KR20170004823A - Method for detecting nucleic acid based on FRET assay using polymerase lacking exonuclease activity - Google Patents

Abstract

The present invention is directed to a nucleic acid encoding a target nucleic acid, at least one primer and probe that binds to the target nucleic acid, a deoxynucleotide 5'-triphosphate, and a DNA polymerase lacking or reducing 5'-> 3'exonuclease activity And a heat-resistant flap endonuclease; And detecting and amplifying FRET-based nucleic acids conveniently using 5'- > 3'exonuclease activity-deficient or reduced DNA polymerase, comprising amplifying said target nucleic acid under amplifiable conditions A possible method is disclosed.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a FRET-based nucleic acid detection method using a polymerase lacking exonuclease activity,

This is a technical field related to nucleic acid molecule amplification and detection.

Along with the development of molecular biology, a method for detecting a specific target nucleic acid present in a specimen and using it for diagnosis of diseases has been widely used. This requires amplification of a small amount of nucleic acid in a sample, and the most widely used method is PCR (Polymerase Chain Reaction).

However, PCR is relatively inconvenient in fields requiring rapid detection such as diagnosis of diseases in an urgent situation due to a relatively long reaction time, which typically takes 2-5 hours only in response to the reaction time. Therefore, various methods have been attempted to reduce the reaction time through improvement of the polymerase.

US Patent Publication No. 2004/0081963 relates to a Sso-7 polymerase conjugate protein, wherein a Sso7d protein, a DNA binding region, is fused to a Taq polymerase from which an N-terminal region has been removed, thereby improving the processivity .

U.S. Patent Application Publication No. 2011/0027833 discloses thermostable type-A DNA polymerases with increased resistance to synthetic forces and inhibitors, and improved polymerases through mutation of specific sequences of wild-type Taq polymerase .

However, in the case of Taq polymerase having the fusion or mutation as described above, the DNA amplification rate or efficiency was improved, but it was not verified whether or not the DNA polymerase possesses both 5 '-> 3' exo / endo-nuclease activity.

However, if the 3 '-> 5' DNA polymerase activity is not retained but the 5 '-> 3' exo / endonuclease activity is not detected, the nucleic acid can be detected using the FRET method as the most commonly used TaqMan probe There is a problem that it can not be done.

Therefore, it is necessary to develop a nucleic acid detection method using the FRET method using a polymerase having 3 '-> 5' DNA polymerase activity.

The present invention provides a method capable of nucleic acid detection and / or amplification based on FRET using a DNA polymerase lacking 5 '-> 3' exonuclease activity.

In one embodiment, the subject matter provides a kit comprising a target nucleic acid, at least one primer and probe that binds to the target nucleic acid, a deoxynucleotide 5'-triphosphate, and a 5'-> 3'exonuclease activity lacking or reducing Providing an amplification reaction composition comprising a DNA polymerase and a heat-resistant flap endonuclease; And amplifying the target nucleic acid. The present invention provides a nucleic acid detection and / or amplification method based on FRET using a DNA polymerase lacking or reducing 5 '-> 3' exo / endonuclease activity. The amplification is performed under amplifiable conditions depending on the length of the target nucleic acid, the probe used, and the primer, the sequence composition, the degree of hybridization, the composition of the buffer to be used, and the like.

The DNA polymerase that lacks or reduces the 5 '-> 3' exo / endo-nuclease activity used in the method according to the present invention may be a pfu originating from an enzyme that is not originally active, such as Pyrococcus furiosus, The mutant-induced enzyme, for example, the N-terminus of Taq (Thermus aquaticus) is modified to have no 5 '-> 3' exo / endo-nuclease activity through mutations such as deletion. Such DNA polymerases include, for example, Methanococcus jannaschii, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermus flavus, Thermococcus literalis, Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus Thermus Thermus rubens, Thermus rubens, Thermus scandiatus, Thermus species sp0 17, Thermus species sps 17, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcus barossi, Thermococcus gorgonarius, Thermococcus kodakaraensis, Pyrococcus furiosus, Pyrococcus horisoshii, But are not limited to, Pyrococcus abyssi, Pyrodictium occultum, Aquifex pyrophilus, or Aquifex aeolieus.

In another embodiment, the nucleic acid polymerase lacking or reducing the 5 ' - > 3 ' exo / endo nuclease activity used in the methods of the present invention may lack N terminus or introduce mutations into the N terminus exonuclease domain Or a polypeptide having DNA-binding activity at the N or C terminus, wherein the polymerase is a modified or wild-type Taq (Thermus aquaticus), Pfu (Pyrococcus furiosus) polymerase, Tth Thermus thermophilus polymerase, or Tf1 (Thermus flavus) polymerase. Sso7d-Taq and Pfu-S (Nucleic Acids Research, 2004, Vol. 32, No. 3, pp. 1197-1207) are examples of polymerases in which a polypeptide having DNA binding activity at the N or C terminus is fused have. Sso7d-Taq has a DNA binding polypeptide, Sso7d, fused to the N-terminus of Taq, and Sso7d is fused to the N or C terminus of Pfu-S.

