WO2011025329A2

WO2011025329A2 - Detecting methods of dna amplification using new intercalating agent

Info

Publication number: WO2011025329A2
Application number: PCT/KR2010/005840
Authority: WO
Inventors: Jun Hee Lee; Hwang Seo Park; Eun Su Han; Jae Don Lee; Han Oh Park
Original assignee: Bioneer Corp
Current assignee: Bioneer Corp
Priority date: 2009-08-31
Filing date: 2010-08-30
Publication date: 2011-03-03
Anticipated expiration: 2012-02-29
Also published as: WO2011025329A3; KR20110023265A; KR101438747B1

Abstract

Disclosed are a fluorescent composition for staining DNA and a method for measuring DNA amplification using the same. More particularly, disclosed are a novel fluorescent compound that emits light by intercalating with DNA and a method for measuring DNA amplification using the same. The novel fluorescent compound is advantageous in that, due to high sensitivity, target-specific amplification products by real-time polymerase chain reaction(PCR) can be accurately quantified without affecting the PCR reaction. In addition, the compound is unharmful to the human body, can be prepared at low cost, and allows monitoring of various DNA and RNA reactions in vitro or in vivo.

Description

Title of Invention: DETECTING METHODS OF DNA AMPLIFICATION USING NEW INTERCALATING AGENT

Technical Field

[1] The present disclosure relates to a novel fluorescent composition for staining DNA and a method for measuring DNA amplification using the same. More particularly, the disclosure relates to a novel fluorescent compound that emits light by intercalating with DNA and a method for measuring DNA amplification using the same.

Background Art

[2] Currently available methods for detecting DNA amplification are polymerase chain reaction(PCR), which is an end-point detection technique, and real-time PCR.

According to the end-point detection technique PCR, after performing PCR on agarose gel, the PCR product is analyzed to attain information of the amplification. Usually, the observation is made with unaided eyes by comparing amplification at specific sites with standard material(DNA size marker).

[3] However, since the observation with eyes may be different among individuals, base sequencing or Southern blotting using an isotope-labeled probe is required if a more accurate analysis is desired. Although this gives information about the band specificity in addition to the length of the amplified sites, the procedure is complicated and requires a lot of time. Further, to confirm the specificity, the degree of

homology(stringency) necessary for probe binding needs to be controlled variously. Further, there are a lot of problems in quantification of DNA in samples.

[4] In order to solve the problems associated with the end-point detection method, the real-time PCR technique was developed in which signals from each PCR cycle are analyzed to accurately calculate the concentration of DNA in sample, and a fluorescent DNA dye that binds specifically to double-stranded DNA is used to attain the PCR result quickly, accurately and with good sensitivity. By combining the advantage of PCR of amplifying a small amount of DNA quickly and specifically with accurate quantification, the real-time PCR gives results accurately and quickly by automating many simple, repeating procedures and reducing errors that may occur.

[5] In the real-time PCR, a fluorescent reporter is used to monitor the overall reaction.

The fluorescent material increases in proportion to amplification products accumulating with increasing amplification cycles. Although the increase in fluorescence intensity is not detected initially, it begins to be detected after a certain number of amplification cycles. The amplification cycle at which the fluorescence from a sample crosses a minimum detectable level (background level) is called the threshold cycle C_τ. [6] The logarithm of an initial amount of a template is linearly proportional to the threshold cycle. Thus, a standard curve may be drawn using a sample with a known initial amount of DNA, and the initial amount of DNA in an unknown sample may be accurately calculated using the standard curve.

[7] As methods for accurately calculating the concentration of DNA in a sample from the fluorescence intensity measured at each cycle of real-time PCR, TaqMan probe chemistry (Holland et al., 1991, Proc. Natl. Acad. Sci. USA 88:7276-7280; Leeetal., 1993, Nucleic Acids /tes.21:3761-3776),use of a molecular beacon(Tyagi & Kramer 1996, Nature Biotech. 14:303-309, USP 5,119,801,USP

5 ,312,728),useofanintercalatingreagentsuchasS YBRGreenlbetweendouble-strandedDN A,useofadjacenthybridizationprobes(USP 5,210,015),or the like are known.

