KR101793911B1 - Primers for distinguishing between Hinoki Cypress and Sawara Cypress and method using the same - Google Patents

Abstract

The present invention relates to a primer set capable of amplifying the genetic variation of chloroplast DNA capable of discriminating between white and white, and a PCR-RFLP technique using the primer set to discriminate between white and white , It is possible to observe the respective restriction enzyme cleavage products specific for the whiteness and the whiteness, thereby eliminating the identification errors that may occur during the experiment and improving the clarity.

Description

TECHNICAL FIELD [0001] The present invention relates to a primer for distinguishing between white and white, and a discriminating method using the primer.

More particularly, the present invention relates to a primer capable of distinguishing between white and white, and a PCR-RFLP (Polymerase Chain Reaction Polymorphism) method using the primer. It is about how to distinguish between white and white.

There are about 125 genera in the genus Cervus spp. In the 19 genera. There are about 10 kinds of spruce trees and species in Korea, and many other imported species are planted (Non-patent document 0001). There are many kinds of native trees, such as japanese tree, japanese tree, japanese tree, and japanese tree, which are native to Korea, and are widely introduced as a typical introduced species (Non-patent document 0002). Shrub and tree species are important forest resources and are widely used as timber and landscape water.

It is known to be the first species planted in Korea in the 17th century, and was introduced from Japan in earnest in the beginning of the 20th century (Non-Patent Document 0003). It grows well in the temperate south (south of Jeonnam Jangseong) and the hanbai area in Korea, and is used as architecture, civil engineering, furniture, sculpture, plywood, etc. (Non-patent document 0004). Recently, phytoncide content is high, so that a large-scale forest is planted for the purpose of healing forests, and it is widely used as household goods such as bathtubs and bedclothes (Non-Patent Document 0005).

It is known to be introduced into Korea in 1920 (Non-Patent Document 0006). The flower is stronger than the whiteness and has a characteristic of growing well in the central region of the temperate region (Non-Patent Document 0007). Paintings are mainly used as landscaping, and they are also used for construction, civil engineering, furniture, and plywood (Non-Patent Document 0008).

It is not difficult to discriminate between white and white flowers by taxonomic characteristics, and it is difficult to discriminate between the species such as the bracken tree and the bamboo trees of the Japanese cabbage tree because of their external shape or wood histological characteristics (Non-Patent Document 0009). On the other hand, it is possible to easily distinguish between white and white leaves in the shape of outer leaves, but the wood is very difficult to identify because of its very similar histological characteristics, and there is a concern that mixed use of wood and illegal circulation is a problem (Non-Patent Document 0010) .

DNA markers can be used as a technique to identify DNA variants present in living organisms and to identify individuals and species. DNA markers have no environmental impacts, such as external morphological features, and can provide objective criteria for identifying organisms. In order to solve the problem of the identification error caused by the histological similarity between the white and white woods using the DNA marker, the present invention provides an object identification and clear coherence based on the DNA mutation which is not influenced by the external form and the environmental mutation And to provide an identification criterion.

 Liguo Fu, Yong-fu Yu, Robert P. Adams & Aljos Farjon. 1999. Cupressaceae. Flora of China. 4: 62-77. 1999.  Yang Jong Chul, Hong Jung Ki, Oh Seung Hwan, Youmi Me, Heo Kwon. 2012. A Study on the Morphological Traits of Korean Plants. Korean Society of Limnology. 2012 Volume 0. 2012 pp.235-237.  Compensation. 2012. 5 species of economic species. Korea Forest Research Institute. ISBN: 978-89-8176-924-6.  Compensation. 2012. 5 species of economic species. Korea Forest Research Institute. ISBN: 978-89-8176-924-6.  Compensation. 2012. 5 species of economic species. Korea Forest Research Institute. ISBN: 978-89-8176-924-6.  However, 2007. 100 species of useful species in Korea. National Forestry Research Institute. ISBN: 978-89-8176-411-1.  However, 2007. 100 species of useful species in Korea. National Forestry Research Institute. ISBN: 978-89-8176-411-1.  However, 2007. 100 species of useful species in Korea. National Forestry Research Institute. ISBN: 978-89-8176-411-1.  Compensation. 2012. 5 species of economic species. Korea Forest Research Institute. ISBN: 978-89-8176-924-6.  However, 2006. Method of tree species identification. National Forestry Academy Open Seminar Kit.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems, and it is an object of the present invention to search species-specific DNA mutations capable of discriminating between white and white, PCR-RFLP).

