One-pot multi-target orthogonal detection system based on nicking endonuclease and its application

Abstract

The present invention discloses a one-pot multi-target orthogonal detection system based on nicking endonuclease and its application, belonging to the field of biological detection. The detection system includes recognition sequences of nicking endonucleases designed for multiple targets, fluorescent reporter sequences that can be completely complementary paired with the recognition sequences, a nicking endonuclease that can recognize the double strand formed by the recognition sequences and the fluorescent reporter sequences and play a nicking role, and a reaction buffer solution; wherein, the recognition sequence is a single-stranded DNA fragment, and the single-stranded DNA fragment includes a fixed region and a variable region required for the nicking endonuclease to play a nicking role. The present invention proves that the nicking activity of nicking endonuclease is affected by the complementarity of its recognition sequence, and uses this characteristic to develop a one-pot multi-target orthogonal detection technology.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and particularly to a one-pot multi-target orthogonal detection system based on nicking enzyme and its application. Background Art

[0002] Nicking enzyme is a type of site-specific endonuclease that can specifically recognize DNA double-strand sequences and nick only a specific site on one of the two strands of the DNA double-strand. Based on its characteristics, nicking enzyme is an indispensable tool in the research of bioengineering fields such as recombinant DNA construction and synthetic biology. For example, nicking enzyme can be used to prepare pre-nicked DNA substrates for DNA repair research. At the same time, nicking enzyme can also be applied to isothermal exponential amplification reactions through its specific nicking effect on DNA. At the nicked site formed by nicking enzyme, through the action of DNA polymerase, using dNTPs as raw materials, polymerization extends from the 3'-end of the nicked site, displacing the allelic DNA strand, thereby forming a new complete DNA sequence containing the fixed region of nicking enzyme again. This double-strand is recognized and nicked by nicking enzyme again, and then the "polymerization - nicking" cycle begins, generating a large number of displaced DNA single-strands, thus achieving isothermal amplification.

[0003] Although nicking enzyme has been widely used in the fields of molecular biology and analytical chemistry, there is no report on the orthogonal reaction performance of nicking enzyme, and there is even less technical report on applying its orthogonal reaction performance to one-pot multi-target detection. Summary of the Invention

[0004] The purpose of the present invention is to provide a one-pot multi-target orthogonal detection system based on nicking enzyme and its application to solve the problems existing in the above-mentioned prior art. The detection system is constructed based on the orthogonal reaction characteristics of nicking enzyme and can achieve orthogonal detection of multiple targets simultaneously.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] The present invention provides a one-pot multi-target orthogonal detection system based on nicking enzyme, including recognition sequences of nicking enzyme designed for multiple targets, fluorescent reporter sequences that can be completely complementary paired with the recognition sequences, nicking enzyme that can recognize the double-strand formed by the recognition sequences and the fluorescent reporter sequences and play a nicking role, and a reaction buffer solution; wherein, the recognition sequence is a single-stranded DNA fragment, and the single-stranded DNA fragment includes a fixed region and a variable region required for the nicking enzyme to play a nicking role.

[0007] Preferably, the 5'-end of the fluorescent reporter sequence is labeled with a fluorescent reporter group, and the 3'-end is labeled with a corresponding fluorescent quenching group.

[0008] Preferably, the target includes double-stranded DNA, single-stranded DNA, RNA, or protein.

[0009] The nicking endonuclease includes Nt.BstNBI, one or more of Nt.BstNBI, Nt.BspQI, Nb.BsrDI, Nt.BsmAI, Nt.AlwI, Nb.BtsI, Nb.BsmI, Nt.CviPII, Nb.BbvCI, Nt.BbvCI, and Nb.BssSI.