[8] The method using a TaqMan probe(TaqMan chemistry) does not provide information about size because gel-based analysis is not performed. However, since the highly specific TaqMan probe is used, sequence information of amplification product can be attained with a single reaction of about 3 hours and the quantity of DNA in sample can be accurately determined.

[9] Yet, because an oligonucleotide having a base sequence at lest 20 bases long is used to manufacture the TaqMan probe, the fluorescence may not be perfectly quenched. Further, the method is costly.

[10] The analysis method using a molecular beacon probe is used to analyze a specific

DNA sequence or to detect RNA in a living cell. However, it is difficult to separate the probe from a target hybrid for example when detecting a target in a living cell(Matsuo, T. 1998. In situ visualization of messenger RNA for basic fibroblast growth factor in living cells. Biochim. Biophys. Acta 1379:178-184). Further, since the probe should be at least 30 to 40 bases long, the probe is difficult to manufacture and costs a lot.

[11] In the method using adjacent hybridization probes, a pair of specially designed

probes are used. The probes are designed such that hybridization in a head-to-tail arrangement is possible on a target sequence of an amplified DNA fragment. However, the procedure is complicated because four primers are required and specificity is low (Heller, MJ. and L.E. Morrison. 1985. Chemiluminescent and fluorescent probes for DNA hybridization, p. 245-256. In D.T. Kingsbury and S. Flakow(Eds.), Rapid Detection and Identification of Infectious Agents. Academic Press, New York).

[12] Another analysis method uses an intercalating dye such as SYBR Green I, Foerst

33228, ethidium bromide(EtBr), etc. The method can be applied to wide range of samples because SYBR Green I can bind to double-stranded DNA without special target sequences. However, the method has a demerit that amplification products of primers only, non-specific amplification products, or the like are easily produced. Because of these background noises, it is needed to measure melting temperatures(T_m) of the amplification products and confirm the PCR products through melting curve analysis(Higuchi, R., Dollinger, G., Walsh, P.S., and Griffith, R. 1992. Simultaneous amplification and detection of specific DNA sequences, Biotechnology 10:413, Higuchi, R., Fockler, C, Dollinger, G., and Watson, R., 1993. Kinetic PCR: Real monitoring of DNA amplification reactions. Biotechnology W:\Q26).

Disclosure of Invention

Technical Problem

[13] The inventors of the present disclosure have carried out researches in order to solve the aforesaid problems. As a result, they have found a novel fluorescent compound which has superior detection sensitivity, can be prepared at low cost, is unharmful to the human body and is applicable to real-time polymerase chain reaction.

[14] The present disclosure is directed to providing a novel fluorescent intercalating

compound exhibiting change in fluorescence when bound to double-stranded DNA and thus allowing detection.

[15] The present disclosure is also directed to providing a method for real-time detection of DNA amplification products using the fluorescent intercalating compound.

Solution to Problem

[16] In one general aspect, there is provided a composition for staining DNA including a fluorescent compound which having an absorption wavelength of 418 to 480 nm, an excitation wavelength of 426 to 510 nm and an emission wavelength of 500 to 625 nm.

[17] In another general aspect, there is provided a composition for staining DNA

including one or more fluorescent compound(s) selected from Compounds 1 to 5:

[18] [Compound 1 ]

[19] [Compound 2 ]

[2°] [Compound 3 ]

[21] [Compound 4 ]

[22] [Compound 5 ]

[23] In another general aspect, there is provided a method for detecting DNA amplification, including: (1) adding a target DNA sample to a buffer solution containing a first oligonucleotide and a second oligonucleotide each having a base sequence complementary to the base sequence of each strand of the target DNA, the fluorescent compound which is intercalated with the double-stranded DNA and exhibits change in fluorescence, nucleotide monomers and a DNA polymerase; (2) amplifying the target DNA using the DNA polymerase under a polymerase chain reaction(PCR) condition; and (3) measuring the fluorescence resulting from the intercalation of the fluorescent compound in the amplified DNA.