It is another object of the present invention to provide a DNA polymerase which is capable of discriminating the objectivity and the definite consistency of discrimination based on DNA mutation which is not influenced by the external morphology and environmental variation by using the primer in order to solve the problem of uncertainty of external form comparison and subjective identification error And to provide a method for discriminating textiles and photographs.

It is still another object of the present invention to provide a kit for identifying white and white flowers having the objectivity and clear consistency of discrimination based on DNA variation without influence of external form and environmental variation by including the primer.

In order to solve the above-described problems, the present invention provides a primer set for distinguishing a facial expression and a facial expression comprising the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

In addition, the present invention provides a method for discriminating the white and white characters using the primer set.

In addition, the present invention provides a kit for identifying white and white flowers, which comprises the primer set.

According to the present invention, it is possible to stably and reliably distinguish between the white back and the white background by using the primer for the white back and the white background, which can be a scientific guarantee for identification of the white back and the white back which are difficult to identify by a microscopic method. In particular, restriction enzyme cleavage products specific for the glue can be observed using primers and restriction enzymes.

In addition, when the above method of the present invention is used, the external shape comparison itself is impossible, and it can also be utilized in the case of identifying a processed wood which is difficult to identify even by a microscopic method.

FIG. 1 shows a fragmentation pattern of a product obtained by amplifying a DNA region containing a DNA variation capable of discriminating between a monoclonal antibody and a monocyte by a polymerase chain reaction (PCR) using a 'Hinoki_1' primer (lane 1 to 4: 8: flower, M: DNA size marker).
Fig. 2 shows fractions (lanes 1 to 4: whiteness, lanes 5 to 8: flower buds, M: DNA size marker) of products obtained by digesting the PCR products of the PCR products of Fig. 1 with restriction enzymes.

The present invention provides a primer set for white-and-white identification comprising a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2.

In the present invention, " primer " is a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming a base pair with a template of a complementary nucleic acid, Lt; RTI ID = 0.0 > nucleic acid < / RTI > The primer can initiate DNA synthesis in the presence of a reagent for polymerization reaction at a suitable buffer solution and temperature. Typically, the primer should be composed of approximately 50-60% GC%, with a uniform distribution of the four bases, and avoiding a continuous array of purines or pyrimidines. When three or more primers G or C are continuously arranged, they are preferably avoided because they can bind to undesired sites, and the formation of a 'primer-dimer' by forming a complementary bond at the 3 'end in a pair of primers is minimized For efficient cloning of the PCR product, the 5 'end of the primer may contain a suitable restriction endonuclease sequence.

The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer depends on the primer usage conditions such as the complexity of the desired DNA target, temperature and ionic strength.

In the present invention, an oligonucleotide used as a primer may also include a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, And may include intercalating angents.

The nucleotide sequence of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2 which constitute the primer set according to the present invention was developed based on the nucleotide sequence of chloroplast DNA of the white and white flowers, will be.

In addition, the present invention provides a method for discriminating the white and white characters using the primer set.

The method for discriminating the monoclonal antibodies and the monoclonal antibodies of the present invention comprises the steps of (a) separating genomic DNA from a monoclonal antibody and a monoclonal antibody sample, (b) using the separated genomic DNA as a template, (C) digesting the amplified product by digesting the amplified product with restriction enzymes, and (d) electrophoresing the digested product to obtain a size of the digested product. And analyzing.

 &Quot; Polymerase chain reaction " is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such polymerase chain reaction methods are well known in the art and the amplified target sequence may be labeled with a detectable labeling substance. The labeling substance may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto.

The reaction solution for the PCR reaction is prepared by mixing 25 ng of the white or white genomic DNA, 1x buffer, 20 uM of the forward primer of SEQ ID NO: 1 , 20 μM of the reverse primer of SEQ ID NO: 2, 0.2 mM dNTPs, 2.5 mM MgCl ₂ , and 1.25 units of Taq DNA polymerase.

The amplification of step (b) in the step (b) of the present invention is carried out after the initial denaturation step at 94 ° C for 5 minutes, followed by denaturation at 94 ° C for 30 seconds, primer binding at 58 ° C for 30 seconds, The procedure can be repeated 35 times and the final polymerization process can be performed at 72 ° C for 10 minutes.

