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WO2020145762A1 - Adn polymérase pour la détection de mutation de jak2, et kit la comprenant - Google Patents

Adn polymérase pour la détection de mutation de jak2, et kit la comprenant Download PDF

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WO2020145762A1
WO2020145762A1 PCT/KR2020/000540 KR2020000540W WO2020145762A1 WO 2020145762 A1 WO2020145762 A1 WO 2020145762A1 KR 2020000540 W KR2020000540 W KR 2020000540W WO 2020145762 A1 WO2020145762 A1 WO 2020145762A1
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pcr
jak2
gene mutation
jak2 gene
kit
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이병철
박일현
변유정
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Genecast Co Ltd
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Definitions

  • the present invention relates to a DNA polymerase for detecting JAK2 mutants and a kit comprising the same, and more specifically, a DNA polymerase, primer set, probe, and kit capable of detecting somatic mutations with high sensitivity in codon 617 of the JAK2 gene. And JAK2 gene mutation detection method using the kit.
  • Cancer refers to a population of abnormal cells caused by continuous division and proliferation by destroying the balance between cell division and death by various causes, and is also called a tumor or a neoplasm. It usually affects more than 100 parts of the body, including organs, white blood cells, bones, and lymph nodes, and develops into serious symptoms through infiltration into surrounding tissues and metastasis to other organs.
  • cancer screening methods are physical. Examples include gastrointestinal X-ray imaging, double contrast, compression imaging, or mucosal imaging, and by using an endoscope to visually check internal organs, very small lesions that do not appear on X-ray examination can be found, as well as cancer. Biopsies can also be performed directly in this suspicious area, increasing the diagnostic rate.
  • this method has disadvantages in terms of hygiene and the need for the patient to suffer during the examination.
  • MPNs Myeloproliferative neoplasms
  • the three main diseases that make up MPN are polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), characterized by unique somatic cell mutations do.
  • PV polycythemia vera
  • ET essential thrombocythemia
  • PMF primary myelofibrosis
  • JAK2 (Janus kinase 2) is a member of the cytosolic tyrosine kinase group and is involved in signal transduction from growth factor receptors. JAK2 plays a key role in the JAK2-STAT (signal transducer and activator of transcription) pathway. In autophosphorylation after activation through ligand binding, JAK2 is phosphorylated by mobilizing the STAT day and translocated to the nucleus to act as a transcription factor. This mutation interferes with the self-inhibition of JAK2 JH2, resulting in the constitutive activity of JAK2, resulting in uncontrolled proliferation.
  • Somatic cell gain-of-function mutations in the JAK2 kinase gene on chromosome 9 were found in more than 50% of those with MPN.
  • the mutation is the constant G to T conversion of exon 14, which causes the valine of codon 617 (JAK V617F) to be substituted with phenylalanine.
  • the JAK2 V617F mutation (G1849T) is present in a number of myeloproliferative neoplasms such as PV ( ⁇ 95%), ET ( ⁇ 60%), PMF ( ⁇ 50%).
  • V617F mutations in patients with bcr-abl-myeloproliferative disease varies, but defining the presence of these mutations is part of current clinical diagnostic algorithms.
  • JAK inhibitors capable of treating diseases caused by mutation of JAK2 V617F were evaluated for efficacy and stability through several clinical trials. It has entered the clinical stage and is recognized for its excellent efficacy and stability.
  • Jakafi (Ruxolitinib Phosphate) is treated with myelofibrosis (Post-polycythemia vera myelofibrosis), post-essential thrombocythemia myelofibrosis, and hydroxyurea in adults. It has been approved for use in treating patients who cannot or cannot improve symptoms.
  • “Liquid biopsy” provides a new source of cancer-derived substances that can better reflect the condition of the disease and contribute to more individualized treatment. It is also important to develop more sensitive techniques for mutation detection.
  • Korean Patent Publication No. 10-2009-0090263 discloses a probe for detecting mutations in the JAK2 gene and its use, and discloses a probe for detecting a mutation in the JAK2 gene, but polymerization for detecting a mutation in the JAK2 gene There is no known reaction buffer for increasing enzymes and their activity.
  • the present inventors developed a kit containing a DNA polymerase capable of detecting mutations in exon 14 of the JAK2 gene with high sensitivity and a reaction buffer for increasing its activity, and completed the present invention.
  • An object of the present invention is to provide a DNA polymerase for detecting JAK2 gene mutation.
  • Another object of the present invention is to provide a primer set for detecting JAK2 gene mutations.
  • Another object of the present invention is to provide a probe for detecting JAK2 gene mutation.
  • Another object of the present invention is to provide a kit for detecting a JAK2 gene mutation, comprising the DNA polymerase and/or primer set described above.
  • Another object of the present invention is to provide a method for detecting JAK2 gene mutations using the kit described above.
  • the present invention is a 507th amino acid residue glutamic acid (E) in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine (K), the 536th amino acid residue arginine (R) is lysine (K)
  • E amino acid residue glutamic acid
  • R amino acid residue arginine
  • K lysine
  • K DNA polymerase for detecting a JAK2 gene mutation, having Taq polymerase substituted with valine (V), wherein arginine (R), the 660th amino acid residue, is substituted with.
  • the present invention also provides a primer set for detecting a JAK2 gene mutation comprising any one or more primers selected from the group consisting of SEQ ID NOs: 3 to 6.
  • the present invention also provides a probe for detecting a JAK2 gene mutation, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7 and 8.
