KR101517493B1 - Efficient Multiplex PCR method using fluorescent markers and mobility markers - Google Patents

Abstract

The present invention relates to an efficient multiplex PCR method using a fluorescent labeling substance and a mobile marker. More specifically, in order to attempt two or more PCRs having different targets in one reaction solution, two or more primer sets are used, In the multiplex PCR in which the above-mentioned primers are labeled with different labeling substances according to the respective primer sets to distinguish the PCR amplification products according to the target, a combination of 6-FAM, JOE, ALEXA 546 and ALEXA 568 Lt; RTI ID = 0.0 > PCR < / RTI >
According to the present invention, the PCR amplification products for each target can be clearly identified even in the multiplex PCR of four or more targets, and it is possible to detect multiple targets such as genetic testing, identification of organisms in the sample, If it is necessary to confirm the presence of DNA or RNA, accurate results can be obtained quickly and easily. In particular, even when a short repeated sequence is targeted, amplification can be effectively confirmed.

Description

(Efficient Multiplex PCR method using fluorescent markers and mobility markers)

The present invention relates to an efficient multiplex PCR method using a fluorescent labeling substance and a mobile marker, and specifically, a method of identifying an amplification product produced by a PCR reaction as a labeling substance by labeling a primer with a labeling substance is used. Characterized in that only one type of labeling substance is used and at least one of the primers used for amplification of one target is labeled with a labeling substance and 6-FAM, JOE, ALEXA 546 and ALEXA 568 are used as labeling substances Multiple PCR methods.

Multiplex PCR (polymerase chain reaction) is a method that can simultaneously amplify multiple target DNAs or RNAs through a single reaction, and can be performed relatively quickly and easily compared with other methods. Therefore, genetic testing, , Microorganisms or viruses.

The most commonly used method in multiplex PCR method is to design amplification product by designing primer so that PCR amplification product size differs according to target, and it is a method to distinguish amplification of the target by amplification product. In this case, The number of genes that can be amplified is generally only three to four. This is due to the competition or interferences between the primers, but since the general DNA polymerase exhibits the optimal amplification efficiency in polymerization within 1 kb, the selection range is very limited in order to be able to distinguish amplification products within this range , And the size of desired gene amplification products may overlap in consideration of the optimum temperature condition among the primers and the concentration of the reactants.

In order to diagnose a microorganism or a virus infection, PCR should be performed on a large number of targets. If only a small number of targets can be identified through one PCR reaction, PCR is repeated several times while changing the target. Time, cost, manpower, and the like are required. This can be a major drawback, especially in the case of infection diagnosis, since the results must be checked quickly.

To solve this problem, Korean Patent Registration No. 10-0277289 discloses a method for expanding the number of targets of multiplex PCR by using silver salt, radioactive, dye, etc. However, these methods are more effective than conventional agarose electrophoresis A lot of time and skill is required for the system, and the system can not be greatly improved in terms of actually increasing the number of targets.

In addition, when multiplex PCR is performed with a short repeating sequence (STR, short tandem repeat) as a target, PCR amplification products are produced not only in a single size but in various sizes, It becomes very difficult.

Therefore, the inventors of the present invention have developed a method for performing multiple PCR on a large number of targets at once by making it possible to more clearly distinguish each PCR amplification product through an easier, quicker, and more efficient method for performing multiplex PCR. Research efforts.

As a result, 6-FAM, JOE, ALEXA 546, and ALEXA 568 were used as the fluorescent material to be applied to the primer according to the target, while the fluorescent material used for labeling DNA, RNA or protein was applied to the primer Multiple PCRs can be used to clearly distinguish at least four target amplifications, even if the PCR products are of the same or similar size, and the mobility markers, ie, the hexaethylenes that can control the molecular weight of the PCR product and have little effect on the PCR reaction It is possible to clearly identify more types of target amplification than when the oxide is applied as a linker of a primer and a fluorescent substance. In particular, when this method is introduced into a multiplex PCR which targets a short repeated sequence, The present inventors have confirmed that amplification can be efficiently identified and the present invention has been completed.

Korean Patent No. 10-0277289

Accordingly, a main object of the present invention is to provide a method for clearly analyzing the results by performing multiplex PCR on many types of targets at a time.

It is another object of the present invention to provide an optimal combination of primer-labeled phosphor combinations.