In one embodiment, the FENl used in the method according to the invention is selected from the group consisting of Sulfolobus solfataricus, Pyrobaculum aerophilum, Thermococcus litoralis, Archaeaglobus veneficus, Archaeaglobus profundus, Acidianus brierlyi, Acidianus ambivalens, Desulfurococcus amylolyticus, Desulfurococcus mobilis, Pyrodictium brockii, Thermococcus gorgonarius, Thermococcus zilligii, Methanopyrus but are not limited to, E. coli, Candida, Methanococcus igneus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeropyrum pernix and Archaeaglobus veneficus.

The method according to the present invention can be used in a quantitative or qualitative PCR reaction.

The method according to the present invention can be used for quantitative or qualitative detection of a target nucleic acid.

In the method according to the present invention, the 5'- > 3'exonuclease activity-deficient or reduced heat-resistant DNA polymerase and the heat-resistant flap endonuclease may be contained in a molar ratio of 1: 0.02 to 1:20.

In another aspect, the present invention provides a kit or composition for use in the methods described herein, wherein the enzyme required for nucleic acid amplification is a DNA polymerase having a 5 ' - > 3 ' exonuclease activity deficient or reduced and a heat resistant flap end A nucleic acid detection kit or composition based on FRET, comprising a nuclease, is provided.

The methods, compositions and kits according to the present invention can be used to detect 5 'to 3' exo / endonuclease activity using 5'-> 3 'exo / endonuclease activity-deficient or reduced DNA polymerase and FEN (Flap Endo Nuclease) It is possible to detect or quantitate FRET-based nucleic acids that have not previously been possible with DNA polymerase that lacks or reduces endonuclease activity. This improves convenience by accurately and promptly diagnosing diseases or infecting pathogens in a variety of applications where detection of nucleic acids is required in a shortened time as well as accuracy.

Figure 1 schematically illustrates the working principle of a method according to one embodiment of the present invention, wherein the polynucleotide can be cleaved by recognizing the flap structure in a 5'- >3'exo, a DNA polymerase that lacks endonuclease activity The Flap endo nuclease is used together to show that Flap endo nuclease can replace the 5 'exonuclease activity of Taq polymerase.
Figure 2 shows the results of real time quantitative PCR (qPCR) based on TaqMan probes using a combination of mutant Taq polymerase without 5 'exonuclease activity and various exo- and endo-nuclease in one embodiment of the invention.
FIGS. 3 and 4 are results of real-time quantitative PCR (qPCR) based on TaqMan probes in which mutant Taq polymerase without 5 'exonuclease activity and FEN1 were used at various mixing ratios or amounts in one embodiment of the present invention.
FIG. 5 is a result of analysis of a cleavage pattern of a probe of 'mutant Taq polymerase and FEN1 mixed enzyme' according to the present invention, which shows equivalent performance to wild-type Taq polymerase in quantitative PCR of TaqMan probe type.
FIG. 6 shows the results of real-time quantitative PCR (qPCR) of the synthesis rate of mutant Taq polymerase and FEN1 mixed enzyme (1: 2 molar ratio) according to the present invention.
Fig. 7 shows the result of performing heat-resistant FEN1 and TaqMan qPCR using a Pfu polymerase-mixed enzyme having a correcting function.

The present invention enables a FRET-based nucleic acid detection and / or amplification method using DNA polymerase lacking 5 '-> 3' exo / endonuclease activity or reduced, and dramatically increases the time required for amplification Respectively.

In general, the two different activities possessed by one enzyme are known to be fully functional when the corresponding activity is present in one enzyme (Bae et al. In other words, the two enzymatic activities contained in the original enzyme are separated into separate enzymes, which are provided as individual enzymes having respective activities (see, for example, JB Biol. Chem. (2012) 277: 26632-41) , It is known that it is difficult to restore the activity of the original enzyme by simply mixing each enzyme.

However, as described in FIG. 1, by providing the 5 '-> 3' exonuclease function as a separate enzyme in the amplification reaction, a FRET-based enzyme that lacks exonuclease function has not been previously available Thereby enabling amplification or detection of nucleic acid.

Thus, in one embodiment, the subject matter provides a method for detecting and / or inhibiting 5 '- > 3' exonuclease activity as a target nucleic acid, at least one primer and probe that binds to the target nucleic acid, deoxynucleotide 5-triphosphate, Providing an amplification reaction composition comprising a DNA polymerase and a heat-resistant flap endonuclease; And amplifying the target nucleic acid. The present invention provides a nucleic acid detection and / or amplification method based on FRET using a DNA polymerase lacking or reducing 5 '-> 3' exo / endonuclease activity. The amplification is performed under amplifiable conditions depending on the length of the target nucleic acid, the probe used, and the primer, the sequence composition, the degree of hybridization, the composition of the buffer to be used, and the like.