  • the nucleotide sequence of SEQ ID NOs: 7 and 8 may be labeled with one fluorescent material selected from the group consisting of FAM, Quasar 670 and CY5 at the 5'-end, respectively 3 At the'-end, one quencher selected from the group consisting of BHQ-1 and BHQ-2 may be labeled.
  • the present invention also provides a kit for detecting a JAK2 gene mutation, comprising the DNA polymerase and/or primer set described above.
  • the kit may further include a probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7 and 8.
  • the nucleotide sequences of SEQ ID NOs: 7 and 8 are labeled with one fluorophore selected from the group consisting of FAM, Quasar 670 and CY5 at the 5'-end, respectively.
  • One of the quenchers selected from the group consisting of BHQ-1 and BHQ-2 may be labeled at the 3'-end.
  • the kit comprises 25 to 100 mM KCl; And 1 to 7 mM (NH 4 ) 2 SO 4 ; and may further include a PCR buffer composition having a final pH of 8.0 to 9.5.
  • the kit comprises 40 to 90 mM KCl; 1 to 5 mM (NH 4 ) 2 SO 4 ; And 5 to 80 mM of TMAC (Tetra methyl ammonium chloride), and may further include a PCR buffer composition having a final pH of 8.0 to 9.5.
  • the present invention also provides a method for detecting a JAK2 gene mutation comprising the following steps:
  • the PCR may be allele-specific PCR or real-time PCR.
  • (d) may further include the step of confirming the amplification result by the PCR by measuring the Ct (cycle threshold) value.
  • the JAK2 gene mutation may include one or more selected from the group consisting of deletion, substitution and insertion mutations in codon 617 of exon 14 of the JAK2 gene.
  • the JAK2 gene mutation may include substitution of valine, which is the amino acid of codon 617 of exon 14 of the JAK2 gene.
  • the JAK2 gene mutation detection method may be applied to diagnose myeloproliferative neoplasm.
  • the myeloproliferative neoplasm may be one or more selected from the group consisting of true erythrocyte hyperplasia, essential thrombocytopenia, and primary myelofibrosis (PMF).
  • the nucleic acid of step (a) may be extracted from bone marrow tissue biopsy or liquid biopsy.
  • the kit of the present invention shows high detection sensitivity (up to 0.01%, 3 mutant copies in 30,000 wild-type copies), high specificity and reproducibility, and is applicable to both liquid biopsy and tissue biopsy.
  • high detection sensitivity up to 0.01%, 3 mutant copies in 30,000 wild-type copies
  • high specificity and reproducibility high specificity and reproducibility
  • Figure 1 shows the production process of Taq DNA polymerase containing each of the R536K, R660V and R536K/R660V mutations, (a) schematically shows fragment PCR and overlap PCR, (b) is amplified in fragment PCR The result of confirming the product by electrophoresis, and (c) shows the result of confirming the amplified product by electrophoresis by amplifying the entire length by overlap PCR.
  • Figure 2 is a result of confirming the overlap PCR product of FIG. 1(c) purified by digestion with the restriction enzyme EcoRI/XbaI and then the SAP-treated pUC19 vector for gel extraction.
  • Figure 3 is a schematic diagram showing fragment PCR and overlap PCR during the preparation of Taq DNA polymerase containing E507K, E507K/R536K, E507K/R660V and E507K/R536K/R660V mutations, respectively.
  • FIG. 4 shows the results of confirming by electrophoresis the overlap PCR product of FIG. 3 purified with the pUC19 vector digested with the restriction enzyme EcoRI/XbaI for gel extraction, and then with SAP.
  • V617F mutant plasmid (30,000, 3,000, 300, 30, 3 and 0 copies) template.
  • the presence of the JAK2 mutation acts as a predictor for the diagnosis of myeloproliferative neoplasms, and thus an effective and rapid detection method of the JAK2 mutation is required for early diagnosis of myeloproliferative neoplasms.
  • the present inventors sought a solution to the above-mentioned problem by providing a kit including a DNA polymerase capable of detecting V617F in exon 14 of the JAK2 gene with high sensitivity and a reaction buffer for increasing its activity.
  • the kit of the present invention exhibits high detection sensitivity, high specificity and reproducibility, and is applicable to both liquid biopsy and tissue biopsy.
  • FAM/CY5 channels enable analysis on all qPCR devices.
  • amino acid refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein.
  • amino acid includes the following 20 natural or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspart Acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan ( Trp or W), tyrosine (Tyr or Y), and valine (Val
  • Amino acids are typically organic acids, which include substituted or unsubstituted amino groups, substituted or unsubstituted carboxy groups, and one or more side chains or groups, or any analogue of these groups.
  • exemplary side chains include, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, Borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thio acid, hydroxylamine, or any combination of these groups.
  • mutant refers to a recombinant polypeptide comprising one or more amino acid substitutions compared to the corresponding naturally occurring or unmodified DNA polymerase.
  • thermo stable polymerase (referring to a heat stable enzyme) is heat resistant, retains sufficient activity to achieve subsequent polynucleotide elongation reactions and is treated with elevated temperature for the time required to achieve denaturation of the double-stranded nucleic acid Does not irreversibly denature (deactivate) when As used herein, it is suitable for use at temperatures cycling reactions such as PCR. Irreversible denaturation herein refers to permanent and complete loss of enzyme activity.
  • enzymatic activity refers to catalyzing a combination of nucleotides in a suitable way to form a polynucleotide extension product complementary to the template nucleic acid strand.
  • thermophilic bacteria include, for example: Thermomoto maritima, thermos aquaticus, thermos thermophilus, thermos flavus, thermomod philipformis, thermos species DNA polymerase derived from Sps17, Thermos species Z05, Thermos Caldophyllus, Bacillus caldotenax, Thermomoto Neopolitana, and Thermosippo africanus.
