KR102371553B1 - Method of DNA isolation using affinity column in sugar honey - Google Patents

Abstract

A novel method for separating residual genes from fermented honey using an affinity column is disclosed. The present invention is a method for isolating residual genes in fermented honey using an affinity column, (a) promoting binding of residual genes present in fermented honey to a silica membrane using a binding buffer. inducing; (b) loading the residual gene-containing sample into the affinity column including a silica membrane; (c) removing impurities using a washing buffer; and (d) recovering the desired residual gene from the affinity column using an elution buffer.

Description

Method of DNA isolation using affinity column in sugar honey

The present invention relates to a method for isolating residual genes from fermented honey, and more particularly, to a method for isolating residual genes from fermented honey using an affinity column.

According to the notification of the Ministry of Food and Drug Safety (2016-43) in Korea, honey is divided into pure honey and specification honey. ), breeding honey also provides a basis for distribution in the market under the name of honey (see Non-Patent Document 2), and there is concern about damage to producers and consumers.

Currently, isotope ratio mass spectrometry (isotope ratio mass spectrometry) is used to confirm the addition of sugar cane and corn syrup to honey in many countries, including Korea and Canada. Based on the fact that the relative ratio of stable isotope C13 to C12 is changed due to the change in the carbon metabolic pathway in the organism, this method converts the Calvin cycle in the C3 plant population (CO ₂ fixation process). Stable isotope ratios of passing plants, beets, maples, most fruits, etc.) are - (22~30)‰, C4 flora (CO ₂ fixation process) in the hatch slack circuit (Hatch) Stable isotope ratios of plant groups passing through the slack cycle, corn, sugarcane, etc.) are - (8~11)‰, CAM (CO 2 fixation process) in the CAM plant group (CO ₂ fixation process) The stable isotope ratio of plant groups, pineapple, etc.) through Crassulacean Acid Metabolism) is distinguished by - (11~13.5)‰. Accordingly, the carbon isotope ratio became a standard for estimating the origin of the sugar contained in honey, and was adopted by the International Organization for Analytical Chemistry (AOAC) as a method for distinguishing C4 sugar in honey. In Korea, this method is also used to discriminate fermented honey (see Non-Patent Documents 3 to 5).

However, this method requires an expensive instrument such as an isotope ratio mass spectrometer equipped with a carbon element analyzer, and the time required for detection and classification is difficult when C3 flora, honey, and fermented honey are mixed. There is a difficult problem.

Recently, there have been efforts to solve this problem through the gene detection method using the PCR method. In line with these efforts, it is very important to isolate the remaining genes in fermented honey. Gene isolation from conventional fermented honey took a long time as there was a pretreatment process using heat treatment or reagents such as CTAB (cetyl trimethylammonium bromide), and there was a problem such as requiring a large amount of sample due to phenol treatment (N. See Patent Documents 6 and 7).

[Non-patent literature]

1. MFDS, 2014, Food Standard Code, Ministry of food and Drug Safety, Cheongju, Korea.

2. AOAC, 1995. Official Method of Analysis of AOAC Intl. 20th ed. Method 998-12. Association of Official Analytical Chemists, Arlington, VA, USA.

3. Somin Kim, Byounghee Kim, Moonjung Kim, Jungmin Kim, Truong A Tai, Byoungsu Yoon, Detection of Sugar Beet (Beta vulgaris) - Specific Gene from Honey Made by Sugar of Sugar Beet, Journal of Apiculture Vol.33 No.3 2018. 09 213 - 219.

4. Byounghee Kim, Somin Kim, Moonjung Kim, Jungmin Kim, Truong A Tai, Byoungsu Yoon, Detection of Sugar Cane (Saccharum officinarum)-specific Gene from Sugar and Sugar-honey, Journal of Apiculture 33(3), 2018. 9 , 221-226.

5. Hwa-Jung Sung, Chuleui Jung, Jiyoung Kwon, Ho-Yong Sohn, Evaluation of Commercial Korean Honey Quality and Correlation Analysis of the Quality Parameters, Journal of Life Science 28(12), 2018.12, 1489-1500.