In the method according to the present invention, the reaction product for digesting the amplification product by treating the restriction enzyme of the step (c) is 1 × reaction buffer solution and 0.5 unit restriction enzyme per 5 μl of the PCR amplification product .

In the method of identification of the white-and-white and the flower of the present invention, the restriction enzyme may use HinP 1I, but is not limited thereto.

In the method of identification of the whitish and white color of the present invention, the treatment of the HinP 1 I restriction enzyme may include a step of activating at 37 캜 for 3 hours and then deactivating at 65 캜 for 20 minutes.

The truncated target sequence of the present invention may be labeled with a detectable labeling substance. In one specific example, the labeling material can be, but is not limited to, a material that emits fluorescence, phosphorescence, or radiation. Preferably, the labeling substance is Ethidium Bromide (EtBr), Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. When the radioactive isotope such as 32P or 35S is added to the PCT reaction solution during the PCR, the amplification product is synthesized and the radiation can be incorporated into the amplification product and the amplification product can be labeled with the radiation.

In addition, the present invention provides a kit for identifying white and white flowers, which comprises the primer set.

In the kit for identifying white and white flowers of the present invention, various reagents required for confirming the test procedure and results necessary for discriminating the white and white flowers using the primer set, for example, PCR reaction mixture, restriction enzyme, agarose, A buffer solution necessary for electrophoresis, and the like may be further included.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

≪ Example 1 > Extraction and purification of template DNA

In order to prepare a sample for template DNA extraction and purification, 200 mg of leaf samples collected from each individual were quantitated and placed in a 2 ml microtube together with ceramic beads. 400 μl of Buffer AP1 of DNeasy Plant Mini Kit (QIAGEN) 4 μl was added thereto, followed by shaking for 10 minutes at a rate of 30 times / sec using a Tissue Lyser (QIAGEN) to disrupt the leaf sample tissue.

DNA was extracted and purified according to the manufacturer's instructions using DNeasy Plant Mini Kit (QIAGEN). The final concentration of extracted DNA was diluted to 5 ng / ㎕.

Example 2: Design of primer

The nucleotide sequence of the region to be amplified was determined using the nucleotide sequence of the chloroplast DNA of the monoclonal antibody and the monoclonal antibody, and the primer was designed using Primer3 software.

Primer sizes ranged from 18 to 22 mers, Tm ranged from 57 to 63 ° C, one GC clamp, and primer amplified products ranging from 200 bp to 350 bp.

Table 1 below shows the nucleotide sequences of the primers Hinoki-1 of the present invention, wherein the combination of the primer base sequences is regarded as a combination of the reverse primer base sequences and the same primer base sequence combination.

≪ Example 3 > Polymerase chain reaction (PCR)

The amplification products were generated by performing PCR using the primer Hinoki-1 designed in Example 2 for the monoclonal and fluorescent samples.

The composition of the PCR reaction mixture is as follows.

The PCR reaction solution contained 25 ng of each of white-washed, white genomic DNA, 1 x buffer, 20 μM primer, 0.2 mM dNTPs, 2.5 mM MgCl ₂ and 1.25 units of DiaStar Taq DNA polymerase per 50 μl.

After the initial denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 30 seconds, primer binding at 58 ° C for 30 seconds, and polymerization at 72 ° C for 30 seconds were repeated 35 times, followed by final polymerization at 72 ° C for 10 minutes Respectively.

<Example 4> Production of soybean product by restriction enzyme fragment polymorphism (RFLP) technique

The amplification product generated in Example 3 was treated with a restriction enzyme to generate a cleavage product of the amplification product. As the restriction enzyme, HinP 1I (NEB) was used. The composition of the reaction product for restriction enzyme digestion of the PCR amplification product was a mixture composed of 1 × reaction buffer and 0.5 U of restriction enzyme per 5 μl of the PCR amplification product . The HinP 1I restriction enzyme treatment was performed at 37 ° C for 3 hours and then at 37 ° C for 20 minutes.

&Lt; Example 5 > Amplification products and fractions of cleavage products

5 μl of the amplification product generated in Example 3 was removed and mixed with 3 μl of loading dye. 2% agarose gel electrophoresis was performed. A 100 bp Ladder (Fermentas Co., 50 μg / μl) The result of checking the product fragment size is shown in Fig.