  • thermoactivity refers to an enzyme that maintains catalytic properties at temperatures (ie 45-80° C.) commonly used for reverse transcription or annealing/extension steps in RT-PCR and/or PCR reactions.
  • Thermostable enzymes are those that are not irreversibly inactivated or denatured when treated at elevated temperatures required for nucleic acid denaturation.
  • the thermoactive enzyme may or may not be thermostable.
  • the thermally active DNA polymerase can be DNA or RNA dependent from thermophilic or mesophilic species, including but not limited to:
  • nucleotide is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that exists in the form of a single strand or a double strand, and is not specifically mentioned otherwise. Unless it can contain analogs of natural nucleotides.
  • nucleic acid or “polynucleotide” refers to a polymer that can correspond to a DNA or RNA polymer, or analogs thereof.
  • Nucleic acids can be, for example, chromosomal or chromosomal segments, vectors (eg, expression vectors), expression cassettes, naked DNA or RNA polymers, products of polymerase chain reaction (PCR), oligonucleotides, probes, and primers. Or may include it.
  • Nucleic acids can be, for example, single-stranded, double-stranded, or triple-stranded, but are not limited to any particular length. Unless otherwise stated, certain nucleic acid sequences include or encode complementary sequences in addition to any sequence specified.
  • primer refers to a polynucleotide that can serve as a starting point for nucleic acid synthesis in the template-direction when placed under conditions where polynucleotide elongation is initiated. Primers can also be used in a variety of other oligonucleotide-mediated synthesis processes, including as initiators of de novo RNA synthesis and in vitro transcription-related processes. Primers are typically single-stranded oligonucleotides (eg, oligodeoxyribonucleotides). The appropriate length of the primer will typically depend on the intended use in the range of 6 to 40 nucleotides, more typically in the range of 15 to 35 nucleotides.
  • Primers are not required to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template for elongation of the primer.
  • the term “primer pair” includes a 5′-sense primer that hybridizes complementarily to the 5′-end of the amplified nucleic acid sequence, and a 3′-antisense primer that hybridizes to the 3′ end of the amplified sequence.
  • Means a set of primers comprising Primers can be labeled, if necessary, by incorporating a label that can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means.
  • useful labels include: 32 P, fluorescent dyes, electron-dense reagents, enzymes (usually used in ELISA assays), biotin, or haptens and proteins in which antiserum or monoclonal antibodies can be used. .
  • 5′-nuclease probe refers to an oligonucleotide comprising at least one luminescent label moiety used in a 5′-nuclease reaction to target nucleic acid detection.
  • the 5'-nuclease hydrolyzate probe comprises only a single luminescent moiety (eg, fluorescent dye, etc.).
  • the 5'-nuclease probe contains a self-complementary region so that the probe can form a hairpin structure under selective conditions.
  • the 5'-nuclease hydrolyzate probe comprises two or more labeling moieties, and one of the two labels is released from the oligonucleotide after being separated or degraded to increase the emission intensity.
  • the 5'-nuclease hydrolase probe is labeled with two different fluorescent dyes, for example a 5'-terminal reporter dye and a 3'-terminal quencher dye or moiety.
  • the 5'-nuclease probe is labeled in addition to, or at one or more positions other than the terminal position. When the probe is intact, energy transfer typically occurs between the two phosphors such that the fluorescence emission from the reporter dye is partially extinguished.
  • a 5'-nucleic acid hydrolase probe bound to the template nucleic acid has an activity such that the fluorescence of the reporter dye is no longer quenched, for example, Taq polymerase or other Degraded by the 5'to 3'-nucleic acid hydrolase activity of the polymerase.
  • a 5'-nuclease probe can be labeled with two or more different reporter dyes and a 3'-terminal quencher dye or moiety.
  • FRET fluorescence resonance energy transfer
  • poster resonance energy transfer refers to the transfer of energy between two or more chromophores, donor chromophores and receptor chromophores (referred to as quenchers).
  • the donor typically transfers energy to the receptor when the donor is excited by emitting light of a suitable wavelength.
  • Receptors typically re-emit energy transferred in the form of light emitted at different wavelengths.
  • the receptor is a “cancer” matting agent, it disperses the energy transferred in a form other than light. Whether a particular fluorescent substance acts as a donor or a receptor depends on the properties of other members of the FRET pair. Commonly used donor-receptor pairs include FAM-TAMRA pairs.
  • Commonly used matting agents are DABCYL and TAMRA.
  • Commonly used cancer matting agents include: BlackHole QuenchersTM (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa BlackTM (Integrated DNA Tech., Inc., Coralville, Iowa), And BlackBerryTM Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).
  • nucleic acid base nucleoside triphosphate, or nucleotide refers to naturally occurring polynucleotides described (ie, for DNA, they are dATP, dGTP, dCTP and dTTP).
  • dATP dGTP
  • dCTP dCTP
  • dTTP dTTP
  • dITP, and 7-deaza-dGTP are frequently used instead of dGTP and can be used instead of dATP in in vitro DNA synthesis reactions such as sequencing.
  • nucleic acid base nucleotide, or nucleotide
  • nucleotide is a conventional base, nucleotide, or modification, derivative, or nucleotide that occurs naturally in a particular polynucleotide, or Analogs.
  • Certain unusual nucleotides are modified at the 2'position of the ribose sugar compared to conventional dNTP.