In another embodiment, the present invention utilizes a combination of DNA polymerase and heat resistant flap endonuclease that lacks or reduces 5'- > 3 'exonuclease activity during nucleic acid or DNA amplification reactions And more particularly to a method for enhancing the amplification rate of a target nucleic acid.

Herein, 5 '->3' exonuclease means an enzyme that specifically recognizes double-stranded nucleic acid and cleaves or cleaves nucleic acid in the 5 'direction or 3' direction. Many types of DNA polymerases have 5 '->3' exonuclease activity in addition to nucleic acid polymeric activity. Examples include E. coli DNA polymerase I, polymerase derived from Thermus aquaticus (Taq), polymerase derived from Thermus thermophilus (Tth), Thermus Brockianus- derived polymerase, Pyrococcus Polyrase derived from furiosus (Pfu) and Thermus flavus (Tfl) polymerase, but are not limited thereto.

The 5 'to 3' exonuclease activity lacking or reduced in the present invention is an enzyme or polymerase for synthesizing a nucleic acid, and the 5 'to 3' exonuclease activity as described above is the original Or that such activity is inactivated or removed in a manner such as by mutation such as deletion, substitution, etc. in various ways. For example, reference may be made to Taq polymerase in which the 5 '-> 3' exonuclease function is deleted or removed, U.S. Patent 5,474,920, U.S. Patent Publications 2004-0081963, 2013-0252309, and 2011-0027833.

The term "5'-flap endonuclease "," FEN ", and "FEN1 ", as used herein, refers to the endo-nuclease activity and nick or gap It has a specific 5'-3 'exonuclease activity against double strands and is found in mice, humans, yeast, prokaryotes and various thermostable bacteria. In prokaryotes, FEN1 activity is possessed by the 5 'nuclease domain of DNA polymerase I. In eukaryotes, archaea, and bacteriophages, they exist as distinct polypeptides. Additional information related to flap endonuclease may be found in Xu et al., J. Biol. Chem. 276: 30167-30177 (2001) and Kaiser et al. J Biol Chem 274: 21387-21394 (1999). FEN catalyzes the hydrolysis of phosphodiester bonds at the junctions of single and double stranded DNA (Harrington and Lieber, EMBO 13: 1235-46 (1994); Harrington and Lieber, J Biol Chem 270: 4503 -8 (1995)). In cells, the flap endonuclease is one of the enzymes necessary for the replication of discontinuous strands in DNA replication, especially for the maturation of Okazaki fragments. Various methods of FEN1 may be used in the method according to the present invention. In particular, a heat resistant FEN1 capable of stably activating at more than 50 degrees, which can be stably used in the FRET-based nucleic acid detection method, can be used.

In one embodiment, heat resistant FEN1 derived from Pfu is used. Because it has heat tolerance, it acts stably in repeated PCR of heating and cooling.

As used herein, a "target nucleic acid" refers to a nucleic acid or nucleic acid fragment (polynucleotide) having a particular nucleotide sequence to be amplified or detected using the method, composition or kit according to the present invention. Such a target nucleic acid is not particularly limited in size (length), and may be present in one or more types of templates provided for amplification. The target nucleic acid may also be double-stranded or single-stranded. In the case of a double strand, it includes all single strand sequences. Bases or nucleotides included in a target nucleic acid according to the present invention include those that are modified for their natural or special purposes.

As used herein, the term amplification refers to any process that increases the number of copies of a nucleic acid of interest or a target nucleic acid or a target nucleotide sequence in vitro (in vitro), in which the nucleotide is incorporated into DNA or RNA. According to an embodiment of the present invention, the amplification reaction repeats the replication process several cycles (cycles), for example, repeats the denaturing-hybrid-replication process 10 to 100 cycles through a PCR (polymerase chain reaction) process. Methods for amplifying nucleic acids such as PCR (Polymerase Chain Reacton) are known in the art. In one embodiment according to the present invention, PCR is used. PCR methods are described, for example, in U.S. Patent Nos. 4,683,195 and 4,683,202. Generally, PCR uses two forward and reverse oligonucleotide primers and a heat-resistant DNA polymerase that specifically bind to a site flanking a region to be amplified of a nucleic acid template, ≪ / RTI > terminus. Typically, PCR involves (i) separating the double strand into a single strand; (ii) binding the primer to the single stranded template; (iii) synthesizing the nucleic acid in the 5-3 'direction from the primer is repeatedly performed to amplify the target nucleic acid, and steps (ii) and (iii) may be performed in one step. PCR is carried out in automated controlled temperature devices. The PCR can be repeated at various cycles of interest and as a result the target nucleic acid is amplified exponentially and both ends of the amplification product are identical to the primers used. The temperature, time, and transition rate between steps used in each step are determined by a number of factors known to those skilled in the art, for example McPherson, M.J. and Quirke, P. (eds) (1991) PCR: and A Practical Approach. IRL Press, London; Mullis, K., Ferre, F. and Gibbs, R.A. (eds) (1994) The Polymerase Chain Reaction. Birkauser, Boston, MA, USA, and the like. The conditions for carrying out each step of the PCR can vary, but typically the nucleic acid is denatured at > 90 ° C, the primer is annealed at about 50-75 ° C, and the synthesis is carried out at 65-75 ° C. PCR can be performed in a variety of modified forms and can be performed in a variety of ways including "RT-PCR," "real-time PCR," "nested PCR," "quantitative PCR," "multiplexed PCR, Asymmetric PCR ", and the like.