According to one aspect of the present invention, in a multiplex PCR in which two or more PCRs having different targets are attempted in one reaction solution, the primer is labeled with a labeling substance to identify the amplification product generated by the PCR reaction as a labeling substance Methods are used, one target uses only one kind of labeling substance, One or more of the primers used for the amplification of one target are labeled with a labeling substance,

As the labeling substance

6-carboxyfluorescein of formula 1;

6-carboxy-2 ', 7'-dimethoxy-4', 5'-dichlorofluorescein of formula (2);

6 - [2-carboxy-5 - [[2 - [(5-carboxypentyl) amino] -2-oxoethyl] thio] -3,4,6-trichlorophenyl] , 4,8,9,10,11-octahydro-2,2,4,8,10,10-hexamethyl-12,14-disulfo-pyrano [3,2-g: 5,6-g ] Diquinolin-13-ium {6- [2-carboxy-5 - [[2 - [(5-carboxypentyl) amino] -2-oxoethyl] thio] -3,4,6-trichlorophenyl] 3,4,8,9,10,11-octahydro-2,2,4,8,10,10-hexamethyl-12,14-disulfo-pyrano [3,2-g: 5,6-g] diquinolin- 13-ium}; And

2, 10, 10-tetramethyl-4,8-bis (sulfomethyl) -2H-pyrano [3,2-g: 5,6-g] di Quinolin-6-yl] -benzene dicarboxylic acid {[1,10-dihydro-2,2,10,10-tetramethyl-4,8-bis (sulfomethyl) -2H- pyrano [3,2- , 6-g] diquinolin-6-yl] -benzenedicarboxylic acid}.

≪ Formula 1 >

(2)

(3)

≪ Formula 4 >

In the multiplex PCR method of the present invention, when one labeling substance is used for two or more targets, the linker is included between the primer and the labeling substance to adjust the molecular weight of the PCR amplification product. In this case, the use of hexaethylene oxide . The molecular weight of the PCR product can be controlled by using hexaethylene oxide as one unit and repeating one or two or more of the units. Represents the molecular weight difference corresponding to about 2.5 bp of DNA per unit of hexaethylene oxide.

The labeling substance of Formula 1 is also known as 6-FAM, the labeling substance of Formula 2 is JOE, the labeling substance of Formula 3 is ALEXA 546, and the labeling substance of Formula 4 is ALEXA 568, have. Methods for labeling these marker substances on primers are well known in the art. Any person skilled in the art can easily carry out labeling through manuals provided by the manufacturer, and thus a detailed description thereof will be omitted.

In order to amplify the target DNA or RNA, two kinds of primers having different sequences are usually used. When the target is amplified by PCR, the primer is included in the amplification product. Therefore, even if only one of the two primers is labeled, It becomes a mark on the product. Therefore, only one primer per target can be used for labeling, and if necessary, both primers can be labeled.

Because multiple PCRs target multiple targets, they often target four or more different targets. When there are four targets, 6-FAM, JOE, ALEXA 546, and ALEXA 568 may be labeled on the primers corresponding to each target. When the target is larger than the target, the primers corresponding to the targets are divided into four groups, It can be labeled with a labeling substance.

In this case, when the target belonging to the same group is amplified, the amplification product is labeled with the same labeling substance, and it is difficult to clearly distinguish whether the target is amplified only by the identification of the labeling substance (see FIG. 5) The amplification product can be separated by using a separate amplification product sorting method such as a method of changing the amplification product. For this purpose, a method of preparing a primer by setting a target region narrower or wider in order to vary the size of the amplification product can be used. However, there are limitations because it is necessary to change the sequence of the primer and to consider the conditions of successful PCR. Therefore, in the present invention, it is preferable to include a linker (a migratory marker) between the primer and the labeling substance. In this case, the linker is used for differentiating the mobility of the amplification product during electrophoresis. In the present invention, it is preferable to use hexaethylene oxide as the linker. Since this hexaethylene oxide molecule has the same mobility as the DNA of about 2.5 bp, the size of the amplified product can be controlled by controlling the number of hexaethylene oxide molecules contained in the primer, and the mobility of the final amplified product can also be predicted 5).

The primer for carrying out the multiplex PCR method of the present invention can be produced as shown in FIG. 6 shows a form in which a fluorescent marker is labeled on a primer and a linker is included.

In the case of targeting multiple short PCR sequences (STR, short tandem repeats), PCR amplification products are produced not only in a single size but in various sizes. Therefore, if there are two or more targets, the identification range of each amplification product overlaps In this case, if the multiple PCR method of the present invention is used, it is possible to clearly distinguish amplification of each STR even if a plurality of STRs are targeted.

According to the present invention, the PCR amplification products for each target can be clearly identified even in the multiplex PCR of four or more targets, and it is possible to detect multiple targets such as genetic testing, identification of organisms in the sample, If it is necessary to confirm the presence of DNA or RNA, accurate results can be obtained quickly and easily. In particular, even when a short repeated sequence is targeted, amplification can be effectively confirmed.

