164175B1 (en) * | 2019-08-29 | 2020-10-12 | 서울대학교산학협력단 | Marker derived from complete sequencing of chloroplast genome of Cynanchum wilfordii and Cynanchum auriculatum, primer set for discrimination of Cynanchum species and uses thereof |
| KR20210027070A (en) * | 2020-07-20 | 2021-03-10 | 서울대학교산학협력단 | Marker derived from complete sequencing of chloroplast genome of Cynanchum wilfordii and Cynanchum auriculatum, primer set for discrimination of Cynanchum species and uses thereof |
| KR20210081498A (en) * | 2019-12-23 | 2021-07-02 | 대한민국 (식품의약품안전처장) | Composition for distinguishing a plant of genus Cynanchum |
| KR20220060492A (en) * | 2020-11-04 | 2022-05-11 | 가천대학교 산학협력단 | Composition for discriminating Cenchrus longispinus, and use thereof |
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|---|---|---|---|---|
| KR20090123113A (en) | 2008-05-27 | 2009-12-02 | 한국 한의학 연구원 | Dna marker for discrimination of polygonum multiflorum thunberg, cynanchum wilfordii max. hemsl. and cynacum auriculatum royle ex wight |
| KR20120062614A (en) | 2010-12-06 | 2012-06-14 | 재단법인 제주테크노파크 | Method for discriminating between cynanchum wilfordii and cynanchum auriculatum and a kit therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090123113A (en) | 2008-05-27 | 2009-12-02 | 한국 한의학 연구원 | Dna marker for discrimination of polygonum multiflorum thunberg, cynanchum wilfordii max. hemsl. and cynacum auriculatum royle ex wight |
| KR20120062614A (en) | 2010-12-06 | 2012-06-14 | 재단법인 제주테크노파크 | Method for discriminating between cynanchum wilfordii and cynanchum auriculatum and a kit therefor |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101866163B1 (en) | 2018-03-20 | 2018-06-08 | 재단법인 전남생물산업진흥원 | InDel markers for discrimination of Cynanchum wilfordii Mas. Hemsley. and Cynanchum auriculatum Royle ex Wight and method for use thereof |
| KR102164175B1 (en) * | 2019-08-29 | 2020-10-12 | 서울대학교산학협력단 | Marker derived from complete sequencing of chloroplast genome of Cynanchum wilfordii and Cynanchum auriculatum, primer set for discrimination of Cynanchum species and uses thereof |
| KR20210081498A (en) * | 2019-12-23 | 2021-07-02 | 대한민국 (식품의약품안전처장) | Composition for distinguishing a plant of genus Cynanchum |
| KR20210027070A (en) * | 2020-07-20 | 2021-03-10 | 서울대학교산학협력단 | Marker derived from complete sequencing of chloroplast genome of Cynanchum wilfordii and Cynanchum auriculatum, primer set for discrimination of Cynanchum species and uses thereof |
| KR20220060492A (en) * | 2020-11-04 | 2022-05-11 | 가천대학교 산학협력단 | Composition for discriminating Cenchrus longispinus, and use thereof |
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