[24] In another general aspect, there is provided a reaction mixture for detection of DNA amplification comprising a first oligonucleotide and a second oligonucleotide each having a base sequence complementary to the base sequence of each strand of the target DNA, the fluorescent compound which is intercalated with the double-stranded DNA and exhibits change in fluorescence, nucleotide monomers and a DNA polymerase.

Advantageous Effects of Invention

[25] The fluorescent compound according to the present disclosure is a fluorescent intercalating compound exhibiting fluorescence when bound to double-stranded DNA and thus enabling detection thereof. It is advantageous in that, due to high sensitivity, target-specific amplification products by real-time polymerase chain reaction(PCR) can be accurately quantified without affecting the PCR reaction.

[26] The fluorescent compound according to the present disclosure is unharmful to the human body, can be prepared at low cost, and allows monitoring of various DNA and RNA reactions in vitro or in vivo.

Brief Description of Drawings

[27] The above and other objects, features and advantages of the present disclosure will become apparent from the following description of certain exemplary embodiments given in conjunction with the accompanying drawings, in which:

[28] Fig. 1 shows a result of scanning absorbance of the novel fluorescent compound 1 according to the present disclosure;

[29] Fig. 2 shows a result of scanning absorbance of the novel fluorescent compound 2 according to the present disclosure;

[30] Fig. 3 shows a result of scanning absorbance of the novel fluorescent compound 3 according to the present disclosure;

[31] Fig. 4 shows a result of scanning absorbance of the novel fluorescent compound 4 according to the present disclosure;

[32] Fig. 5 shows a result of scanning absorbance of the novel fluorescent compound 5 according to the present disclosure;

[33] Fig. 6 shows a result of measuring excitation wavelengths and emission wavelengths of the novel fluorescent compound 1 according to the present disclosure;

[34] Fig. 7 shows a result of measuring excitation wavelengths and emission wavelengths of the novel fluorescent compound 2 according to the present disclosure;

[35] Fig. 8 shows a result of measuring excitation wavelengths and emission wavelengths of the novel fluorescent compound 3 according to the present disclosure;

[36] Fig. 9 shows a result of measuring excitation wavelengths and emission wavelengths of the novel fluorescent compound 4 according to the present disclosure;

[37] Fig. 10 shows a result of measuring excitation wavelengths and emission

wavelengths of the novel fluorescent compound 5 according to the present disclosure;

[38] Fig. 11 shows a result of monitoring change in fluorescence resulting from the intercalation of the novel fluorescent compound 1 according to the present disclosure with DNA;

[39] Fig. 12 shows a result of monitoring change in fluorescence resulting from the intercalation of the novel fluorescent compound 2 according to the present disclosure with DNA;

[40] Fig. 13 shows a result of monitoring change in fluorescence resulting from the intercalation of the novel fluorescent compound 3 according to the present disclosure with DNA;

[41] Fig. 14 shows a result of monitoring change in fluorescence resulting from the intercalation of the novel fluorescent compound 4 according to the present disclosure with DNA;

[42] Fig. 15 shows a result of monitoring change in fluorescence resulting from the intercalation of the novel fluorescent compound 5 according to the present disclosure with DNA;

[43] Fig. 16 shows a result of identifying the effect of the addition of the novel fluorescent compounds according to the present disclosure on polymerase chain reaction(PCR) by means of electrophoresis(l: Compound 1, 2: Compound 2, 3: Compound 3, 4:

Compound 4, 5: Compound 5);

[44] Fig. 17 shows a result of melting curve analysis for real-time PCR using the novel fluorescent compound 1 according to the present disclosure;

[45] Fig. 18 shows a result of melting curve analysis for real-time PCR using the novel fluorescent compound 2 according to the present disclosure;