[0010] The present invention also provides a method for constructing the one-pot multi-target orthogonal detection system based on the nicking endonuclease, including the following steps:

[0011] (1) According to multiple targets to be detected, a fixed region containing a recognition sequence with a nicking endonuclease recognition site is designed and introduced respectively;

[0012] (2) According to the recognition sequence, a fluorescent reporter sequence that can be completely complementary paired with different recognition sequences is added to the reaction system, so that the recognition sequence and the fluorescent reporter sequence combine to form double-stranded DNA, and at the same time the fluorescence remains quenched; the reaction system includes a reaction buffer solution and a nicking endonuclease;

[0013] The above-mentioned fluorescent reporter sequence is labeled with a fluorescent reporter group at the 5'-end and a corresponding quenching group at the 3'-end, so that the fluorescent signal emitted by the reporter group is absorbed by the quenching group. The fluorescent reporter sequence cannot bind to an incompletely complementary paired recognition sequence or can only form an unstable binding.

[0014] (3) In the reaction system, the nicking endonuclease recognizes the fixed region of the orthogonal reaction sequence and performs nicking and signal amplification in the variable region, causing the fluorescent reporter sequence to break into two independent fragments, so that the fluorescent signal can be restored. Based on the orthogonality of the nicking endonuclease, different targets output different fluorescent signals, enabling simultaneous detection of multiple targets.

[0015] Preferably, the recognition sequence is selected from any one of the following sequences:

[0016] (a) A segment of the target itself;

[0017] (b) A segment of the sequence that the target has through a molecular recognition transformation pathway. For example, antigen-ssDNA-labeled antibody recognition.

[0018] Preferably, the fixed regions of the recognition sequences are exactly the same, and the base difference in the variable regions is ≥40%.

[0019] Preferably, the fluorescent reporter groups labeled at the 5'-end of the fluorescent reporter sequence include FAM, TEXAS RED, JOE, TAMRA or ROX, and the fluorescent quenching groups labeled at the 3'-end include BHQ1 or BHQ2.

[0020] The present invention discloses the following technical effects:

[0021] The present invention demonstrates that the nicking activity of nicking endonuclease is affected by the complementarity of its recognition sequence, and utilizes this property to develop a one-pot multi-target orthogonal detection technique. Nicking endonuclease can exert its maximum nicking ability only when the double-stranded DNA substrate is completely complementary paired. If there is incomplete complementarity between the double-stranded DNAs of the recognition sequence, that is, there are certain mismatches, its nicking activity will be significantly reduced or even disappear. Therefore, the present invention demonstrates the orthogonal reaction property of nicking endonuclease, and constructs a multi-target orthogonal detection technique based on this property, providing a new detection method for simultaneous detection of multiple targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 It is a schematic diagram for one-pot multi-target orthogonal detection using nicking endonuclease. A: Schematic diagram of the structure of the coding sequence and the probe sequence; B: Orthogonal reaction regulation is achieved by changing the double-stranded complementary recognition ability between the probe sequence and the coding sequence.

[0024] Figure 2 It is a result diagram showing the influence of the nicking effect of nicking endonuclease when the fixed region is fixed as the recognition site of Nt.BstNBI and the variable region is any base and the coding sequence is completely complementary paired with the probe sequence, taking Nt.BstNBI nicking endonuclease as an example;

[0025] Figure 3 It is a result diagram showing the influence of single-base mismatch of the coding sequence on the nicking effect of nicking endonuclease, taking Nt.BstNBI nicking endonuclease as an example;

[0026] Figure 4 It is a result diagram showing the influence of double-base mismatch of the coding sequence on the nicking effect of nicking endonuclease, taking Nt.BstNBI nicking endonuclease as an example;

[0027] Figure 5 Taking Nt.BstNBI nickase as an example, it is the result graph of the influence of three-base mismatches in the coding sequence on the nicking effect of the nickase;

[0028] Figure 6 Taking Nt.BstNBI nickase as an example, it is the result graph of the influence of four-base and five-base mismatches in the coding sequence on the nicking effect of the nickase;

[0029] Figure 7 Taking Nt.BstNBI nickase as an example, it is the influence of mismatches at the 1st and 5th positions in the variable region on the coding ability;