6. Sobrino-Gregorio, L., S. Vilanova, J. Prohens and I. Escriche. 2018, Detection of honey adulteration by conventional and real-time PCR, Food Control, 2015, 95:57-62.

via Thalita de Jesus; Giulia Manso Marchioro; Ed

lson Divino de Ara

jo. Extraction of DNA from honey and its amplification by PCR for botanical identification. Food Sci. Technol (Campinas) vol.33 no.4 753-756.7. Sona Arun Jain; Fl

via Thalita de Jesus; Giulia Manso Marchioro; Ed

lson Divino de Ara

jo. Extraction of DNA from honey and its amplification by PCR for botanical identification. Food Sci. Technol (Campinas) vol.33 no.4 753-756.

The present invention intends to present a new method of separating residual genes from fermented honey using an affinity column.

In order to solve the above problem, the present invention is a method for isolating residual genes using an affinity column in fermented honey, (a) using a binding buffer, wherein residual genes present in fermented honey are silica membrane (silica membrane) inducing the promotion of binding; (b) loading the residual gene-containing sample into the affinity column including a silica membrane; (c) removing impurities using a washing buffer; and (d) recovering the desired residual gene from the affinity column using an elution buffer.

In addition, the binding buffer (binding buffer) is guanidine hydrochloride (guanidine hydrochloride, GnHCl) provides a method for separating residual genes using an affinity column from honey, characterized in that.

In addition, the washing buffer includes salt and ethanol, but the concentration of the salt is 10 to 20 mM with respect to 80 to 90% (v/v) of ethanol using an affinity column. A method for isolating residual genes is provided.

In addition, the elution buffer is 5 to 15 mM Tris-hydrogen chloride (Tris-HCl), or a mixture of 5 to 15 mM Tris-hydrogen chloride (Tris-HCl) and 0.1 to 1 mM Ethylenediaminetetraacetic acid (EDTA), distilled water It provides a method for isolating residual genes using an affinity column in the specification honey, characterized in that it is.

The present invention is a method for isolating residual genes present in fermented honey. Unlike gene separation in conventional honey, by using an affinity column, it is faster and easier to operate than conventional methods to separate genes from a large number of samples. It has the advantage of being able to isolate the gene, and this advantage has the effect of making the application of the gene detection method more advantageous in the evaluation of honey quality.