  • nucleotides for RNA are ribonucleotides (i.e., ATP, GTP, CTP, UTP, collective rNTP), since nucleotides have hydroxyl groups at the 2'position of the sugar, this is compared to the absence of dNTP,
  • ribonucleotides are unusual nucleotides as substrates for DNA polymerases.
  • unusual nucleotides include, but are not limited to, compounds used as terminators for nucleic acid sequencing.
  • Exemplary terminator compounds include, but are not limited to, compounds having a 2',3'- dideoxy structure, referred to as dideoxynucleoside triphosphate.
  • Dideoxynucleoside triphosphate ddATP, ddTTP, ddCTP and ddGTP are collectively referred to as ddNTP.
  • Additional examples of terminator compounds include 2'-PO 4 analogues of ribonucleotides.
  • Other unusual nucleotides are phosphorothioate dNTP ([[ ⁇ ]-S]dNTP), 5'-[ ⁇ ]-borano-dNTP, [ ⁇ ]-methyl-phosphonate dNTP, and ribonucleosides Triphosphate (rNTP).
  • Uncommon bases include radioactive isotopes such as 32 P, 33 P, or 35 S; Fluorescent labels; A label for chemiluminescence; Bioluminescent markers; Hapten labels such as biotin; Or it can be labeled with an enzyme label such as streptavidin or avidin.
  • Fluorescent labels can include negatively charged dyes, such as the dyes of the fluorescein family, or neutrally charged dyes, such as the dyes of the rhodamine family, or positively charged dyes, such as the dyes of the cyanine family. Dyes of the fluorescein family include, for example, FAM, HEX, TET, JOE, NAN and ZOE.
  • Rhodamine family dyes include Texas Red, ROX, R110, R6G, and TAMRA.
  • Various dyes or nucleotides labeled FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, Texas Red and TAMRA are Perkin-Elmer (Boston, MA), Applied Biosystems (Foster City, CA), or Invitrogen /Molecular Probes (Eugene, OR).
  • the cyanine family dyes include Cy2, Cy3, Cy5, and Cy7, and are marketed by GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).
  • the 507th amino acid residue, glutamic acid (E), is substituted with lysine (K), the 536th amino acid residue, arginine (R), is replaced with lysine (K), and the 660th amino acid residue.
  • a DNA polymerase for detecting a JAK2 gene mutation including Taq polymerase in which phosphorus arginine (R) is substituted with valine (V).
  • the "Taq polymerase” was a thermophilic DNA polymerase named after the thermophilic bacterium Thermus aquaticus and was first isolated from the bacteria.
  • Thermos Aquaticus is a bacterium inhabiting hot springs and hot water jets, and Taq polymerase has been identified as an enzyme capable of withstanding the protein denaturation conditions (high temperature) required in PCR.
  • the optimum activity temperature of Taq polymerase is 75-80 °C, has a half-life of 9 hours at 22.5 hours at 92.5 °C, 40 minutes at 95 °C, 9 minutes at 97.5 °C, and replicates 1000 base pair DNA within 10 seconds at 72 °C Can.
  • PCR can be performed at high temperatures (above 60°C).
  • the amino acid sequence shown in SEQ ID NO: 1 for Taq polymerase is used as a reference sequence.
  • the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine (K) in glutamic acid (E)
  • the 536th amino acid residue is substituted with lysine (K) in arginine (R)
  • the 660th amino acid The Taq polymerase in which the residue was substituted with arginine (R) to valine (V) was designated as “E507K/R536K/R660V”, and its amino acid sequence and nucleotide sequence were shown in SEQ ID NO: 2 and SEQ ID NO: 17, respectively.
  • the present invention also provides a primer set for detecting a JAK2 gene mutation comprising one or more primers selected from the group consisting of SEQ ID NOs: 3-6.
  • the primer set for JAK2 gene mutation detection of the present invention may be, for example, those described in Table 16 of Example 3, but is not limited thereto.
  • the polymerase according to the present invention has excellent sensitivity to detect JAK2 mutations, especially when used together with the primer sequences in Table 16.
  • the present invention also provides a probe for detecting a JAK2 gene mutation, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7 and 8.
  • the nucleotide sequence of SEQ ID NOs: 7 and 8 may be labeled with a fluorophore at the 5'-end, and a quencher at the 3'-end.
  • nucleotide sequences of SEQ ID NOs: 7 and 8 are labeled with one fluorescent material selected from the group consisting of FAM, Quasar 670 and CY5 at the 5'-end, respectively, and BHQ-1 and BHQ at the 3'-end.
  • One quencher selected from the group consisting of -2 may be labeled.
  • the present invention also provides a kit for detecting a JAK2 gene mutation, comprising the DNA polymerase and/or primer set described above.
  • the kit of the present invention can be used for research (Research Use Only, RUO) or in-vitro diagnostic (IVD).
  • the kit of the present invention may be a PCR kit, and may contain any reagents or other elements recognized by those skilled in the art as being used in the primer extension process.
  • the PCR kit of the present invention includes (a) nucleoside triphosphate; (b) a reagent for quantification that binds double-stranded DNA; (c) polymerase blocking antibodies; (d) one or more control values or control sequences; And (e) one or more templates; may further include one or more selected from the group consisting of.
  • the kit comprises 25 to 100 mM KCl; And 1 to 7 mM (NH 4 ) 2 SO 4 ; and may further include a PCR buffer composition having a final pH of 8.0 to 9.5.
  • the kit comprises 40 to 90 mM KCl; And 1 to 5 mM (NH 4 ) 2 SO 4 ; and may further include a PCR buffer composition having a final pH of 8.0 to 9.0.