[0030] Figure 8 It is the relationship between the change in the nicking efficiency of Nt.BstNBI and the mismatched bases;

[0031] Figure 9 Taking Nt.BstNBI nickase as an example, it is the result graph of the orthogonal reaction ability of 117 groups of probe sequences and the coding sequence;

[0032] Figure 10 Taking Nt.BstNBI nickase as an example, it is the sequence graph of 117 groups of probe sequences and the coding sequence;

[0033] Figure 11 Taking Nt.BstNBI nickase as an example, it is the three-dimensional fluorescence spectrum result graph of the one-pot detection of 5 targets: A: The three-dimensional fluorescence spectrum result graph of the one-pot detection when 5 targets coexist; B: The background three-dimensional fluorescence spectrum result graph of the one-pot detection system without targets; C: The three-dimensional fluorescence spectrum result graph when only target 1 exists in the one-pot detection system; D: The three-dimensional fluorescence spectrum result graph when only target 2 exists in the one-pot detection system; E: The three-dimensional fluorescence spectrum result graph when only target 3 exists in the one-pot detection system; F: The three-dimensional fluorescence spectrum result graph when only target 4 exists in the one-pot detection system; G: The three-dimensional fluorescence spectrum result graph when only target 5 exists in the one-pot detection system. Detailed implementation manners

[0034] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0035] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0037] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention's specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0038] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0039] Example 1

[0040] In this example, taking the nicking endonuclease Nt.BstNBI as an example, a one-pot multi-target orthogonal detection is carried out using the nicking endonuclease.

[0041] The probe sequence that Nt.BstNBI can recognize is a sequence containing a fixed region of 5 bases in length (5’→3’: GAGTC) and a variable region of 5 bases in length (5’→3’: NNNNN). On the premise of containing a completely complementary coding sequence, Nt.BstNBI can recognize the fixed region and generate a single-strand nick at the 3' end of the 4th base in the variable region. Thus, as shown in Figure 1 Figure A, the coding sequence and the codable region of the probe sequence are mainly an identification region of 5 bases in length (i.e., the fixed region) and a variable region of 5 bases. According to this characteristic, by changing the five bases in the variable region, the base difference between sequences can be made no less than 2 bases, so that only the sequence that is completely complementary paired with the probe sequence can undergo nicking, while the sequence that is not completely complementary paired with the probe sequence cannot exert the nicking ability of the nicking endonuclease (see Figure 1in B).

[0042] To verify the above theory, the nicking ability of the nicking enzyme was verified by urea-denatured polyacrylamide gel electrophoresis. The specific experimental steps of urea-denatured polyacrylamide gel electrophoresis (Urea-PAGE) are as follows:

[0043] (1) Prepare 10% ammonium persulfate (APS): Dissolve 1 g of APS in 10 mL of water and shake well until fully dissolved.

[0044] (2) Prepare 8 M urea solution: Dissolve 4.8 g of urea in water and make up to 10 mL, and dissolve fully.

[0045] (3) Prepare 1× urea Loading buffer: Mix 6× Loading buffer and urea at a volume ratio of 1:5 for later use.

[0046] (4) Prepare 20× TBE solution: Dissolve 1× TBE powder (1 L) in 50 mL for later use (i.e., 20×).

[0047] (5) Prepare 15% Urea-PAGE premix: Mix 240 g of urea, 25 mL of 20× TBE buffer, and 250 mL of acrylamide / methylene bisacrylamide 30% solution (29:1), add water to make up to 500 mL. Sonicate to fully dissolve the urea, or stir magnetically at 50 °C to fully dissolve the urea.

[0048] (6) According to the conventional method of preparing the gel, prepare the gel plate in advance, clamp the glass, and align the rear bayonet.

[0049] (7) Take a 15 mL centrifuge tube, add 10 mL of 15% Urea-PAGE premix, 30 μL of 10% APS, and 10 μL of tetramethylethylenediamine (TEMED), and gently invert and mix about ten times.