1 is a graph showing the results of ultrafast PCR according to the number of copies of sugarcane-specific genetic recombinant DNA (pSc) template in an experimental example of the present invention. (a) Amplification curve and melting point analysis graph to confirm the detection limit for serial dilution of 10' pSc template copy number and specificity of PCR amplicons, (B) Quantitative curves were prepared through Ct values for pSc serial dilution to ensure linearity and slope The correlation coefficient of slope -0.2856 and R ² =0.998 was obtained by investigation, and the reliability of the quantitative line was secured.
Figure 2 is a graph showing the residual gene detection results in honey according to the DNA binding buffer in the experimental example of the present invention. Add 10 ⁸ sugar cane-specific recombinant DNA (pSc) to 100 μl honey, add 500 μl of different concentrations of GnHCl binding buffer, and analyze the PCR amplicon amplification curve for the recovered pSc and melting point analysis to confirm the specificity of this amplicon shows the graph.
Figure 3 is a graph showing the residual gene detection results in honey according to the DNA binding buffer in the experimental example of the present invention. Add 10 ⁸ sugar cane-specific recombinant DNA (pSc) to 100 μl honey, add 500 μl of GuSCN binding buffer of different concentrations, and amplification curve of PCR amplicon for the recovered pSc and melting point analysis graph for confirming the specificity of this amplicon represents
Figure 4 is a graph showing the results of comparing the DNA binding capacity between the commercially available DNA binding column products in the Experimental Example of the present invention. Add 10 ⁸ sugar cane-specific recombinant DNA (pSc) to 100 μl honey and use 500 μl of 5 M GnHCl binding buffer to confirm the amplification curve and specificity of PCR amplicon for pSc recovered through 5 different columns The melting point analysis graph for
5 is a graph showing the Ct value and the Tm value of the amount of DNA recovered according to the washing buffer composition in the experimental example of the present invention. From the recovered DNA sample obtained by washing the column with a buffer in which ethanol was fixed at 80% (v/v) through the PCR amplification graph and the salt concentration was changed to 10 mM, 15 mM and 20 mM Tris-HCl (pH 7.6) It can be confirmed that the sugar cane-specific gene was amplified, and it was confirmed that the PCR amplicon amplified in all samples was a sugar cane gene-specific product through the melting point analysis graph.
6 is a graph showing the Ct value and the Tm value of the amount of DNA recovered according to the washing buffer composition in the experimental example of the present invention. From the recovered DNA sample obtained by washing the column with a buffer in which ethanol was fixed at 85% (v/v) through the PCR amplification graph and the salt concentration was changed to 10 mM, 15 mM and 20 mM Tris-HCl (pH 7.6) It can be confirmed that the sugar cane-specific gene was amplified, and it was confirmed that the PCR amplicon amplified in all samples was a sugar cane gene-specific product through the melting point analysis graph.
7 is a graph showing the Ct value and the Tm value of the amount of DNA recovered according to the composition of the elution buffer in the experimental example of the present invention. Through the PCR amplification graph, it was confirmed that the sugar cane-specific gene was amplified in the recovered DNA sample obtained by elution with 10 mM Tris-HCl (pH 8.5), TE buffer (10 mM Tris-HCl and 0.5 mM EDTA, pH 8.5) and DW. Through the melting point analysis graph, it was confirmed that the PCR amplicon amplified in all samples was a product specific to the sugar cane gene.
8 is a graph showing the Ct value and the Tm value of the amount of DNA recovered according to the composition of the elution buffer in the experimental example of the present invention. Through the PCR amplification graph, it was confirmed that the sugar cane-specific gene was amplified in the recovered DNA sample obtained by elution with 10 mM Tris-HCl (pH 9.0), TE buffer (10 mM Tris-HCl, 0.5 mM EDTA, pH 9.0) and DW. Through the melting point analysis graph, it was confirmed that the PCR amplicon amplified in all samples was a product specific to the sugar cane gene.
9 is a graph showing the results of detection and quantification of sugar cane-specific genes by a gene detection method performed by isolating residual genes of honey using a commercially available affinity column in an experimental example of the present invention.

Hereinafter, the present invention will be described in detail through examples. Prior to this, the terms or words used in the present specification and claims should not be construed as being limited to their ordinary or dictionary meanings, and the inventor should properly understand the concept of the term in order to best describe his invention. Based on the principle that can be defined, it should be interpreted as meaning and concept consistent with the technical idea of the present invention. Accordingly, since the configuration of the embodiments described in the present specification is only the most preferred embodiment of the present invention and does not represent all the technical spirit of the present invention, various equivalents and modifications that can be substituted for them at the time of the present application It should be understood that there may be

The present invention is a new method for separating residual genes from fermented honey using an affinity column, (a) using a binding buffer to prevent residual genes from fermented honey using a silica membrane ) inducing binding promotion to; (b) loading the residual gene-containing sample into the affinity column including a silica membrane; (c) removing impurities using a washing buffer; and (d) recovering the desired residual gene from the affinity column using an elution buffer; discloses a method for isolating residual genes using an affinity column in fermented honey comprising a.

Step (a) is a step of generating a bridge such that a pH suitable for the desired residual gene and the desired residual gene promote binding to the silica of the column.

In the present invention, the binding buffer is not particularly limited, but it was confirmed that guanidine hydrochloride (GnHCl) was most suitable for efficiently separating residual genes through an affinity column in fermented honey.

Step (b) is a step of attaching the desired residual gene by flowing a sample containing the desired residual gene to the equilibrated column.

In the present invention, by using a column including a silica membrane as an affinity column, a desired residual gene can be bound to the silica membrane.

Step (c) is a step for removing impurities except for the desired residual gene.