[46] Fig. 19 shows a result of melting curve analysis for real-time PCR using the novel fluorescent compound 3 according to the present disclosure;

[47] Fig. 20 shows a result of melting curve analysis for real-time PCR using the novel fluorescent compound 4 according to the present disclosure;

[48] Fig. 21 shows a result of melting curve analysis for real-time PCR using the novel fluorescent compound 5 according to the present disclosure;

[49] Fig. 22 shows a result of analyzing linearity, threshold cycles and efficiency of realtime PCR using the novel fluorescent compound 1 according to the present disclosure;

[50] Fig. 23 shows a result of analyzing linearity, threshold cycles and efficiency of realtime PCR using the novel fluorescent compound 2 according to the present disclosure;

[51] Fig. 24 shows a result of analyzing linearity, threshold cycles and efficiency of realtime PCR using the novel fluorescent compound 3 according to the present disclosure;

[52] Fig. 25 shows a result of analyzing linearity, threshold cycles and efficiency of realtime PCR using the novel fluorescent compound 4 according to the present disclosure;

[53] Fig. 26 shows a result of analyzing linearity, threshold cycles and efficiency of realtime PCR using the novel fluorescent compound 5 according to the present disclosure;

[54] Fig. 27 shows denaturation curves following real-time PCR using the novel fluorescent compound 1 according to the present disclosure;

[55] Fig. 28 shows denaturation curves following real-time PCR using the novel fluorescent compound 1 according to the present disclosure;

[56] Fig. 29 shows denaturation curves following real-time PCR using the novel fluorescent compound 2 according to the present disclosure;

[57] Fig. 30 shows denaturation curves following real-time PCR using the novel flu- orescent compound 2 according to the present disclosure;

[58] Fig. 31 shows denaturation curves following real-time PCR using the novel fluorescent compound 3 according to the present disclosure;

[59] Fig. 32 shows denaturation curves following real-time PCR using the novel fluorescent compound 3 according to the present disclosure;

[60] Fig. 33 shows denaturation curves following real-time PCR using the novel fluorescent compound 4 according to the present disclosure;

[61] Fig. 34 shows denaturation curves following real-time PCR using the novel fluorescent compound 4 according to the present disclosure.

[62] Fig. 35 shows denaturation curves following real-time PCR using the novel fluorescent compound 5 according to the present disclosure;

[63] Fig. 36 shows denaturation curves following real-time PCR using the novel fluorescent compound 5 according to the present disclosure.

Best Mode for Carrying out the Invention

[64] Hereinafter, the embodiments of the present disclosure will be described in detail.

However, the present disclosure is not limited by the following embodiments. Those skilled in the art will appreciate that various changes or modifications may be made thereto within the spirit and scope of the disclosure.

[65] Unless defined otherwise, all terms(including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art. In the following description and the attached drawings, details of well-known features and techniques are omitted to avoid unnecessarily obscuring the presented embodiments.

[66] The present disclosure provides a novel fluorescent compound which exhibits change in fluorescence when intercalated with double-stranded DNA and a method for realtime detection of DNA amplification products using the novel fluorescent compound.

[67] The disclosure provides a novel fluorescent compound which exhibits change in fluorescence when intercalated with double-stranded DNA.

[68] The novel fluorescent compound according to the present disclosure has an absorption wavelength of 418 to 480 nm, an excitation wavelength of 426 to 510 nm and an emission wavelength of 500 to 625 nm.

[69] The inventors measured absorbance with a UV spectrometer(Shimadzu, Japan) at

250 to 700 nm for commercially available compound libraries. As a result, they could screen five novel fluorescent compounds exhibiting highest absorbance at 418 nm(Compound 1), 420 nm(Compounds 2 and 3), 419 nm(Compound 4) and 480 nm(Compound 5). See Fig. 1.