The term " nucleotide " as used herein is a nucleic acid that is a polymer to which a base-sugar-phosphate is attached, i.e., a monomer that constitutes DNA or RNA. The nucleotides used in the synthesis of nucleic acids in the method of the present invention include dATP, dCTP, dGTP or dTTP, which is a deoxyribonucleotide triphosphate, and may, if necessary, contain dUTP. A nucleoside is a base-sugar conjugate that is used interchangeably with a nucleotide.

As used herein, the terms " oligonucleotide " and " polynucleotide " are used interchangeably, and both include both, including nucleic acid (RNA or DNA), aptamer, and the like.

The term " primer " as used herein refers to a nucleic acid in which a nucleotide is extended by covalent bonding at its 3 'end in a nucleic acid amplification or synthesis reaction using a polymerase as a single strand oligonucleotide.

As used herein, the term " probe " is used in combination with a primer according to various types of fluorescence detection methods in real time quantitative PCR, for example, in a concept distinct from a primer. The oligonucleotides are labeled with fluorescent dyes or scavengers at the 5 'and 3' ends, respectively.

The fluorescence detection method in which such a probe is used may be, for example, a Molecular beacon probe or a strand based on a proximity probe or a 5 ' neuritic probe method based on Fluorescent Resonance Energy Transfer (FRET), or contact quenching But not limited to, star-ended displacement probes (Recent Pat DNA Gene Seq. 2007; 1 (2): 145-7 Mol Biotechnol. 2003 Nov; 25 (3): 267-74. ). Such a method is well-known. For example, a proximity probe method is a principle in which a fluorescence signal disappears when two oligonucleotides labeled with a fluorescent dye and an eliminator come close to each other, and the 5 ' A TaqMan probe is a principle in which the 5 'and 3' portions of the nucleotide are labeled with a fluorescent dye and a scavenger (or vice versa), and the fluorescent signal is released with the 5 'portion cleaved by the nucleases. The contact elimination method is a principle in which a fluorescent dye and an erasing agent are combined by hydrogen bonding or the like, and a fluorescence signal is emitted by binding to a complementary sequence or substitution of a strand. The specific manner of use may vary depending on the detection equipment, the specific fluorescent dye and abolisher to be used, the nucleic acid sequence, and the like, and those skilled in the art will be able to select an appropriate method in view of this.

The term " fluorophore " (fluorochrome) as used herein refers to a material capable of absorbing energy of a first wavelength and emitting energy of another second wavelength. For example, fluororesin or derivatives thereof such as fluorescein isothiocyanate, carboxyfluorescein such as FAM, fluorescein amidite, cyanine such as Cy3, Cy5, Cy7 or derivatives thereof, tetramethylrhodamine (TAMRA) But are not limited to, rhodamine or derivatives thereof, coumarin or derivatives thereof, and the like. In one embodiment according to the present application, for example, FAM, HEX, Cy3, TMR, ROX, Texas red, LC red 640, Cy5, or LC red 705 are used as fluorescent dyes. These materials are commercially available from Applied Biosystems, . For example, FAM, TET, JOE, HEX, TAMRA, ROX, Cy5, Cy3, CalRed, Quasar, VIC, Texas Red.

The term " quencher " as used herein is a fluorophore quencher or a dark quencher that accepts excitation energy generated in a fluorescent dye. In the case of the electron phosphorus scavenger, the energy transferred is emitted as light of a specific wavelength, or in the latter case, it becomes heat. When they are located close to or within a certain distance from the fluorescent dye, the emission of the fluorescent dye is suppressed, and a fluorescent signal is detected when the fluorescent signal is not detected and is located at a distance long enough not to be inhibited . For example, TAMRA among fluorescent materials can be used as an eliminator of a substance called FAM. Examples of scavengers include Black Hole Quencher  (BHQ; Biosearch Technologies, Novato, Calif.), Dabsyl (dimethylaminoazosulphonic acid), Qxl ^TM quenchers (AnaSpec Inc., San Jose, Calif.), Iowa black FQⓡ, Iowa black RQⓡ IRDye QC-1, QYS quenchers (Life technologies, USA), and the like. In addition, the fluorescent substance may be used as an erasing agent depending on the type of the substance to be used together.