In the present invention, the washing buffer is not particularly limited, but includes salt and ethanol in efficiently separating residual genes through an affinity column in raw honey, 80 to 90% (v/v) of ethanol It was confirmed that the salt concentration of 10 to 20 mM is most suitable.

Step (d) is a step of recovering the desired residual gene from the affinity column using the elution buffer.

In the present invention, the elution buffer is not particularly limited, but 5 to 15 mM Tris-hydrogen chloride (Tris-HCl) or 5 to 15 mM Tris- It was confirmed that a mixture of hydrogen chloride (Tris-HCl) and 0.1 to 1 mM ethylenediaminetetraacetic acid (EDTA) or distilled water was most suitable.

Hereinafter, the present invention will be described in more detail by way of experimental examples.

experimental method

(1) Creation of specific gene detection limit and quantitative line through pGEM-sugarcane-cp(pSc) plasmid DNA dilution

In order to find out the optimal conditions for DNA isolation in fermented honey, add a certain number of molecules to pGEM-sugarcane-cp(pSc) plasmid DNA, isolate the DNA using the QIAquick PCR purification kit, and compare it with the amount of the recovered DNA copy number. Optimal conditions were confirmed.

The pSc used in this experiment is a maturase K (matK, GenBank accession No. LN 849913) gene located in the chloroplast of sugar cane (see Non-Patent Document 4). As primers for detecting this, Cane-dF (SEQ ID NO: 1) 5'-CACCGCAATTATTTTTATTCTGAG-3' and Cane-dR (SEQ ID NO: 2) 5'-GAACATCTTGAATCCGGTATTC-3' were used. The PCR reaction solution was prepared by mixing 1 μl 10 pmole primer (F, R), 5 μl 2x Rapi:Detect Master mix (Genesystem, Korea), 2 μl distilled water, and 1 μl pSc, into a 10 μl reaction volume, and heated at 95°C for 30s. After 1 cycle of initial denaturation reaction, a total of 50 cycles of 95°C 3s, 55°C 3s, 72°C 3s were performed as 1 cycle using GENECHECKER (Genesystem, Korea). Thereafter, the analysis was performed using the change in Ct value, the Tm value, and the dCt ₁₀ value.

(2) Residual gene separation using DNA affinity column

In order to isolate residual genes present in honey, a DNA affinity column method different from CTAB method was applied to honey and the suitability was investigated.

1) Selection of an appropriate binding buffer for residual DNA isolation from honey

After adding 1 μl of 1x 10 ⁸ pSC to 100 μl of honey, DNA is isolated using GI buffer (5 M GnHCl, 30% IPA), GSI buffer (5 M GuSCN, 30% IPA) and QIAGEN Kit, and the recovery rate is compared. and the experimental method is as follows. After adding 1 μl of 1x 10 ⁸ pSC to 100 μl of honey sample, 500 μl of each buffer is added. After mixing well using a vortexer, put it in a QIAGEN column, and centrifuge at 13,000 rpm for 30s. 700 μl of washing buffer is added to the column and centrifuged at 13,000 rpm for 30 s. Elution was performed with 50 μl of 3' DW (distilled water), and for comparison with GI buffer and GSI buffer, one sample using the QIAGEN kit was DNA obtained using 50 μl of the elution buffer in the kit. PCR was performed using 1 μl of this to analyze the results.

2) Comparison of appropriate DNA binding column for residual DNA isolation from honey

To select an appropriate affinity column, add 1 µl of 1x 10 ⁸ pSC to 100 µl of honey for columns of 5 different products, add 500 µl of GI buffer, mix well, and put in 5 different columns Centrifugation was performed at 13,000 rpm. Experiments were performed in duplicate, and after washing with washing buffer, elution was performed using 50 μl 3'DW. PCR was performed using 1 μl of the 50 μl thus obtained, and the results were analyzed.

3) Selection of appropriate washing buffer for residual DNA isolation from honey

To compare washing buffers A and B for selection of appropriate washing buffer, 1 μl of 1x 10 ⁸ pSC was added to 100 μl of honey, 500 μl of GI buffer was added, mixed well, placed in P column, and centrifuged at 13,000 rpm. After washing with washing buffers A and B, elution was performed using 50 μl 3'DW. PCR was performed using 1 μl of the 50 μl thus obtained, and the results were analyzed.