[70] They also measured optimal emission and excitation wavelengths of the five screened fluorescent compounds. Compound 1 had an excitation wavelength of 445 nm to 455 nm and an emission wavelength 510 to 625 nm. Compound 2 had an excitation wavelength of 426 nm to 446 nm and an emission wavelength of 500 to 550 nm. Compound 3 had an excitation wavelength of 470 to 490 nm and an emission wavelength of 520 to 600 nm. Compound 4 had an excitation wavelength of 470 to 488 nm and an emission wavelength 500 to 570 nm. Compound 5 had an excitation wavelength of 490 to 5iO nm and an emission wavelength 550 to 580 nm. See Fig. 2.

[71] The screened novel fluorescent compounds are Compounds 1 to 5.

[72] [Compound 1 ]

[73] [Compound 2]

[74] [Compound 3]

[75] [Compound 4]

[76] [Compound 5 ]

[77] Compounds 1 to 5 may be used for staining, labeling or detection of DNA. The novel compounds are intercalated with double-stranded DNA.

[78] More specifically, Compounds 1 to 5 may be intercalated with double-stranded DNA by a procedure wherein (1) a DNA sample is denatured at high temperature and split into single strands; (2) the single-stranded DNA sample is hybridized with a first oligonucleotide and a second oligonucleotide; and (3) as the single-stranded DNA sample is replicated into double-stranded DNA by a DNA polymerase, Compounds 1 to 5 are intercalated between the bases of the DNA. The change in fluorescence resulting from the intercalation is measured for the purpose of staining, labeling or detection of DNA.

[79] Typically, the first oligonucleotide and the second oligonucleotide are a pair of

primers.

[80] The present disclosure also provides a method for detecting DNA amplification, including: (1) adding a target DNA sample to a buffer solution containing a first oligonucleotide and a second oligonucleotide each having a base sequence complementary to the base sequence of each strand of the target DNA, the fluorescent compound which is intercalated with the double-stranded DNA and exhibits change in fluorescence, nucleotide monomers and a DNA polymerase; (2) amplifying the target DNA using the DNA polymerase under a polymerase chain reaction (PCR) condition; and (3) measuring the fluorescence resulting from the intercalation of the fluorescent compound in the amplified DNA.

[81] Real-time PCR may be performed using Compounds 1 to 5 by a procedure wherein

(1) a target DNA sample is added to a buffer solution containing a first oligonucleotide and a second oligonucleotide each having a base sequence complementary to the base sequence of each strand of the target DNA, Compounds 1 to 5 which are intercalated with the double-stranded DNA and exhibit change in fluorescence, nucleotide monomers and a DNA polymerase; (2) the DNA sample is split into single strands and hybridized with the first oligonucleotide and the second oligonucleotide; (3) as the hybridized DNA sample is replicated by the DNA polymerase; and (4) fluorescence is measured at each cycle of the replication. Based on the real-time PCR result, a standard curve may be drawn. [82] In case RNA is desired to be detected, a reverse transcriptase may be used to synthesize cDNA, which is then hybridized with the first oligonucleotide and the second oligonucleotide. After performing replication using a DNA polymerase, the replicated DNA is split into single strands and the change in fluorescence resulting from the intercalation with Compounds 1 to 5 is detected. Accordingly, the present disclosure is also capable of quantitatively analyzing the amplification of RNA.

[83] Thus, the present disclosure provides a novel fluorescent compound allowing homogeneous analysis, detection and quantification of DNA amplification by a DNA polymerase in real-time PCR. As the By fluorescent compound is intercalated between the bases of DNA during replication thereof from single strands, the fluorescence emission increases in proportion to the quantity of the DNA amplification products. Hence, the DNA amplification can be detected quantitatively.

Mode for the Invention

[84] The examples and experiments will now be described. The following examples and experiments are for illustrative purposes only and not intended to limit the scope of the present disclosure.