Those skilled in the art will be able to select suitable fluorescent dyes and scavengers in consideration of the fluorescence energy transfer method, the wavelength of each material, and the detection equipment.

The probe according to one embodiment of the present invention is single stranded DNA labeled with a fluorescent dye at 5 'and an erasing agent at 3', for example, labeled with a non-fluorescent molecular sterol (dark quencher) It can be used in the FRET system. The principles of FRET are described in Vladimir V. Didenko, Biotechniques. 2001 November; 31 (5): 1106-1121.

The method according to the invention is used in one embodiment in particular for real-time quantitative PCR. In this case, the method according to the present invention further comprises a probe as described above.

In the method according to the present invention, a lack of exonuclease activity results in a lack of 5 '-> 3' exonuclease activity or a mixture of reduced DNA polymerase and heat resistant flap endonuclease at a certain ratio, resulting in TaqMan qPCR , But it has become possible to apply it to TaqMan qPCR while maintaining these advantages by mixing other functions, such as speed and accuracy, with polymerase, FEN1.

In one embodiment, the detection time can be reduced by about 13 cycles with 24.88 cycles using the mixed enzyme according to the present invention at a high rate of 38.14 cycles when using the existing enzyme.

In the method according to the present invention, the DNA polymerase lacking 5 '-> 3' exonuclease activity or the reduced DNA polymerase and the heat resistant flap endonuclease are used in a molar ratio of 1: 0.005 to 1:50. Preferably in a molar ratio of 1: 0.02 to 1:20, more preferably in a molar ratio of 1: 0.1 to 1:10. In one embodiment, it is used in a molar ratio of 1: 2.

In one embodiment, the DNA polymerase and the heat-resistant flap endonuclease lacking 5 ' - > 3 ' exonuclease activity in the method according to the present invention can be used at the same molar ratio level, 0.5 to 1: 2 or especially 1: 1. Although not limited to this theory, when one molecule of polymerase lifts a TaqMan probe to form a flap, FEN1, which can actually act here, is also a molecule. When the number of molecules of FEN1 increases, it is impossible to exclude the possibility that several FEN1s bind to one flap, and thus the competitive effect may be rather suppressed. When the number of FEN1 molecules is decreased, The reaction efficiency becomes low due to a small amount and a limiting factor.

In another aspect, the disclosure provides a reaction composition or kit for nucleic acid amplification for use in rapid nucleic acid amplification as used in the methods herein. The compositions and kits according to the present invention may comprise other components necessary for nucleic acid amplification, such as PCR, for example, probes, primers, dNTPs, divalent cations and the like, in combination with enzymes according to the present invention.

The method according to the present invention can be used in real time PCR using, for example, FRET in one embodiment. In this case, the nucleic acid amplification process can be performed in real time using a PCR apparatus equipped with a fluorescence detector capable of detecting and / As shown in FIG.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Example 1. Quantitative PCR using a combination of mutant Taq polymerase and various Nuclease

A mutant Taq polymerase (nTaq, Cat # P025 enginemix) without 5 'nuclease activity was combined with various enzymes having 5' nuclease activity to prepare a polymerase-nucleicase suitable for quantitative PCR implementation of the TaqMan probe type The following experiments were conducted to select the combinations. Forward (5'-ACGGATTTGGTCGTATTGGGC-3 ') and Reverse (5'-TTGACGGTGCCATGGAATTTG-3') primers and fluorescent TaqMan probes (5 'FAM-CCTGGTCACCAGGGCTGCTTTTAA-3') were used to amplify the GAPDH gene with 10 ng of human cDNA as a template. TAMRA 3 ') (Genentech, Korea). For gene amplification, 1 unit (50 ng / unit) each of wild-type Taq polymerase and mutant Taq polymerase was added to standard PCR buffer (10 mM Tris-HCl / pH 8.3, 1.5 MgCl ₂ , 50 mM KCl, 0.2 mM dNTP) Respectively. 30 units of RecJ (NEB, USA), 1 unit of Nuclease BAL-31 (NEB, USA), 10 units of T7 Exonuclease (NEB, USA) 10 units of Exonuclease VII (NEB, USA), 10 units of Exonuclease V (NEB, USA), and 50 ng of heat resistant FEN1 (Ennomix, Korea) were used. PCR was performed using a real-time PCR instrument (CFX96, Bio-Rad). After a denaturation step at 95 ° C for 10 minutes, a cycle consisting of 95 ° C for 30 seconds and 60 ° C for 60 seconds was performed for 45 cycles. Relative fluorescence values were measured after 60 seconds of 60 sec reaction per cycle.