4) Selection of an appropriate elution buffer for residual DNA isolation from honey

To select an appropriate elution buffer, 3'DW, and to compare Elution buffers A and B, add 1 µl of 1x 10 ⁸ pSC to 100 µl of honey, add 500 µl of GI buffer, mix well, put in P column, and centrifuge at 13,000 rpm separated. After washing with washing buffer A, elution was performed using 50 μl 3'DW and Elution buffers A and B. PCR was performed using 1 μl of the 50 μl thus obtained, and the results were analyzed.

5) Recovery rate and number of recovered molecules according to the amount of honey sample

In order to determine the increase in the amount of DNA separated when the amount of honey sample is increased, the amount of honey sample in one column was changed to separate DNA, and PCR was performed to compare the recovery rate. The experimental method is as follows. Add 38 µl of 1x10 ⁸ molecules of pSC to 3.8 ml of honey. Add 19 ml of GI buffer to each tube and mix well. Pass 0.6 ml, 1 ml, 6 ml, and 12 ml of honey sample solution through each column. Wash with 700 μl of Washing Buffer A. Elution is performed by adding 50 μl of Elution buffer A. PCR was performed with 1 μl of the obtained 50 μl DNA and the results were analyzed.

(3) Detection of sugarcane-specific genes in commercial products

10 μl of GI buffer was added to 2 μl of 5 product samples, mixed well, placed on a Pro column, and centrifuged. 700 μl of washing buffer A was added to the column, centrifuged, and then elution was performed with 50 μl Elution buffer A. PCR was performed using 1 μl of this, and the results were analyzed.

Experiment result

(1) Creation of specific gene detection limit and quantitative line through pGEM-sugarcane-cp(pSc) plasmid DNA dilution

1x10 ¹¹ Molecules 10 ⁹ , 10 ⁸ , 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10 ¹ , 10 ⁰ Number of molecules by 10' serial dilution of pGEM-sugarcane-cp(pSc) plasmid DNA, And as a negative control (negative, DW), ultra-fast PCR was performed with a total of 10 samples as follows, and the results are shown in Table 1 and FIG. 1 below.

pSc molecules Ct(Cycle) Tm(℃) dCt ₁₀ (cycle) 1x10 ⁸ 17.13 76.51 3.07 1x10 ⁷ 20.20 76.51 3.97 1x10 ⁶ 24.17 76.18 3.99 1x10 ⁵ 28.16 76.18 3.00 1x10 ⁴ 31.16 76.18 2.99 1x10 ³ 34.15 76.18 3.96 1x10 ² 38.11 75.52 76.05±0.41

Referring to Table 1 and FIG. 1 , the detection limit of pSc was 1× ^{10 2} , and the average melting point temperature was 76.05°C. Using this result, a calibration curve was prepared and the linearity and inclination were investigated. As a result, y=-0.2856x + 12.879, which is within the reliable slope value range of -0.25 to -0.33, and a high correlation coefficient value (R ² =0.998) was obtained. This was used for quantitative experiments.

(2) Residual gene isolation using DNA affinity column

1) Select an appropriate binding buffer for residual DNA isolation from honey

The binding strength of guanidine hydrochloride (GnHCl) and guanidine thiocyanate (GuSCN) was compared. According to the report that the higher the concentration of the chaotropic agent used in the DNA binding buffer, the better the binding power. Two samples were tested according to each concentration, and qPCR was performed with two recombinant DNA samples to compare Ct values according to the number of externally added recombinant DNA copies, and the results for GnHCl are shown in Table 2 and FIG. 2 below. was shown, and the results for GuSCN are shown in Table 3 and FIG. 3 below.