[85] [Example 1] Screening of DNA fluorescent compounds

[86] Compound libraries were purchased from InterBioScreen, ASINEX, Specs,

ChemDiv, etc. The compounds were completely dissolved in DMSO (Sigma, USA) and diluted in Ix polymerase solution, and absorbance was measured using a UV spectrometer (Shimadzu, Japan).

[87] The absorbance measurement was made as follows. After mixing the compound(0.2 mL) with Ix polymerase reaction buffer(20 mM Tris-HCl, 30 mM KCl, 1.5 mM MgCl 2, 1.8 mL), absorbance was scanned using the UV spectrometerinawavelength range from 250 to 700 run.

[88] As seen from Fig. 1, five fluorescent compounds could be screened in the absorption wavelength range from 418 to 480 nm, as described in Table 1. The fluorescent compounds showed highest absorbance at 418 nm(Compound 1), 420 nm(Compounds 2 and 3), 419 nm(Compound 4) and 480 nm(Compound 5), respectively.

[89] [Table 1 ] Novel fluorescent compounds

[90] Optimal emission and excitation wavelengths of the five screened fluorescent

compounds were identified as follows. In a 1.5 mL tube, Ix polymerase reaction buffer(20 mM Tris-HCl, 30 mM KCl, 1.5 mM MgCl₂, 1.8 mL),each

compound(100μL)and TE buffer(10 mM Tris-HCl, 1 mM EDTA,100 μL)were added to make a final volume of 2 mL. Then, excitation and emission wavelengths(nm) of the compounds were measured using a spectrofluorophotometer. In Fig. 2, the X-axis represents a scan wavelength(nm) and the Y-axis represents fluorescence intensity measured at the corresponding scan wavelength. Peaks 1 denote excitation and peaks 2 denote emission.

[91] As seen from Fig. 2, Compound 1 had an excitation wavelength of 445 to 455 nm and an emission wavelength of 510 to 625 nm. Compound 2 had an excitation wavelength of 426 to 446 nm and an emission wavelength of 500 to 550 nm.

Compound 3 had an excitation wavelength of 470 to 490 nm and an emission wavelength of 520 to 600 nm. Compound 4 had an excitation wavelength of 470 to 488 nm and an emission wavelength of 500 to 570 nm. Compound 5 had an excitation wavelength of 490 to 510 nm and an emission wavelength of 550 to 580 nm.

[92] [Example 2] Measurement of change in fluorescence resulting from intercalation with DNA

[93] The change in emission energy resulting from the intercalation of the five novel fluorescent compounds screened in Example 1 was measured using a spectrofluo- rophotometer as follows.

[94] Two 1.5 mL tubes were prepared. In tube 1, Ix polymerase reaction buffer (20 mM

Tris-HCl, 30 mM KCl, 1.5 mM MgCl₂, 1.8 mL), each compound(100 μL)and TE buffer(10 mM Tris-HCl,l mM EDTA, 100 μL)were added to make a final volume of 2 mL. In tube 2, Ix polymerase reaction buffer (10 mM Tris-HCl, 60 mM KCl, 1.5 mM MgCl₂, 1.8mL),each compound(100μL)and lambda DNA(Bioneer, Korea, 100 μL(50 ng/μL))were added to make a final volume of 2 mL. Each tube was subjected to de- naturation at 94°C for 5 minutes and then cooled slowly at room temperature for hybridization.

[95] Then, excitation and emission wavelengths (nm) of the compounds were measured using a spectrofluorophotometer. In Fig. 3, the X-axis represents a scan

wavelength(nm) and the Y-axis represents fluorescence intensity measured at the corresponding scan wavelength.

[96] Peaks 1 are for a sample without lambda DNA added thereto. Peaks 1-1 denote excitation and peaks 1-2 denote emission.

[97] Peaks 2 are for a sample with lambda DNA added thereto. Peaks 2-1 denote excitation and peaks 2-2 denote emission.

[98] As seen from Fig. 3, the sample with lambda DNA added thereto showed increased emission energy as compared to the sample without lambda DNA added thereto.