As shown in FIG. 2, in the case of wild-type Taq polymerase (WT), it was confirmed that a fluorescence amplification curve appears by amplification of the target gene and subsequent TaqMan probe cleavage (FIG. 2A, red curve) In the case of the Taq polymerase (MT) having no activity, it was confirmed that the TaqMan probe could not be cleaved and the fluorescence amplified signal was not detected (Fig. 2A, black curve). To complement the lacking 5 'nuclease activity of the mutant Taq polymerase (MT), a variety of 5' nuclases expected to aid in the quantitative PCR implementation of the TaqMan probe type were incubated with the mutant Taq polymerase (MT) As a result, it was confirmed that when mixed with heat-resistant FEN1, a fluorescence amplification curve similar to that of wild-type Taq polymerase and a cycle threshold (Ct) were derived (FIG. 2A; purple curve, FIG. 2B).

These results indicate that a variety of thermostable polymerases (eg, genetically modified rapid Taq polymerase, which has a considerable effect in terms of rapidity, accuracy, etc., but which is not applicable to TaqMan quantitative PCR due to lack of 5 ' Pfu polymerase) and heat-resistant FEN1 to produce a mixed enzyme suitable for TaqMan quantitative PCR.

Example 2. Quantitative PCR using a combination of mutant Taq polymerase and FENl

The following experiment was carried out in order to find out the combination ratio of mutant Taq polymerase (angiomix) and heat-resistant FEN1 without 5 'nuclease activity in TaqMan quantitative PCR. Basically, the experiment was carried out in the same manner as in Example 1, and 1 unit of wild-type Taq polymerase and mutant Taq polymerase were increased to 1: 0 and 1: 0.01 to 1: 1, respectively, in the case of heat-resistant FEN1 Respectively.

As shown in FIG. 3, when Taq polymerase (MT) having no 5 nuclease activity and heat resistant FEN1 were increased in weight ratio of 1: 0, 1: 0.01, 1: 0.1, 1: 1, the amount of FEN1 And the slope of the fluorescent graph gradually increased as the number of samples increased (Fig. 3A; (3), (4), (5), and (6)). When the ratio of Taq polymerase (MT) to FEN1 was 1: 1, the slope of the graph was the largest, and it was confirmed that the graph shows a fluorescence graph similar to that of the wild type Taq polymerase (WT) (FIG. 3A; ⑦). No amplification signal was detected in Taq polymerase (MT) + FEN1 and Taq polymerase (WT) when no template DNA was added as a negative control (Fig. 3A;

When an amplified signal was detected, an agarose gel assay was performed on the amplified product to see if the target of the correct size was amplified. As a result, a PCR product was generated at the expected 157 bp when a fluorescent amplified signal was detected (Fig. 2B). The cycle threshold value and the relative fluorescence intensity (RFU) value in each experiment are shown graphically in C and D of FIG. 2, respectively.

Example 3. Determination of optimal combination ratio and amount of mutant Taq polymerase and FEN1

First, the optimal combination ratio of mutant Taq polymerase and FEN1 was determined in more detail. Basically, the experiment was carried out in the same manner as in Example 2. The amount of mutant Taq polymerase was 1 unit and the mixing ratio of heat-resistant FEN1 was 1: 0.2 to 1: 200 (molar ratio).

The results are shown in Fig. 4A. When the mutant Taq polymerase was immobilized and the amount of FEN1 was increased, the Ct difference according to the mixing ratio was as small as 1.5 cycles (maximum 28.69 to minimum 27.38) until 1:20 to 1:10 (molar ratio) The fluorescence signal was not detected from 1:40 (indicated by 40 Ct on the graph). Therefore, in order to suitably implement the present invention, the combination ratio of mutant Taq polymerase and FENl should be lower than 1:40. Considering the results of Example 2 together, it can be seen that it is preferably used in a ratio of 1: 0.02 to 1:20 (molar ratio).

Next, optimal amounts of mutant Taq polymerase and heat resistant FEN1 were determined. The basic experimental procedure was the same as in Example 1, and the mixing ratio of the mutant Taq polymerase and the heat-resistant FEN1 was 1: 2 (molar ratio).

The results are shown in Fig. 4B. When the amount of mixed enzyme was increased from 0.005 to 50 units based on the amount of Taq polymerase, the fastest Ct value was obtained with an average of 26 cycles using 2.5 to 10 units. When 0.5 and 50 units were used, the average Ct values were 30.44 and 28.22, respectively. Gene amplification did not occur when the amount of mixed enzyme was less than 0.1 unit. Accordingly, when the mutant Taq polymerase and FEN1 are used in a molar ratio of 1: 2, they can be used in an amount of more than 0.1 unit and less than 50 units based on the amount of Taq polymerase, especially 0.5 to 50 units, more particularly 2.5 to 10 units Should be used.

Example  4. Heat resistance FEN1  of TaqMan  Breaking pattern analysis

The following experiment was conducted to confirm the difference in the cleavage pattern of the probe of 'mutant Taq polymerase-FEN1 mixed enzyme' which has equivalent performance to the wild type Taq polymerase in the quantitative PCR of the TaqMan probe type. The basic experiment was carried out in the same manner as in Example 1. After the completion of the quantitative PCR, the amplified product was analyzed by 15% denaturing PAGE.