No. Sample Ct (Cycle) number of DNA molecules Tm(℃) One 5 M GnHCl (1) 23.37 76.03 2 5 M GnHCl (2) 23.34 76.03 23.36±0.02 1.6x10 ⁶ 3 6 M GnHCl (1) 24.30 75.70 4 6 M GnHCl (2) 23.38 75.70 23.4±0.65 1.2x10 ⁶ 5 10 ⁷ pSc 20.36 76.36 6 10 ⁵ pSc 28.08 76.03 75.98±0.25

No. Sample Ct(Cycle) number of DNA molecules Tm (℃) One 5 M GuSCN (1) 27.00 75.70 2 5 M GuSCN (2) 26.94 75.70 3 5 M GuSCN (3) 26.95 75.70 26.96±0.03 1.5x10 ⁵ 4 4 M GuSCN (1) 26.25 75.70 5 4 M GuSCN (2) 27.95 75.70 6 4 M GuSCN (3) 29.98 75.70 28.06±1.52 7.3x10 ⁴ 7 5 M GI　(1) 25.95 75.80 8 5M GI　(2) 26.01 76.03 25.98±0.03 2.9x10 ⁵ 9 10 ⁷ pSc 20.90 76.36 10 10 ⁵ pSc 26.92 76.36 75.86±0.27

Referring to Table 2 and Figure 2, the number of gene copies recovered between 5 M and 6 M GnHCl did not show an effective difference between 1.6x10 ⁶ and 1.2x10 ⁶ .

Referring to Table 3 and Figure 3, in the case of the binding buffer prepared with GuSCN, the number of molecules of the gene recovered with 5 M GuSCN binding buffer (1.5x10 ⁵ ) The number of molecules of the gene recovered with 4 M GuSCN binding buffer (7.3x10) ⁴ ), but it was not found to be more effective than 5 M GnHCl binding buffer. Therefore, in the residual gene separation method using the affinity column method, the use of a 4 to 6 M level of GnHCl binding buffer was found to be suitable, and in the subsequent experiment, it was decided to use a 5 M GnHCl binding buffer.

2) Comparison of appropriate DNA binding column for residual DNA isolation from honey

In the separation of residual DNA, the DNA binding capacity of the column is important. Therefore, the difference in DNA binding capacity was confirmed using 5 kinds of affinity columns currently commercially available in Korea, and the results are shown in Table 4 and FIG. 4 below.

No. column Ct(Cycle) Aver. Ct Tm(℃) number of DNA molecules One probio 23.51 75.70 2 25.25 24.38±1.23 75.70 8.3x10 ⁵ 3 viralGene 24.40 76.03 4 23.50 23.95±0.64 76.03 1.1x10 ⁶ 5 pathogen 25.26 76.03 6 25.22 25.24±0.03 75.70 4.7x10 ⁵ 7 Intron-pla 25.20 75.70 8 24.34 24.77±0.61 75.70 6.4x10 ⁵ 9 biosolution 24.30 75.70 10 24.27 24.29±0.02 75.70 8.8x10 ⁵ 75.80±0.16

Referring to Table 4 and Figure 4, the columns of the five manufacturers showed a difference in the number of molecules recovered from 4.7x10 ⁵ to 1.1x10 ⁶ It was confirmed that the column is an important item to be considered when isolating a trace amount of DNA from honey.

3) Selection of appropriate washing buffer for residual DNA isolation from honey

For the purity of the recovered DNA, after binding the DNA to the column, the composition of the washing buffer was changed as follows to confirm the change in the amount of recovered DNA. Washing buffer consists of salt and ethanol, and the results for ethanol 80% (v/v) are shown in Table 5 and Figure 5 below, and the results for ethanol 85% (v/v) are shown in Table 6 (PE) The sample (comparative group) was ethanol 80% (v/v)) and is shown in FIG. 6 .