  • the kit comprises 25 to 100 mM KCl; 1 to 7 mM (NH 4 ) 2 SO 4 ; And 5 to 50 mM of TMAC (Tetra methyl ammonium chloride), and may further include a PCR buffer composition having a final pH of 8.0 to 9.5.
  • the kit comprises 40 to 90 mM KCl; 1 to 5 mM (NH 4 ) 2 SO 4 ; And 10 to 40 mM of TMAC (Tetra methyl ammonium chloride), and may further include a PCR buffer composition having a final pH of 8.0 to 9.0.
  • the kit according to the present invention may further include TrisCl (pH 8.0 to 9.5), MaCl 2 , Tween 20, and Bovine serum albumin (BSA) in addition to KCl, (NH 4 ) 2 SO 4 , and TMAC, and these
  • TrisCl pH 8.0 to 9.5
  • MaCl 2 MaCl 2
  • Tween 20 Bovine serum albumin
  • BSA Bovine serum albumin
  • concentration of the composition may be used by a person skilled in the art adjusting to an appropriate range.
  • the PCR buffer composition as described above enables reliable gene mutation-specific amplification by remarkably improving the activity of the DNA polymerase of the present invention.
  • the PCR kit may be applied to general PCR (1st generation PCR), real-time PCR (2nd generation PCR), digital PCR (3rd generation PCR) or mass array (MassARRAY).
  • the digital PCR may be cast PCR (Competitive allele-specific TaqMan PCR) or Droplet digital PCR (ddPCR), and more specifically, allele specific cast PCR or allele It may be a specific droplet digital PCR, but is not limited thereto.
  • the “cast PCR” is a method for detecting and quantifying rare mutations in a sample containing a large amount of normal wild type gDNA, allele-specific TaqMan ® qPCR to inhibit non-specific amplification from wild type alleles. Combination with gene-specific MGB blockers can produce specificity superior to traditional allele-specific PCR.
  • the “Droplet digital PCR” is a system for counting target DNA by splitting and amplifying a PCR reaction of 20 ⁇ l into 20,000 droplets, and depending on whether or not the target DNA is amplified in the droplet, positive droplets (1) It is counted as a digital signal with a negative drop (0), counts the copy number of the target DNA through Poisson distribution, and finally, the result can be confirmed by the number of copies per ⁇ l of the sample. Rare mutation detection, very small amount of gene It can be used when amplification, mutation type, etc. are to be simultaneously confirmed.
  • the “mass array” is a multiplexing analysis method applicable to various genomic studies such as genotyping using a MALDI-TOF mass spectrometer, and rapidly analyzes multiple samples and targets at a low cost. It can be used when you want to do it, or if you want to do customized analysis only for a specific target.
  • the JAK2 gene mutation detection kit of the present invention may further include a probe, a fluorophore, and/or a quencher.
  • the fluorophore may be VIC, HEX, JOE, FAM, CAL Flour Orange 560, Quasar 670, CY5 EverGreen dye, etc., but is not limited thereto.
  • the probe sequence, the type of the fluorophore and the quencher may be the same as in Table 21 of Example 5, but is not limited thereto.
  • the kit of the present invention adopts AS-PCR (Allele-specific PCR) and real-time PCR technology, and includes a specific primer and a fluorescent probe for detecting JAK2 V617F mutations in peripheral blood or whole blood samples.
  • AS-PCR Allele-specific PCR
  • the targeted mutant DNA matches the base at the 3'end of the primer, is selectively and efficiently amplified, and then the mutant amplicon is detected by a fluorescent probe labeled with FAM. Wild-type DNA cannot match specific primers, and no amplification occurs.
  • the kit of the present invention may be composed of JAK2 V617F Master Mixture, ADPS TM smart DNA polymerase, JAK2 V617F positive control and nuclease-free distilled water.
  • the JAK2 gene mutation detection kit of the present invention may be configured as shown in Table 1, but is not limited thereto.
  • Table 3 shows the detection information of the JAK2 V617F Master Mixture.
  • the present invention also provides a method for detecting a JAK2 gene mutation comprising the following steps:
  • the PCR may be allele-specific PCR or real-time PCR.
  • the JAK2 gene mutation detection method of the present invention may further include (d) confirming the amplification result by the PCR by measuring a cycle threshold (Ct) value.
  • the cycle threshold (Ct) value means the number of cycles in which the fluorescence generated in the reaction exceeds a threshold, which is inversely proportional to the logarithm of the initial copy number. Therefore, the Ct value assigned to a particular well reflects the number of cycles in which a sufficient number of amplicons have accumulated in the reaction.
  • the Ct value is the cycle in which the increase in ⁇ Rn was first detected.
  • Rn means the magnitude of the fluorescence signal generated during PCR at each time point
  • ⁇ Rn means the fluorescence emission intensity (standardized reporter signal) of the reporter dye divided by the fluorescence emission intensity of the reference dye.
  • the Ct value is also referred to as a crossing point (Cp) in LightCycler.
  • the Ct value represents the point in time at which the system begins to detect an increase in the fluorescence signal associated with the exponential growth of the PCR product in the log-linear phase. This period provides the most useful information about the reaction.
  • the slope of the log-linear phase represents the amplification efficiency (Eff) (http://www.appliedbiosystems.co.kr/).
  • the TaqMan probe specifically hybridizes to the template DNA in the annealing step, but fluorescence is inhibited by quenching on the probe.
  • the TaqMan probe hybridized to the template is decomposed by the 5'to 3'nuclease activity of the Taq DNA polymerase, and the fluorescent dye is released from the probe.