The results are shown in FIG. The band that appears on the gel after imaging on UV after polyacrylamide gel electrophoresis is either a complete TaqMan probe labeled with FAM at 5 'or a truncated form of the probe. When the wild-type Taq polymerase was used, it was confirmed that a part of the TaqMan probe was cleaved in the presence of template DNA, resulting in two different small-sized bands (Fig. 5, Lane 3). 5 (Lane 4). However, when heat resistant FEN1 was added thereto, a cleavage pattern similar to the case of using wild-type Taq polymerase was obtained. In contrast, when Taq polymerase (MT) (Fig. 5; Lane 5). This result implies that the enzymatic activity of heat tolerant FEN1 can replace the 5 'nuclease activity of wild-type Taq polymerase in PCR.

Example 5. Rapid quantitative PCR using a combination of mutant Sso7d-ΔTaq polymerase and FEN1

In this example, Sso7d-ΔTaq (disclosed in US 2004/0081963) was used as a mutant Taq polymerase. The experiment was basically carried out in the same manner as in Example 1. The mixing ratio of Sso7d-ΔTaq and heat-resistant FEN1 was 1: 2 (molar ratio). TaqMan quantitative PCR was performed under different normal and fast conditions to confirm the rapid PCR reaction using mutant Taq polymerase. The normal conditions were the same as in Example 1. Fast conditions were 95 cycles of 10 minutes at 95 DEG C, 3 cycles of 95 DEG C and 10 seconds at 60 DEG C for 45 cycles.

The results are shown in FIG. When PCR was performed under the usual conditions (95 ° C / 30 seconds, 60 ° C / 60 seconds, 45 cycles in total) as a target with GAPDH (TaqMan probe: FAM dye) as shown in FIGS. 6A and 6B, The Ct and RFU values of lase (Sso7d-ΔTaq + FEN1) and wild-type polymerase (WT Taq) were similar. However, when the experiment was performed under fast conditions (95 ° C / 3 sec, 60 ° C / 10 sec, 45 cycles), it was confirmed that the mixed polymerase showed a Ct value of about 13 cycles faster than the wild type polymerase.

These results show that when the MERS-CoV (up-region, TaqMan probe: Cy5 dye) is used as a target, the Ct value of the hybrid polymerase according to the present invention is 15 cycles or more faster than that of the wild-type polymerase, (C and D in Fig. 6).

Therefore, the above results show that although a mutation polymerase such as Sso7d-ΔTaq, which has a rapid synthesis rate in itself, but lacks 5 'exonuclease activity and can not be applied to quantitative PCR using a FRET-based probe TaqMan When mixed with thermostable FEN1 according to the method of the present invention, it can be used in quantitative PCR using TaqMan, and convenience is greatly improved.

Example 6. Fast, Highly Reliable Quantitative PCR Mixed with Mutant Pfu Polymerase and Heat Resistant FEN1

Basically, the experiment was carried out in the same manner as in Example 1. Rapid PCR conditions were used for rapid PCR, and 1 unit (50 ng) of Pfu polymerase was used in the same manner as Taq polymerase for comparison.

Pfu polymerase possesses 3 '-> 5' exonuclease activity instead of 5 '-> 3' exo / endonuclease activity of Taq polymerase. This makes it possible to correct erroneously cloned nucleotides and is mainly used for the synthesis of nucleic acids requiring high fidelity. However, it has disadvantages that it can not be applied to TaqMan qPCR because there is no activity of 5 '-> 3' nucleases.

In order to accurately diagnose a new virus, rapid diagnosis is performed by quantitative PCR using a probe such as TaqMan, and the mutation of the gene is analyzed by sequencing the amplified product in the corresponding quantitative PCR, An operation to perform an operation may be required. Especially in the case of MERS-CoV which is popular in recent years, it is necessary to analyze the sequence of ORF1b and N gene for accurate confirmation. However, in the case of the TaqMan quantitative PCR product using the conventional Taq polymerase, there is a problem that the Taq polymerase itself is not suitable for sequencing due to its low accuracy. Therefore, for sequence analysis, the sample must be amplified again with Pfu polymerase, which has a calibration function. If the sample is not sufficient or there is a risk of infection, accurate and safe diagnosis becomes difficult.

However, in the method according to the present invention, it was confirmed that heat-resistant FEN1 was mixed with Pfu polymerase to enable TaqMan qPCR through the activity of FEN1 despite the absence of 5 '-> 3' nuclease activity.