In addition, 5 μl of loading dye was mixed with the cleavage product produced in Example 4, followed by 2% agarose gel electrophoresis. A 100 bp ladder (Fermentas Co., 50 μg / μl) The result of checking the size of the fragment is shown in Fig.

As shown in FIG. 1, when the fractional pattern was observed by electrophoresis on the agarose gel, a DNA fragment of about 220 bp was commonly observed in both the white and flower samples (1 to 4: M: DNA size marker).

Further, as shown in FIG. 2, the cleavage product of the amplification product using the primer Hinoki-1 by HinP II restriction enzyme was electrophoretographed on agarose gel and the fragmentation pattern was observed. In the case of the whitening, two cuts of about 70 bp and about 150 bp As shown in FIG. 1, a PCR amplification product of about 220 bp was observed in the same manner (1 to 4: untreated, 5 to 8: colored DNA, M: DNA size marker) .

The sizes of the DNA fragments observed in the above are visually observed values based on the DNA size markers of the electrophoresis. From the nucleotide sequences used for the primer synthesis, the sizes of the base sequences amplified by the primers and the restriction enzymes The approximate size can be estimated based on the calculated length based on the recognition site.

When observing a DNA fragment through the electrophoresis, it is possible to display a slightly different value according to an observer by visual observation. However, the target DNA region can be accurately amplified and cut, have.

Although the present invention has been described in connection with the specific embodiments of the present invention, it is to be understood that the present invention is not limited thereto. 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. In addition, the materials of each component described herein can be readily selected and substituted for various materials known to those skilled in the art. Those skilled in the art will also appreciate that some of the components described herein can be omitted without degrading performance or adding components to improve performance. In addition, those skilled in the art may change the order of the method steps described herein depending on the process environment or equipment. Therefore, the scope of the present invention should be determined by the appended claims and equivalents thereof, not by the embodiments described.

According to the present invention, it is possible to stably and reliably distinguish the white background and the white background by using a primer set for discriminating the white background and the white background, so that it can be utilized as a molecular marker for scientific guarantee for the white background and the white background.

In addition, when the method of the present invention is used, it is possible to solve unrecognized factors as compared with the conventional methods, and particularly to identify the processed wood which is difficult to identify by the microscopic method even when the external shape comparison itself is impossible have.

<110> KOREA FOREST RESEARCH INSTITUTE <120> Primers for distinguishing between Hinoki Cypress and Sawara          Cypress and method using the same <130> 16-10441 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer of Hinoki_1 <400> 1 caaaaattga ccggataggg 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer of Hinoki_1 <400> 2 gatccaatcc ataatgatac gc 22

Claims (8)

A primer set for PCR-RFLP analysis for discriminating between the flat and the reverse streaks consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 (a). Separating the genomic DNA from the monoclonal and fluorescent samples;
(b). Amplifying the genomic DNA by a polymerase chain reaction using the separated genomic DNA as a template and using the primer set of claim 1;
(c). Treating the amplified product with a restriction enzyme to cleave the amplification product; And
(d). And analyzing the size of the cut product by electrophoresis of the cleavage product. The method according to claim 2, wherein the reaction solution for the polymerase chain reaction comprises 25ng of white or white genome DNA, 1x buffer, 20mM primer, 0.2mM dNTPs, 2.5mM MgCl2 and 1.25 Unit Taq DNA polymerase per 50 mu l of the reaction solution A method for discriminating between white and white characters The method of claim 2, wherein the amplification of step (b) comprises repeating the denaturation at 94 ° C for 30 seconds, the primer binding at 58 ° C for 30 seconds, and the polymerization at 72 ° C for 30 seconds for 35 times after the initial denaturation at 94 ° C for 5 minutes Followed by final polymerization at 72 ° C for 10 minutes. [Claim 3] The method according to claim 2, wherein the reaction product for digesting the restriction enzyme of step (c) comprises 1 * reaction buffer solution and 0.5 unit restriction enzyme per 5 l of the PCR amplification product How to distinguish between white and white The method according to claim 2, wherein the restriction enzyme is HinP II. 7. The method according to claim 6, wherein the treatment of the HinP II restriction enzyme comprises activating at 37 DEG C for 3 hours and then inactivating at 65 DEG C for 20 minutes A kit for identifying white and white flowers comprising the primer set of claim 1

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