[0050] (8) Use a 5 mL pipette to add the solution prepared in (5) above to the gel plate.

[0051] (9) After adding, insert the gel comb and wait for 20 - 30 minutes for the gel to solidify. The preparation of the sample in step (11) can be completed during the waiting period.

[0052] (10) After the gel solidifies, it can be transferred to the electrophoresis tank for later use or stored in 1× TBE buffer at 4 °C. If used immediately, pre-electrophoresis at 120 V for 30 min is required. The preparation of the sample in step (11) can be completed during the waiting period.

[0053] (11) Sample preparation: Add the coding sequence to ddH2O with a total volume of 50 μL to make the final concentration 500 nM; add 5 μL of NEBuffer r3.1 and 0.5 U / μL of Nt.BstNBI. After reacting in a metal bath at 37 °C for 2 h, raise the temperature to 85 °C to inactivate the enzyme. Generally, mix 10 μL of the sample with 10 μL of 1× urea Loading buffer; if a Marker is needed, mix 2 μL of the Marker with 10 μL of 1× urea Loading buffer.

[0054] (12) After pre-electrophoresis, before loading, rinse the loading wells again with a pipette or syringe and load the sample immediately after rinsing.

[0055] (13) Loading: Add 10 μL of the sample or Marker obtained in (11) above to the loading wells in the predetermined order.

[0056] (14) Electrophorese at 120 V for 60 min.

[0057] (15) Prepare 1× GelRed solution: Dilute 2 μL of GelRed (10000×) with ultrapure water to 1× for standby.

[0058] (16) After electrophoresis, take out the gel, place it in the 1× GelRed solution, and incubate it on a shaker at room temperature for 20 min to fully stain the DNA bands.

[0059] (17) Image under a gel imaging system. And perform fluorescence analysis based on the bands, (where the fluorescence intensity of the band is the fluorescence intensity after removing the background). The stronger the fluorescence intensity of the band of the cleaved sequence and the weaker the fluorescence intensity of the band of the non-cleaved sequence, the more cleavage products of the restriction enzyme. Therefore, the cleavage efficiency of the restriction enzyme for this pair of sequences can be obtained by calculating through this fluorescence intensity.

[0060] The results are as Figure 2 shown. When the recognition site of Nt.BstNBI is fixed in a fixed region, and the variable region is any base and the coding sequence is completely complementary paired with the probe sequence, Nt.BstNBI can efficiently cleave each pair of DNA sequences regardless of the base and arrangement of the variable sequence. And when the probe sequence remains unchanged and the bases of the coding sequence are changed to cause a mismatch between the two, there are two cases: ① When the mismatch site is in the fixed region, Nt.BstNBI can hardly cleave this pair of sequences; ② When the mismatch site is in the variable region and the number of mismatched bases is one, the cleavage ability of the restriction endonuclease is weakened to varying degrees (see Figure 3 ). It should be noted that the change in the cleavage efficiency of Nt.BstNBI has nothing to do with the type of mismatch of the mismatched base (seeFigure 8 ). Then, when the mismatch site is in the variable region and is a double base mismatch or above, except for the mutation of 1 / 5 position in the variable region, the performance of the nicking endonuclease almost completely disappears (see Figures 4 - 6 ). The above results show that nicking endonucleases are expected to be used for orthogonal probe sequence cleavage and signal release. It was also proved that the mismatches at positions 1 and 5 in the variable region are not suitable for coding, but when the mismatches at positions 1 and 5 are combined with mismatches at positions 2-4, they still have good coding performance (see Figure 7 ). Figures 2 - 7 In the sequences involved, the probe sequence is based on the sequence in the figure, with 3 additional T bases "TTT" at the 5' end and 27 additional T bases "TTTTTTTTTTTTTTTTTTTTTTTTTT" at the 3' end; the coding sequence is based on the sequence in the figure, with 1 additional T base "T" at the 3' end; in addition Figures 3 - 7 The probe sequences in are labeled with a FAM modification at the 3' end. Figure 7 The sequences involved do not change the probe sequence, and only a single base is changed in the non-mismatch coding sequence as shown in the figure. The 1st and 5th bases close to the fixed region are defined as the 1st and 5th positions, respectively.