Figure 1 is the chemical structure of 6-carboxyfluorescein, i.e., 6-FAM, used for labeling primers in the present invention.
FIG. 2 is a graph showing the fluorescence spectrum of 6-carboxy-2 ', 7'-dimethoxy-4', 5'-dimethoxy-4 '',5'-dichlorofluorescein, that is, the chemical structure of JOE.
FIG. 3 is a graph showing the fluorescence spectrum of 6- [2-carboxy-5- [[2 - [(5-carboxypentyl) amino] -2-oxoethyl] thio] Trichlorophenyl] -1,2,3,4,8,9,10,11-octahydro-2,2,4,8,10,10-hexamethyl-12,14-disulfo-pyrano [3 , 2-g: 5,6-g] diquinolin-13-ium {6- [2-carboxy-5 - [[2 - [(5-carboxypentyl) amino] -2-oxoethyl] thio] , 6-trichlorophenyl] -1,2,3,4,8,9,10,11-octahydro-2,2,4,8,10,10-hexamethyl-12,14-disulfo-pyrano [3,2- g: 5,6-g] diquinolin-13-ium}, i.e. the chemical structure of ALEXA 546.
FIG. 4 is a graph showing the fluorescence spectra of [1,10-dihydro-2,2,10,10-tetramethyl-4,8-bis (sulfomethyl) -2H- -g: 5,6-g] diquinolin-6-yl] -benzene dicarboxylic acid {[1,10-dihydro-2,2,10,10-tetramethyl-4,8-bis (sulfomethyl) 6-g: 5,6-g] diquinolin-6-yl] -benzenedicarboxylic acid}, i.e. the chemical structure of ALEXA 568.
FIG. 5 is a graph showing an analysis result of Example (A) in which multiplex PCR was performed without including a linker in a primer and Example (B) in which multiplex PCR was performed with a linker.
6 is a schematic diagram showing a primer labeled with a fluorescent substance and a linker according to an embodiment of the present invention.
FIG. 7 is a graph showing the results of analysis of multiplex PCR using 6-FAM, JOE, TAMRA, and ALEXA 568 as fluorescent materials.
FIG. 8 is a graph showing the results of analysis of multiplex PCR (one embodiment of the present invention) using 6-FAM, JOE, ALEXA 546, and ALEXA 568 as fluorescent materials.
FIG. 9 is a graph showing the results of analysis of multiplex PCR using 6-FAM, HEX, TAMRA, and ALEXA 568 as fluorescent materials.
10 is a graph showing the results of analysis of multiplex PCR using 6-FAM, HEX, ALEXA 546, and ALEXA 568 as fluorescent materials.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

<Examples>

1. Preparation of primer

Multiplex PCR was performed on 16 target DNAs.

Sixteen target DNAs were screened for STR sites that do not encode specific traits. Primers according to each target are shown in Table 1.

Sixteen targets were divided into four groups and primers for amplifying the targets of each group were labeled with each fluorescent substance according to the combination of Table 2.

Each fluorescent material was purchased from Invitrogen (USA), and a primer labeled with a fluorescent substance was prepared by IDT.

Only one primer of each primer set was labeled with a fluorescent substance at the 5 'end, and the remaining primers were not labeled.