  • the 5'-end of the TaqMan probe should be located downstream of the 3'-end of the extension primer. That is, when the 3'-end of the extension primer is extended by a template-dependent nucleic acid polymerase, the 5'to 3'nuclease activity of this polymerase cuts the 5'-end of the TaqMan probe, thereby Fluorescence signal is generated.
  • the reporter molecule and quencher molecule bound to the TaqMan probe include fluorescent and non-fluorescent materials.
  • Fluorescent reporter molecules and quencher molecules that can be used in the present invention can use any of those known in the art, and examples thereof are as follows (numbers in parentheses are the maximum emission wavelength in nanometers): Cy2 TM 506, YOPRO TM -1 (509), YOYO TM -1 (509), Calcein (517), FITC (518), FluorX TM (519), Alexa TM (520), Rhodamine 110 (520), 5-FAM (522), Oregon Green TM 500 (522), Oregon Green TM 488 (524), RiboGreen TM (525), Rhodamine Green TM (527), Rhodamine 123 (529), Magnesium Green TM (531), Calcium Green TM (533), TO-PRO TM -1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY
  • the non-fluorescent material used in the reporter molecule and the quencher molecule bound to the TaqMan probe may include a minor groove binding (MGB) moiety.
  • MGB minor groove binding
  • TaqMan MGB-conjugate probe refers to a TaqMan probe conjugated with MGB at the 3'-end of the probe.
  • MGB is a substance that binds to the minor groove of DNA with high affinity, such as netropsin, distamicin, lexitropsin, mitramycin, chromomycin A3, and olibo. Olivomycin, anthramycin, sibiromycin, pentamidine, stilbamidine, berenil, CC-1065, Hoechst 33258, DAPI (4-6- diamidino-2-phenylindole), CDPI dimers, trimers, tetramers and pentamers, MPC (N-methylpyrrole-4-carbox-2-amide) and dimers thereof, trimers, tetramers and pentamers It does not work.
  • the conjugation of the probe and MGB significantly increases the stability of the hybrid formed between the probe and its target. More specifically, increased stability (ie, increased degree of hybridization) results in increased melting temperature (Tm) of the hybrid duplex formed by MGB-conjugated probes compared to normal probes.
  • Tm melting temperature
  • MGB stabilizes the van der Waals force, thereby increasing the melting temperature (Tm) of the MGB-conjugate probe without increasing the probe length, resulting in shorter probes (e.g. no more than 21 nucleotides) in Taqman real-time PCR under more stringent conditions. Enables the use of.
  • the MGB-conjugated probe removes background fluorescence more efficiently. Therefore, the probe of the present invention may be in the form of a TaqMan MGB-conjugate, wherein the length of the probe includes 15-21 nucleotides, but is not limited thereto.
  • the JAK2 gene mutation may include one or more selected from the group consisting of deletion, substitution and insertion mutation of codon 617 in exon 14 of the JAK2 gene.
  • the JAK2 gene mutation may include substitution of valine, which is the amino acid of codon 617 of exon 14 of the JAK2 gene.
  • the JAK2 gene mutation detection method of the present invention can detect the V617F mutation described in Table 4 below in the codon 617 of the JAK2 gene.
  • the target sequence may be present in the sample of step (a), and includes DNA, cDNA or RNA, preferably genomic DNA.
  • the test sample may be included in an animal, preferably a vertebrate, more preferably a human subject.
  • the biological sample in step (a) may be sputum, blood, saliva, or urine, and the nucleic acid in step (a) may be extracted from bone marrow tissue biopsy or liquid biopsy.
  • the JAK2 gene mutation detection method of the present invention may include melting temperature analysis using a double-strand specific dye.
  • Melt temperature curve analysis can be performed in real-time PCR devices such as ABI 5700/7000 (96 well format) or ABI 7900 (384 well format) devices with onboard software (SDS 2.1). Alternatively, melt temperature curve analysis can be performed as an endpoint analysis.
  • Double binding to double-stranded DNA or “double-strand specific dye” can be used when it has a higher fluorescence when bound to double-stranded DNA than to the unbound state.
  • dyes are SOYTO-9, SOYTO-13, SOYTO-16, SOYTO-60, SOYTO-64, SYTO-82, Etidium Bromide (EtBr), SYTOX Orange, TO-PRO-1, SYBR Green I, TO-PRO-3 or EvaGreen. These dyes, except EtBr and EvaGreen (Quiagen), have been tested in real-time applications.
  • the JAK2 gene mutation detection method of the present invention includes real-time PCR (real-time PCR) or quantitative PCR (qPCR), analysis on agarose gel after standard PCR, gene mutation specific amplification or allele-specific amplification through real-time PCR , Tetra-primer amplification-refractory mutant systems can be performed by PCR or isothermal amplification.
  • the "standard PCR” is a technique for amplifying single or several copies of DNA or cDNA known to those skilled in the art. Almost all PCR uses thermostable DNA polymerases such as Taq polymerase or Klen Taq. DNA polymerases use single-stranded DNA as a template and enzymatically assemble new DNA strands from nucleotides by using oligonucleotides (primers). The amplicon generated by PCR can be analyzed, for example, on an agarose gel.
  • the "real-time PCR” can monitor the process in real time when performing PCR. Therefore, data is collected throughout the PCR process, not when the PCR ends.
  • the reaction is characterized by a time point during the cycle when amplification is first detected, rather than the target amount accumulated after a fixed number of cycles.
  • Two methods mainly dye-based detection and probe-based detection, are used to perform quantitative PCR.