[0061] Based on the above-discovered coding characteristics, taking Nt.BstNBI nicking endonuclease as an example, the present invention designed a total of 117 pairs of highly orthogonal coding sequences and probe sequences ( Figure 9 ), the paired sequences are completely complementary and the number of mismatches between the unpaired sequences is not less than 2 bases (inclusive). Figure 9 Among the sequences involved, the probe sequence is based on the illustrated sequence, with an additional T base "T" at the 5' end and 19 additional T bases "TTTTTTTTTTTTTTTTTT" at the 3' end; the coding sequence is based on the illustrated sequence, with an additional T base "T" at the 3' end.

[0062] According to the sample preparation method in step (11) above, 117 pairs of completely complementary sequence pairs and mismatched sequence pairs with mismatches of not less than 2 bases were cross-incubated, and the cleavage efficiency was verified by urea-denatured polyacrylamide gel electrophoresis. When incubating the mismatched sequences, ten coding sequences were co-incubated with a probe as a group, where the probe was mismatched with the ten coding sequences. For all completely complementary sequence pairs, they were incubated separately to verify the cleavage ability. After analysis by the aforementioned electrophoresis experimental scheme, the following results were obtained: Figure 10 The heat map shown shows that the nicking endonuclease has extremely strong orthogonal encoding performance.

[0063] Example 2

[0064] Taking the simultaneous detection of five target models using the nicking endonuclease Nt.BstNBI as an example, a total of 5 pairs of probe sequences modified with different fluorescent molecules and quenching groups were designed for each target. The 5 groups of target sequences and probe sequences are shown in Table 1 respectively:

[0065] Table 1 5 groups of target sequences and probe sequences

[0066] Name Sequence (5’-3’) Sequence ID Coding sequence 1 (Target 1) GCATCGACTCT SEQ ID NO:1 Coding sequence 2 (Target 2) GGGAGGACTCT SEQ ID NO:2 Coding sequence 3 (Target 3) GATCAGACTCT SEQ ID NO:3 Coding sequence 4 (Target 4) ATTACGACTCT SEQ ID NO:4 Coding sequence 5 (Target 5) TGCCCGACTCT SEQ ID NO:5 Probe sequence 1 BHQ1-TGAGTCGATGCT-FAM SEQ ID NO:6 Probe sequence 2 BHQ2-TGAGTCCTCCCT-TEXAS RED SEQ ID NO:7 Probe sequence 3 BHQ1-TGAGTCTGATCT-JOE SEQ ID NO:8 Probe sequence 4 BHQ2-TGAGTCGTAATT-TAMRA SEQ ID NO:9 Probe sequence 5 BHQ2-TGAGTCGGGCAT-ROX SEQ ID NO:10

[0067] In 50 μL of ddH2O with a total volume, any one of the coding sequences or all five coding sequences (SEQ ID NO: 1 - 5) was added to make the final concentration 100 nM; the probe sequence (SEQ ID NO: 6 - 10) was added to make the final concentration 500 nM; 5 μL of NEBuffer r3.1; 0.5 U / μL of Nt.BstNBI. After reacting in a metal bath at 37 °C for 2 h, the temperature was raised to 85 °C to inactivate the enzyme. There were 7 samples in total. Except for the different content of the added coding sequences, the other components were exactly the same. The sequence content in the 7 samples included: a negative control without adding any coding sequence, a positive control with all five coding sequences added, and the cases of adding one of the five coding sequences respectively. After complete incubation, the three-dimensional fluorescence spectrum was detected using a fluorescence spectrometer. The excitation light wavelength was set to 350 nm - 700 nm, the emission light wavelength was set to 400 nm - 900 nm, the scanning speed was 6000 nm / min, the excitation bandwidth was 3.0 nm, and the emission bandwidth was 3.0 nm. The results are as Figure 11 shown, and the results show that the nicking endonuclease allows for one-pot orthogonal detection.