No. Sample Buffer composition (80% EtOH) Ct cycle number of DNA molecules Tm(℃) One PE (1) 10 mM Tris-HCl (pH7.6) 25.25 76.03 2 PE (2) 26.16 76.03 25.71±0.46 3.4x10 ⁵ 3 A (1) 15 mM Tris-HCl (pH7.6) 25.20 76.03 4 A (2) 25.24 76.03 5 A (3) 25.25 76.03 25.23±0.02 4.7x10 ⁵ 6 B (1) 20 mM Tris-HCl (pH7.6) 25.23 76.03 7 B (2) 25.23 75.70 8 B (3) 26.16 75.70 25.54±0.44 3.8x10 ⁵ 9 1x10 ⁷ Positive DNA 21.23 76.36 10 1x10 ⁵ Positive DNA 27.11 76.36 76.03±0.2

No. Sample Buffer composition (85% EtOH) Ct cycle number of DNA molecules Tm(℃) One PE (1) 10 mM Tris-HCl (pH7.6)
+ 80% EtOH 24.27 75.85 2 PE (2) 26.07 75.85 25.17±0.9 4.9x10 ⁵ 3 A (1) 15mM Tris-HCl (pH7.6) 24.26 75.85 4 A (2) 25.19 75.85 5 A (3) 25.22 75.85 24.89±0.45 5.9x10 ⁵ 6 B (1) 20 mM Tris-HCl (pH7.6) 25.20 75.85 7 B (2) 25.21 75.52 8 B (3) 24.90 75.85 24.90±0.43 5.9x10 ⁵ 9 1x10 ⁷ Positive DNA 20.26 76.51 10 1x10 ⁵ Positive DNA 27.18 76.18 75.91±0.2

Referring to Table 5, Table 6, Figures 5 and 6, when ethanol was fixed at 80% (v/v) and the salt concentration was changed, ethanol was fixed at 85% (v/v) and the salt concentration was As a result of comparison when changed, the salt concentration showed a high recovery rate in 85% (v/v) ethanol at 15 mM. In the residual gene separation method using the affinity column method, the washing buffer contains salt and ethanol, but For 90% (v/v) ethanol, it was determined that the washing buffer with a salt concentration of 10 to 20 mM was suitable, and in subsequent experiments, washing with a salt concentration of 15 mM with respect to 85% (v/v) ethanol was found to be suitable. It was decided to use the buffer.

4) Selection of an appropriate elution buffer for residual DNA isolation from honey

For DNA elution, the last step of the column affinity method, the elution effect between 10 mM Tris-HCl (pH 8.5), TE buffer (10 mM Tris-HCl and 0.5 mM EDTA, pH 8.5) and DW was compared. It is shown in Table 7, Table 8, FIGS. 7 and 8.

No. Sample Elution Buffer Composition Ct cycle number of DNA molecules Tm(℃) One D1 DW 24.27 76.18 2 D2 DW 24.31 76.18 24.29±0.02 8.8x10 ⁵ 3 A1 10 mM Tris-HCl (pH8.5) 24.31 76.18 4 A2 24.30 76.51 5 A3 25.22 76.18 24.61±0.43 7.1x10 ⁵ 6 B1 10 mM Tris-HCl (pH8.5)
+ 0.5 mM EDTA 24.28 76.18 7 B2 24.20 75.85 8 B3 24.23 76.18 24.24±0.03 9.1x10 ⁵ 9 1x10 ⁷ Positive DNA 20.75 76.51 10 1x10 ⁵ Positive DNA 28.20 76.51 76.25±0.2

No. Sample Elution Buffer Composition Ct cycle number of DNA molecules Tm(℃) One D1 DW 25.16 76.03 2 D2 DW 26.20 76.03 25.68±0.52 3.5x10 ⁵ 3 A1 10 mM Tris-HCl (pH9.0) 26.15 76.03 4 A2 25.20 76.03 5 A3 26.13 76.03 25.83±0.44 3.2x10 ⁵ 6 B1 10 mM Tris-HCl (pH9.0)
+ 0.5 mM EDTA 25.20 76.03 7 B2 25.18 75.70 8 B3 25.20 76.03 25.19±0.01 4.8x10 ⁵ 9 1x10 ⁷ Positive DNA 23.11 76.68 10 1x10 ⁵ Positive DNA 30.11 76.03 76.06±0.23

Referring to Table 7 and Figure 7, when elution was performed with only 10 mM Tris-HCl (pH 8.5), the smallest number of molecules was recovered among the three elution buffers used as quantified as 7.1x10 ⁵ DNA, and TE buffer (10 mM Tris- In the case of HCl and 0.5 mM EDTA, pH 8.5) and DW, DNA quantitative values were 8.8x10 ⁵ and 9.1x10 ⁵ , showing no effective difference.