The results are shown in FIG. 7 and disclose the results of experiments in which TaqMan qPCR was performed using Taq polymerase + FEN1 and Pfu polymerase + FEN1. Polymerases used in this experiment were polymerase conjugated with Sso7d in order to enable fast PCR. Both Fast Taq (Sso7d-ΔTaq + FEN1) and Fast Pfu (Sso7d-Pfu + FEN1) showed similar amplification curves and Ct values (Fig. The PCR products thus amplified were cloned, and the mutation occurrence probability was analyzed by sequence analysis. As a result, it was confirmed that the use of Fast Pfu was about 20 times lower than that in the case of using Fast Taq (see FIG. 7 B). The target sequence used in this experiment is shown in Fig. 7C, and the site where the mutation mainly occurs is shown in red (underline is forward / reverse primer sequence, underlining + italic is TaqMan probe sequence).

These results demonstrate that mixing Sso7d-Pfu polymerase with heat-stable 5 'flap endonuclease (FEN1) with both rapid synthesis properties and corrective function enables the implementation of FRET-based quantitative PCR, , It is possible to detect various nucleic acids with high accuracy as well as fast amplification.

In the present application, experiments using nTaq, Sso7d-Taq, and Sso7d-Pfu as various polymerases lacking 5 'nuclease activity have shown that these enzymes can be successfully applied to FRET-based amplification . Thus, enzymes lacking or reducing various origins and / or types of 5 ' exonuclease function or activity can be successfully applied to the methods of the present application.

The present invention relates to a research (No.2014DD017) carried out by ENGNOMICS Co., Ltd., funded by the government (future creation science department) of the government in 2014, under the support of the "R & D Special Zone Technology Development Project" .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (11)

At least one primer and probe that binds to the target nucleic acid, deoxynucleotide 5'-triphosphate, and a DNA polymerase that lacks or reduces 5 '->3' exonuclease activity as an enzyme, Providing an amplification reaction composition comprising a flap endonuclease; And
A method for detecting or amplifying a nucleic acid based on FRET using a DNA polymerase lacking 5 '->3' exonuclease activity or amplifying the target nucleic acid under amplifiable conditions.
The method according to claim 1, wherein the 5 '- >3' exonuclease activity-deficient or reduced DNA polymerase is originally an enzyme with no activity or a mutation has been induced so as not to have the activity.
3. The method of claim 2,
The DNA polymerase may be selected from the group consisting of Methanococcus jannaschii, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermus flavus, Thermococcus literalis, Thermus antranikianii, Thermus caldophilus, Thermus chliarophilus, Thermus flavus, Thermus igniterrae, Thermus lacteus Thermus oshimai, Thermus Pyrococcus horikoshii, Pyrococcus abyssinus, Pyrococcus horizon, Pyrococcus furiosus, Thermococcus furiosus, Thermococcus furiosus, Thermus rubens, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sps 17, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Thermococcus barossi, Thermococcus gorgonarius, Pyrodictium occultum, Aquifex pyrophilus or Aquifex aeolieus.
The method according to claim 1,
The nucleic acid polymerase lacking or reducing the 5 '- >3' exonuclease activity may be a polymerase lacking the N terminus or having a mutation introduced into the exonuclease domain at the N terminus, (Thermus aquaticus), Pfu (Pyrococcus furiosus) polymerase, Tth (Thermus thermophilus) polymerase, or Tf1 (Thermus flavus) polymerase. Jane, how.
The method according to claim 1,
The thermostable flap endonuclease may be selected from the group consisting of Sulfolobus solfataricus, Pyrobaculum aerophilum, Thermococcus litoralis, Archaeaglobus veneficus, Archaeaglobus profundus, Acidianus brierlyi, Acidianus ambivalens, Desulfurococcus amylolyticus, Desulfurococcus mobilis, Pyrodictium brockii, Thermococcus gorgonarius, Thermococcus zilligii, Methanopyrus kanseri, Methanococcus igneus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeropyrum pernix and Archaeaglobus veneficus.
The method according to claim 1,
Wherein the amplification is PCR and the detection is quantitative or qualitative detection.
The method according to claim 1,
Wherein the 5 '- >3' exonuclease activity is deficient or reduced and the heat resistant DNA polymerase and the heat resistant flap endonuclease are contained in a molar ratio of 1: 0.02 to 1:20.
The method according to claim 1,
Wherein said 5 '- >3' exonuclease activity is lacking or reduced and said heat resistant DNA polymerase and said heat resistant flap endonuclease are contained in the same molar amount.
The kit for use in any one of claims 1 to 8, wherein the kit is an enzyme required for nucleic acid amplification, wherein the 5 '- >3' exonuclease activity is lacked or the DNA polymerase having a reduced heat- A nucleic acid detection kit based on FRET, comprising a nuclease.
A composition for detection of nucleic acid based on FRET, which comprises an enzyme required for nucleic acid amplification, a DNA polymerase lacking 5 '->3' exonuclease activity or reduced and a heat resistant flap endonuclease.
A composition for detection of nucleic acid based on FRET, which comprises an enzyme required for nucleic acid amplification, a polymerase lacking 5 '-> 3' exonuclease activity and corrective function, and a heat resistant flap endonuclease.

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