[0068] As can be seen from the above embodiments, the present invention utilizes the orthogonal reaction performance of the nicking endonuclease. A part of the recognition sequence of the nicking endonuclease is a fixed region, and the other part is a variable region. The orthogonal reaction performance is achieved by adjusting the double-stranded complementary recognition ability of the variable region. Among them, the recognition sequence of the nicking endonuclease is the inherent recognition sequence of the nicking endonuclease in the target molecule, or through molecular recognition technology, a single target or multiple targets are converted into the recognition sequence of the nicking endonuclease.

[0069] The above embodiments only take the nicking endonuclease Nt.BstNBI as an example for illustration. The orthogonal detection system based on the nicking endonuclease of the present invention can also be used. Simply by changing the molecular label of the detection probe to an enzyme label, one-pot multi-target orthogonal detection can be carried out; among them, the detection probe is a molecular beacon, and the output signal is a fluorescence signal. Or by simply changing the type of nicking endonuclease, or by the combined application of multiple nicking endonucleases, one-pot multi-target orthogonal detection can be carried out.

[0070] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A one-pot multi-target orthogonal detection system based on nicking endonuclease, characterized in that It includes the recognition sequence of the nickase designed for multiple targets, the fluorescent reporter sequence capable of completely complementary pairing with the recognition sequence, the nickase capable of recognizing the double strand formed by the recognition sequence and the fluorescent reporter sequence and playing a nicking role, and a reaction buffer solution; wherein, the recognition sequence is a single-stranded DNA fragment, and the single-stranded DNA fragment includes a fixed region and a variable region required for the nickase to play a nicking role. The nickase is Nt.BstNBI.

2. The one-pot multi-target orthogonal detection system based on nickase as claimed in claim 1, wherein The 5' end of the fluorescent reporter sequence is labeled with a fluorescent reporter group, and the 3' end is labeled with a corresponding fluorescent quenching group.

3. The one-pot multi-target orthogonal detection system based on nickase as claimed in claim 1, wherein The targets include double-stranded DNA, single-stranded DNA, RNA or protein.

4. A method for constructing an endonuclease-based one-pot multi-target orthogonal detection system according to any one of claims 1-3, characterized in that, It includes the following steps: (1) According to multiple targets to be detected, the fixed regions of the recognition sequences containing the nickase recognition sites are respectively designed and introduced. (2) According to the recognition sequence, a fluorescent reporter sequence capable of completely complementary pairing with different recognition sequences is added to the reaction system to enable the recognition sequence and the fluorescent reporter sequence to bind to form double-stranded DNA, while the fluorescence remains in a quenched state; the reaction system includes a reaction buffer solution and a nickase. (3) In the reaction system, the nickase recognizes the fixed region of the orthogonal reaction sequence and performs nicking and signal amplification in the variable region, causing the fluorescent reporter sequence to break into two independent fragments, so that the fluorescence signal can be restored. Based on the orthogonal reactivity of the nickase, different targets output different fluorescence signals to enable simultaneous detection of multiple targets.

5. The construction method according to claim 4, characterized in that The recognition sequence is selected from any one of the following sequences: (a) A sequence segment of the target itself. (b) A sequence segment that the target has through a molecular recognition transformation pathway.

6. The construction method according to claim 4, characterized in that, The fixed regions of the recognition sequences are completely the same, and the base difference in the variable region is ≥40%.

7. The construction method according to claim 4, characterized in that, The fluorescent reporter groups labeled at the 5' end of the fluorescent reporter sequence include FAM, TEXAS RED, JOE, TAMRA or ROX, and the fluorescent quenching groups labeled at the 3' end include BHQ1 or BHQ2.

Images