Target site Forward / reverse / [fluorescent labeling] 8S1179 5'-TAACACAAGTTGGGGGAAGCAT-3 '
5 '- [first fluorescent substance] -GATAGCAAGAAGGGCTTTCC-3' 21S11 5 '- [first fluorescent substance] -AGCAAACTTCATCCACTTGG-3'
5'-TTCCTGTCCTCTAATGTAACTTACC-3 7S820 5 '- [first fluorescent substance] -AACAGGATCAATGGATGCAT-3'
5'-TGGCTTTTAGACCTGGACTG-3 ' CSF1PO 5'-CCTTGGGGGATATAGCCTAA-3 '
5 '- [first fluorescent substance] -TGAGTGACAGAGTGATACCATG-3' D3S1358 5 '- [second fluorescent substance] -CAAGGATGGGTGGATCAATA-3'
5'-TTGTATGAGCCACTTCCCAT-3 ' THO1 5 '- [second fluorescent substance] -TGTGAGCCAAGGCCTTATAG-3'
5'-GTCTGGGTTCTCCAAAGAAA-3 ' 13S317 5 '- [Second fluorescent substance] -ACAAAACCCCAAGTACGTGA-3'
5'-TATGACAGGCATCTGGGAGT-3 ' 16S539 5 '- [second fluorescent substance] -GAGCCAGACCTTGTCTCAAA-3'
5'-AAGGGATTTTAGGAACTGATACG-3 ' D2S1338 5 '- [second fluorescent substance] -GGGTGATTTTCCTCTTTGGT-3'
5'-TGATTCCAATCATAGCCACA-3 ' D19S433 5 '- [Third fluorescent substance] -ACAGAAGTCTGGGATGTGGA-3'
5'-GCCCAAAAAGACAGACAGAA3 ' vWA 5 '- [Third fluorescent substance] -ATAGACAGACAGACGGACTG-3'
5'-GGGAGTGGAGATTACCCCT-3 ' TPOX 5 '- [Third fluorescent substance] -AGATAGCCTTTACAGTCACA-3'
5'-AAGTCAGTCACCATGCAAAAAGTA-3 ' D8S51 5 '- [Third fluorescent substance] -TCCCATGCCAAAATTCTTAG-3'
5'-GAAAGAAAGAGAAAGAAGGAAGG-3 ' Amelogenin 5 '- [fourth fluorescent substance] -GTGGATAGACACATGACAGATAGG-3'
5'-TATTTGGCAAGGATAGATACAGG-3 ' D5S818 5 '- [fourth fluorescent substance] -CATCATCTGCACTCCACAAA-3'
5'-TGTCCATCGACAGATGAATG-3 ' FGA 5 '- [fourth fluorescent substance] -TGGTCTCTTCCTCATAACGAC-3'
5'-TGGGGGAGTCTTTGTGTGATT-3 '

Combination 1 Combination 2 Combination 3 Combination 4 The first fluorescent material 6-FAM 6-FAM 6-FAM 6-FAM The second fluorescent material JOE JOE HEX HEX The third fluorescent material TAMRA ALEXA 546 TAMRA ALEXA 546 The fourth fluorescent material ALEXA 568 ALEXA 568 ALEXA 568 ALEXA 568

2. PCR

Multiplex PCR was performed with primers according to each combination prepared in the above 1 using human genomic DNA as a template. The composition of one reaction mixture was as shown in Table 3 and the PCR conditions were as shown in Table 4.

The composition of the reaction mixture density Tris Cl, pH 8.3 10 mM KCl 50 mM MgCl2 1.5mM BSA 60 쨉 g / ml dNTP 200 [mu] M Sodium Azide 0.05% Primers 0.4 μM per primer

Thermal cycler temperature time Number of repeats 95 ℃ 11 minutes 1 time 94 ° C 30 seconds 30 times 58 ℃ 1 minute 72 ℃ 30 seconds 60 ° C 45 minutes 1 time

3. PCR product analysis

The amplified products were separated and detected by electrophoresis to evaluate amplified genes.

The amplification products were mixed with 15 μl of Flourescent loading buffer mixture (Formamide: LIZ500, 24: 1) according to the manual of the instrument. Min, left on ice for 3 minutes, and analyzed by electrophoresis.

As a result, the five targets showed that the size range of each amplification product overlaps with the amplification product of the other target.

4. Multiple PCR with primers containing fluorescent material and linker

In order to solve the problem of overlapping amplification product size according to the result of the above 3, a primer was prepared by including a hexaethylene oxide (HEO) linker in the corresponding primer as shown in Table 5. The linker was included in the fluorescent substance-labeled primer, and was positioned between the fluorescent substance and the primer (see FIG. 6).

The hexaethylene oxide used as a linker was labeled with the desired migration interval since the size of the electrophoresis image was observed to be the same as the increase of the average 2.5 bp DNA per label.

Target site Move interval Number of HEO molecules in the primer CSF1PO 25bp 10 16S539 20bp 8 2S1338 15bp 6 13S317 12bp 5 TPOX 7bp 3

Using the prepared primers, multiplex PCR was carried out in the same manner as in the above 2, and the results of analysis by the same method as in the above 3 are shown in FIGS. 7 to 10.

As can be seen from this result, there is a problem that it is difficult to distinguish the peak of the fluorescence substance actually from the amplification product of the target because the interference occurs between the fluorescent materials in the combination of the labeling substance 1, 3 and 4, In the case of combination 2, the amplification products for each target can be clearly distinguished because no interference phenomenon occurs between the fluorescent materials.

In addition, it was confirmed that the size range of the amplification product was difficult to distinguish by using the linker.