[99] [Example 3] Polymerase chain reaction(PCR) using the novel fluorescent compounds

[100] (I) Preparation of template DNA and designing of primers

[101] First, a template DNA was prepared to perform real-time PCR using the novel fluorescent compounds. The 51st, 830 bp sequence in Plasmodium vivax 18S ribosomal RNA gene(GenBank Accession No.U93233) including primer and probe sequences was genetically synthesized(B iochem. Biophys. Res. Commun. 1998, 248, 200-203 )and cloned into pGEM-T-Easyvector(Cat:A1360,Promega,USA). [102] Specifically, 2x rapid ligation buffer(Promega, USA, 5 μL), T-Easy vector(Promega, USA, 1 μL), T4 DNA ligase(Promega, USA, 1 μL) and genetic synthesis product(3 μL, 8 ng) were mixed in a tube and kept at 37 ⁰C for 1 hour. Then, after adding the resulting solution (5 μL) to E.coli competent cells(50 μL) and placing on ice for 30 minutes, the cells were cultured at 42⁰C for 90 seconds and then placed on ice for 2 minutes. After placing the solution on an LB plate containing ampicillin, IPTG and X- GaI, the cells were incubated at 37°C for 16 hours.

[103] A white colony was taken from the LB plate and cultured in a liquid LB medium for about 16 hours. After centrifuge, the supernatant was discarded and plasmid DNA was extracted from the remaining pellet using AccuPrep plasmid prep kit(Bioneer, Korea).

[104] Concentration and purity of the extracted plasmid DNA were measured using a UV spectrometer(Shimadzu, Japan). Purity was between 1.8 to 2.0. Number of DNA copies was calculated according to the following equation based on the concentration measurement.

[105] 6.02 x 1023 x concentration [concentration(g/mL) measured with the UV spectrometer] / (3015 + 830) x 660

[106] [T-easy vector: 3015 bp, malaria template DNA: 830 bp]

[107] The template DNA was serially diluted 10-fold using Ix TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) and then stored at -70⁰C until use.

[108] Primers that can complementary bind to the template DNA were designed as in Table 2.

[109] [Table 2] Primers

[110] (2) PCR

[111] As a PCR test group, 1 Ox reaction buffer( 100 mM Tris-HCl,600 mM KCl, 15 mM

MgCl₂,5μL)and 10 mM dNTP mixture solution(2.5 mM dATP, 2.5 mM dGTP,2.5 mM dCTP, 2.5 mM dTTP, 5μL)were added to a PCR reaction tube. Then, Top DNA polymerase(Bioneer, Korea, 1 unit) was added.

[112] After adding the template DNA prepared in (1) and the novel fluorescent

compound(l μL) to the PCR reaction tube containing the 10x reaction buffer, 10 mM dNTP mixture solution and Top DNA polymerase, sterilized distilled water was added to make a final volume of 50 μL. As a control group, 10x reaction buffer, 10 mM dNTP mixture solution, Top DNA polymerase, the template DNA prepared in (1) and sterilized distilled water were added to a PCR reaction tube, without the novel flu- orescent compound.

[113] The PCR reaction tubes of the control group and the test group were loaded in a PCR system(Mygenie 96, Bioneer, Korea) and subjected to PCR under the condition of de- naturation at 95°C for 10 minutes followed by 45 cycles of 20 seconds at 95°C, 20 seconds at 55⁰C and 30 seconds at 72°C. After the reaction was completed, bands were identified by electrophoresis on 1% agarose gel.

[114] As seen from Fig. 4, there was no difference in bands of the reaction product between the test group and the control group. This result reveals that none of the five novel fluorescent compounds affect PCR.