In addition, referring to Table 8 and FIG. 8, when the pH of the salt was elution only with 10 mM Tris-HCl (pH 9.0) prepared in accordance with basicity, it was found to be 3.2x10 ⁵ . In the comparison of TE buffer (10 mM Tris-HCl and 0.5 mM EDTA, pH 9.0) and DW, DNA quantitative values were 4.8x10 ⁵ and 3.5x10 ⁵ , which was different from TE at pH 8.5.

From this, although there was a slight difference in the DNA quantification value depending on the composition and pH of the elution buffer used, it was confirmed that it was suitable for confirming sugar cane-specific gene amplification in the separation of residual genes using the affinity column method.

(3) Detection of sugarcane-specific genes in commercial products

Genes were isolated using 2 ml of 5 product samples as described above and a gene detection method using the same was performed, and the results are shown in Table 9 and FIG. 9 below.

commercial product Ct (Cycle) Tm(℃) Detection Quanti. (μl)
(y=-3.4939x+45.053) A 37.31 75.70 + 8.2x10 ³ B 39.30 67.85 - C ^* 38.27 75.37 + 4.3x10 ³ D 38.25 75.37 + 4.4x10 ³ E 25.63 75.70 + 1.8x10 ⁷

^* Honey made from 100% cane sugar at Gyeonggi University Apiary

Referring to Table 9 and FIG. 9, sugar cane-specific genes were detected and quantified by a gene detection method performed by isolating residual genes of honey using a commercially available affinity column. As a result, four PCR amplicons except for sample B had similar Tm value, and it was confirmed that at least 10 ³ molecules were detected only by detection PCR.

In the present invention, a method for separating the most appropriate remaining genes in honey was established by putting the recombinant DNA produced in honey and examining the recovery rate of each step of gene separation using specific primers of the artificially incorporated recombinant DNA. This is expected to facilitate quality evaluation and management by applying a fast and accurate gene detection method to honey in the future.

Preferred embodiments of the present invention described above are disclosed to solve the technical problem, and various modifications, changes, additions, etc. will be possible within the spirit and scope of the present invention by those of ordinary skill in the art to which the present invention pertains. , such modifications and changes should be regarded as belonging to the following claims.

<110> Kyonggi University Industry & Academia Cooperation Foundation <120> Method of DNA isolation using affinity column in sugar honey <130> NP20-08061 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Cane-dF Primer <400> 1 caccgcaatt atttttattc tgag 24 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Cane-dR Primer <400> 2 gaacatcttg aatccggtat tc 22

Claims (4)

As a residual gene separation method using an affinity column in fermented honey,
(a) using a binding buffer (binding buffer) to induce binding of the residual gene present in the honey to a silica membrane (silica membrane);
(b) loading the residual gene-containing sample into the affinity column including a silica membrane;
(c) removing impurities using a washing buffer; and
(d) recovering the desired residual gene from the affinity column using an elution buffer;
Residual gene separation method using an affinity column in fermented honey containing According to claim 1,
The binding buffer (binding buffer) is guanidine hydrochloride (guanidine hydrochloride, GnHCl), characterized in that the residual gene separation method using an affinity column from the honey. According to claim 1,
The washing buffer includes salt and ethanol, but the concentration of the salt is 10 to 20 mM with respect to 80 to 90% (v/v) of ethanol Residue using an affinity column in fermented honey Gene isolation method. According to claim 1,
The elution buffer is 5 to 15 mM Tris-hydrogen chloride (Tris-HCl), or a mixture of 5 to 15 mM Tris-hydrogen chloride (Tris-HCl) and 0.1 to 1 mM Ethylenediaminetetraacetic acid (EDTA), or distilled water. Residual gene separation method using an affinity column in specification honey, characterized in that.

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