  • ASA allele specific amplification
  • the "gene mutation specific amplification or allele-specific amplification through real-time PCR” detects gene mutation or SNP in a very efficient manner. Unlike most other methods for detecting gene mutations or SNPs, preliminary amplification of the target genetic material is not required.
  • ASA combines amplification and detection in a single reaction based on the distinction of matched and mismatched primer/target sequence complexes.
  • the increase in DNA amplified during the reaction can be monitored in real time with an increase in the fluorescence signal caused by dyes such as SYBR Green I, which emit light upon binding to double-stranded DNA.
  • Gene mutation-specific amplification or allele-specific amplification through real-time PCR shows delay or absence of a fluorescent signal for mismatched cases. In detecting genetic variation or SNP, it provides information on the presence or absence of genetic variation or SNP.
  • the "tetra-primer amplification-refractory mutation system PCR” amplifies both wild type and mutant alleles with control fragments in a single tube PCR reaction.
  • Non-allele specific control amplicons are amplified by two common (outer) primers flanking the mutation region.
  • the two allele specific (inner) primers are designed in the opposite direction to the common primer, and can be amplified both wild-type and mutant amplicons simultaneously with the common primer. Consequently, the two allele-specific amplicons have different lengths and can be easily separated by standard gel electrophoresis because the mutations are located asymmetrically with respect to the common (outer) primer.
  • the control amplicon provides internal control for false negatives as well as amplification failures, and at least one of the two allele-specific amplicons is always present in the tetra-primer amplification-refractory mutation system PCR.
  • the "isothermal amplification” does not depend on the thermocycler, and preferably means that the amplification of the nucleic acid takes place at a lower temperature without the need to change the temperature during amplification.
  • the temperature used in isothermal amplification can be between room temperature (22-24 °C) to about 65 °C, or at room temperature between about 60-65 °C, 45-50 °C, 37-42 °C or 22-24 °C.
  • the products of the isothermal amplification results are gel electrophoresis, ELISA, ELOSA (Enzyme linked oligosorbent assay), real-time PCR, ECL (improved chemiluminescence), RNA, DNA, and chip-based capillary electrophoresis devices that analyze protein or turbidity It can be detected with an analyzer (bioanalyzer).
  • ELOSA Enzyme linked oligosorbent assay
  • ECL improved chemiluminescence
  • RNA DNA
  • chip-based capillary electrophoresis devices that analyze protein or turbidity It can be detected with an analyzer (bioanalyzer).
  • the JAK2 gene mutation detection method of the present invention is performed with qPCR, it can be performed under the conditions of Tables 19 to 22, for example.
  • the DNA polymerase, primer set, probe and/or kit for detecting JAK2 gene mutations of the present invention can be used for the diagnosis of myeloproliferative neoplasms.
  • the present invention can provide a DNA polymerase, primer set, probe and/or kit for detecting JAK2 gene mutations for the diagnosis of myeloproliferative neoplasms.
  • the present invention can also provide a method for providing information for the diagnosis of myeloproliferative neoplasm, using the aforementioned DNA polymerase, primer set, probe, and/or kit.
  • the myeloproliferative neoplasm may be one or more selected from the group consisting of true erythrocyte hyperplasia, essential thrombocytopenia, and primary myelofibrosis (PMF).
  • the JAK2 gene mutation detection method of the present invention provides information for early diagnosis of myeloproliferative neoplasms by detecting JAK2 V617F mutations to establish treatment strategies for each patient, thereby contributing to more effective treatment of patients. do.
  • the DNA polymerase, primer set, probe, and/or kit for detecting JAK2 gene mutations of the present invention can be used in the diagnosis, prognosis and drug reactivity prediction methods of all diseases applicable by detecting JAK2 V617F mutation.
  • prognosis refers to the act of predicting the course and outcome of a disease in advance. More specifically, the prognosis prediction can be interpreted to mean any action that predicts the course of the disease after treatment by comprehensively taking into account the patient's physiological or environmental conditions. Can be.
  • the prognosis prediction may be interpreted as an action of predicting the disease-free survival rate or survival rate of a patient in advance by predicting the progress and complete cure of the disease after treatment of a specific disease. For example, predicting that "the prognosis is good” means that the patient has a high probability of being treated without disease or having a high survival rate after treatment of the disease, and predicting that the "prognosis is bad” is a disease After treatment, the patient's disease-free survival rate or survival rate is low, indicating that the disease is likely to recur or die from the disease.
  • no disease survival rate of the present invention means the possibility that a patient can survive without recurrence of the disease after treatment of the specific disease.
  • survival rate of the present invention means the possibility that a patient can survive regardless of whether or not the disease recurs after treatment of a specific disease.
  • Taq DNA polymerase in which the 536th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine to lysine (hereinafter referred to as "R536K”), Taq DNA polymerase in which the 660th amino acid residue is substituted with valine in arginine. (Hereinafter referred to as “R660V”) and Taq DNA polymerase (hereinafter referred to as “R536K/R660V”) in which the 536th amino acid residue is substituted from arginine to lysine and the 660th amino acid residue is substituted from arginine to valine is prepared as follows. Did.
  • Taq DNA polymerase fragments (F1 to F5) were amplified by PCR using the mutant specific primers listed in Table 5, as shown in Figure 1(a). The reaction conditions are shown in Table 6.
  • Each fragment amplified in 1-1 was used as a template to amplify the full length using primers at both ends (Eco-F and Xba-R primers).
  • the reaction conditions are shown in Tables 7 and 8.
  • pUC19 was digested with the restriction enzyme EcoRI/XbaI at 37°C for 4 hours under the conditions of Table 9 below, and then the DNA was purified and the purified DNA was treated with SAP for 1 hour at 37°C under the conditions of Table 10 to prepare a vector. .