[115] [Example 4] Real-time PCR using the novel fluorescent compounds

[116] The novel fluorescent compound of Example 1(0.5 μL), the primers synthesized in Example 3(10 nM each), 10x reaction buffer(100 mM Tris-HCl, 600 mM KCl, pH 9.0, 15 mM MgCl₂,5μL),10 mM dNTP mixture solution(2.5 mM dATP,2.5 mM dGTP,2.5 mM dCTP,2.5 mM dTTP, 5μL)and 20 mM MgCl₂ were added to a real-time PCR tube. Then, after adding Top DNA polymerase(Bioneer, Korea, 1 unit) and the template DNA prepared in Example 3, sterilized distilled water was added to make a final volume of 50 μL.

[117] Real-time PCR was carried out using IQ 5 Real-Time PCR(Bio-Rad, USA). SYBR Green I, which has excitation and emission wavelengths similar to those identified in Example 1, was used. Real-time PCR condition was denaturation at 95°C for 10 minutes followed by 45 cycles of 20 seconds at 95°C and 30 seconds at 55°C.

[118] As seen from Fig. 5, signals increased with the real-time PCR cycles.

[119] Also, Fig. 6 shows that R²,which is the correlation coefficient of the standard curve for real-time PCR, is close to 1. This means that the PCR proceeded normally.

[120] The fluorescent compounds resulted in slopes -2.7 or greater and R²0.980orlarger.

This reveals that the novel fluorescent compounds according to the present disclosure do not affect PCR and can be practically used for real-time quantification of DNA staining reagents.

[121] For the real-time PCR products, denaturation curve analysis was made by measuring change in fluorescence intensity at every second while increasing temperature from 55 to 95°C by 1°C.

[122] As seen from Fig. 7, denaturation was identified, which is due to the intercalation with the double-stranded DNA. A single denaturation curve reveals that the same target was amplified in all samples. Two or more denaturation curves result if there are amplification products of primers only, non-specific amplification products, or the like.

[123] The present application contains subject matter related to Korean Patent Application No. 10-2009-0081031, filed in the Korean Intellectual Property Office on August 31, 2009, the entire contents of which is incorporated herein by reference. Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present disclosure. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the disclosure as set forth in the appended claims.

Claims

Claims [Claim 1] A composition for staining DNA comprising a fluorescent compound which having an absorption wavelength of 418 to 480 nm, an excitation wavelength of 426 to 510 nm and an emission wavelength of 500 to 625 nm. [Claim 2] A composition for staining DNA comprising one or more fluorescent compound(s) selected from Compounds 1 to 5:

[Compound 1]

[Compound 2]

[Compound 4]

[Compound 5 ]

[Claim 3] The composition for staining DNA according to claim 1 or 2, wherein the fluorescent compound is used for staining, labeling or detection of DNA.

[Claim 4] The composition for staining DNA according to claim 3, wherein the fluorescent compound is intercalated with double-stranded DNA.

[Claim 5] A method for detecting DNA amplification comprising:

adding a target DNA sample to a buffer solution containing a first oligonucleotide and a second oligonucleotide each having a base sequence complementary to the base sequence of each strand of the target DNA, the fluorescent compound according to claim 1 which is intercalated with the double-stranded DNA and exhibits change in fluorescence, nucleotide monomers and a DNA polymerase;

amplifying the target DNA using the DNA polymerase under a polymerase chain reaction (PCR) condition; and measuring the fluorescence resulting from the intercalation of the fluorescent compound according to claim 1 in the amplified DNA.

[Claim 6] The method for detecting DNA amplification according to claim 5, wherein the fluorescent compound is one or more selected from

Compounds 1 to 5:

[Compound 1]

[Compound 2]

[Compound 3]

[Compound 4]

[Compound 5]

[Claim 7] The method for detecting DNA amplification according to claim 5, wherein the fluorescent compound exhibits change in fluorescence as it is intercalated with double-stranded DNA.

[Claim 8] A reaction mixture for detection of DNA amplification comprising a first oligonucleotide and a second oligonucleotide each having a base sequence complementary to the base sequence of each strand of the target DNA, the fluorescent compound according to claim 1 which is intercalated with the double-stranded DNA and exhibits change in fluorescence, nucleotide monomers and a DNA polymerase.