<110> Specialty Lab Solution Co., Ltd. <120> Efficient Multiplex PCR method using fluorescent markers and          mobility markers <130> PA120918-C01 <160> 32 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for 8s1179 <400> 1 taacacaagt tgggggaagc at 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for 8s1179 <400> 2 gatagcaaga agggctttcc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for 21S11 <400> 3 agcaaacttc atccacttgg 20 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer for 21S11 <400> 4 ttcctgtcct ctaatgtaac ttacc 25 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for 7S820 <400> 5 aacaggatca atggatgcat 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for 7S820 <400> 6 tggcttttag acctggactg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for CSF1PO <400> 7 ccttggggga tatagcctaa 20 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer for CSF1PO <400> 8 tgagtgacag agtgatacca tg 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for D3S1358 <400> 9 caaggatggg tggatcaata 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for D3S1358 <400> 10 ttgtatgagc cacttcccat 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for THO1 <400> 11 tgtgagccaa ggccttatag 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for THO1 <400> 12 gtctgggttc tccaaagaaa 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for 13S317 <400> 13 acaaaacccc aagtacgtga 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for 13S317 <400> 14 tatgacaggc atctgggagt 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for 16S539 <400> 15 gagccagacc ttgtctcaaa 20 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for 16S539 <400> 16 aagggatttt aggaactgat acg 23 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for D2S1338 <400> 17 gggtgatttt cctctttggt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for D2S1338 <400> 18 tgattccaat catagccaca 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for D19S433 <400> 19 acagaagtct gggatgtgga 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for D19S433 <400> 20 gcccaaaaag acagacagaa 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for vWA <400> 21 atagacagac agacggactg 20 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer for vWA <400> 22 gggagtggag attacccct 19 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for TPOX <400> 23 agatagcctt tacagtcaca 20 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for TPOX <400> 24 aagtcagtca ccatgcaaaa agta 24 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for D8S51 <400> 25 tcccatgcca aaattcttag 20 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for D8S51 <400> 26 gaaagaaaga gaaagaagga agg 23 <210> 27 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amelogenin <400> 27 gtggatagac acatgacaga tagg 24 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer for Amelogenin <400> 28 tatttggcaa ggatagatac agg 23 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for D5S818 <400> 29 catcatctgc actccacaaa 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer for D5S818 <400> 30 tgtccatcga cagatgaatg 20 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for FGA <400> 31 tggtctcttc ctcataacga c 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer for FGA <400> 32 tgggggagtc tttgtgtgat t 21

Claims (2)

In a multiplex PCR method in which two or more PCRs having different targets are attempted in one reaction solution,
The primers of SEQ ID NOS: 1 to 32 were used as primers,
A primer selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2,
A primer selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 4,
Any one of primers selected from SEQ ID NO: 5 and SEQ ID NO: 6 and
Any one primer selected from SEQ ID NO: 7 and SEQ ID NO: 8 is labeled with 6-carboxyfluorescein of formula (1)
A primer selected from SEQ ID NO: 9 and SEQ ID NO: 10,
A primer selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12,
A primer selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14,
Any one of primers selected from SEQ ID NO: 15 and SEQ ID NO: 16 and
A primer selected from SEQ ID NO: 17 and SEQ ID NO: 18 is labeled with 6-carboxy-2 ', 7'-dimethoxy-4', 5'-dichlorofluorescein of formula (2)
A primer selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20,
A primer selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22,
A primer selected from SEQ ID NO: 23 and SEQ ID NO: 24, and
SEQ ID NO: 25 and SEQ ID NO: 26 are used in combination with 6- [2-carboxy-5 - [[2 - [(5-carboxypentyl) amino] -2-oxoethyl] thio] 4,6-trichlorophenyl] -1,2,3,4,8,9,10,11-octahydro-2,2,4,8,10,10-hexamethyl-12,14-disulfo- Pyra [3,2-g: 5,6-g] diquinolin-13-ium,
A primer selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 28,
A primer selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 30,
SEQ ID NO: 31 and SEQ ID NO: 32 are used in combination with [1,10-dihydro-2,2,10,10-tetramethyl-4,8-bis (sulfomethyl) [3,2-g: 5,6-g] diquinolin-6-yl] -benzene dicarboxylic acid,
A primer selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8,
A primer selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14,
Any one of the primers selected from SEQ ID NO: 15 and SEQ ID NO: 16,
Any one of primers selected from SEQ ID NO: 17 and SEQ ID NO: 18 and
Wherein the primer selected from the group consisting of SEQ ID NO: 23 and SEQ ID NO: 24 comprises a linker having hexaethylene oxide as a unit between the primer and the labeling substance.
&Lt; Formula 1 >

(2)

(3)

&Lt; Formula 4 >

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