  • the overlap PCR product of Example 1-2 was purified, digested with restriction enzyme EcoRI/XbaI at 37°C for 3 hours under the conditions of Table 11, and then gel extracted with the prepared vector (FIG. 2 ). ).
  • E. coli DH5 ⁇ was transformed to select from the medium containing ampicillin.
  • the plasmid prepared from the obtained colonies was sequenced to obtain Taq DNA polymerase mutants ("R536K”, “R660V” and “R536K/R660V”) into which the desired mutation was introduced.
  • Taq polymerase activity of "R536K”, “R660V” and “R536K/R660V” prepared in Example 1 was tested to confirm that the activity was poor (data not shown), R536K, R660V, R536K/R660V, respectively
  • an E507K mutation substituted the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 with glutamic acid for lysine
  • WT wild-type Taq DNA polymerase
  • Taq DNA polymerase fragments (F6 to F7) were amplified by PCR using the mutant specific primers listed in Table 13. The reaction conditions are shown in Table 14.
  • Each fragment amplified in 2-1 was used as a template, and the full length was amplified using primers (Eco-F and Xba-R primers) at both ends.
  • the reaction conditions are shown in Table 15.
  • pUC19 was digested with the restriction enzyme EcoRI/XbaI at 37°C for 4 hours under the conditions of Table 9, and then the DNA was purified, and the purified DNA was treated with SAP for 1 hour at 37°C under the conditions of Table 10 to prepare a vector. .
  • the overlap PCR product of Example 2-2 was purified, digested with restriction enzyme EcoRI/XbaI at 37° C. for 3 hours under the conditions of Table 11, and then gel extracted with the prepared vector (FIG. 4 ). ).
  • E. coli was transformed into DH5 ⁇ or DH10 ⁇ , and was selected in a medium containing ampicillin.
  • the plasmid prepared from the obtained colonies was sequenced to obtain Taq DNA polymerase mutants introduced with E507K mutations (“E507K/R536K”, “E507K/R660V” and “E507K/R536K/R660V”).
  • Primer size using the OligoAnalyzer Tool https://sg.idtdna.com/calc/analyzer ) program to design primers that generate PCR products for the mutation of the JAK2 gene (V617F) but not for the wild type JAK2 gene , Tm value, primer GC content, whether there is a self-complementary sequence in the primer, and the like to exclude the possibility of formation of a secondary structure (secondary structure), and then the primer as shown in Table 16 Designed.
  • the wild type DNA of JAK2 a primer set capable of amplifying the peripheral region of the target mutation in Table 4 was applied to prepare a wild type clone of the codon 617 region. Mutagenesis was performed on the V617F mutation using the prepared wild-type DNA, and transformed into E.Coli DH5 ⁇ cells to obtain a mutant clone. Wild type clones and mutant clones were identified by direct sequencing. Wild-type DNA and mutant DNA of codon 617 extracted through clones were used as standards to evaluate the performance of the JAK2 V617F mutation detection kit.
  • samples were prepared by adding 30,000, 3,000, 300, 30, or 3 copies of the V617F mutant plasmid per 30,000 copies of HEK293T cell genomic DNA, and the WT group without addition of the mutant plasmid was used as a control.
  • JAK2 V617F mutations were detected from each group in Table 17 using Taq polymerase (SEQ ID NO: 2) and the primer set of Example 3, each containing the "E507K/R536K/R660V” variant obtained in Example 2.
  • Table 18 shows mutation information targeted by JAK2 V617F Master Mixture.
  • One PCR tube was prepared for the positive control as follows: 10.0 ⁇ L of the JAK2 V617F reaction mixture was divided for the PCR tube, and 5.0 ⁇ L of the positive control was added to the sample tube, and then the PCR tube was capped. Nuclease-free distilled water was added to all PCR tubes to 20.0 ⁇ L, and PCR strips were briefly centrifuged to collect all liquids at the bottom of each PCR tube. The PCR strip tube was placed in a real-time PCR (real-time PCR) instrument, and PCR was performed after setting the PCR protocol using the cycling parameters in Table 22. After the PCR was completed, the FAM/Quasar 670 ⁇ Ct value of each sample was recorded to analyze the data, and the Ct value per well was calculated as follows:
  • ⁇ Ct value sample Ct value (FAM)-positive control Ct value (FAM)
  • the kit of the present invention shows high detection sensitivity (up to 0.01%, 3 mutant copies in 30,000 wild-type copies), high specificity and reproducibility, and is applicable to both liquid biopsy and tissue biopsy.
  • high detection sensitivity up to 0.01%, 3 mutant copies in 30,000 wild-type copies
  • high specificity and reproducibility high specificity and reproducibility

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Abstract

La présente invention concerne une ADN polymérase pour la détection de mutation de JAK2 et un kit la comprenant, et plus spécifiquement une ADN polymérase, un ensemble d'amorces, une sonde et un kit, qui sont capables de détecter une mutation somatique du gène JAK2 au niveau du codon 617 avec une sensibilité élevée, ainsi qu'un procédé de détection de mutation de gène JAK2 à l'aide du kit.
PCT/KR2020/000540 2019-01-11 2020-01-10 Adn polymérase pour la détection de mutation de jak2, et kit la comprenant Ceased WO2020145762A1 (fr)

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KR101312241B1 (ko) * 2010-04-27 2013-09-27 사회복지법인 삼성생명공익재단 증폭억제시발체를 이용하는 유전자 돌연변이 검출 방법
KR20120119571A (ko) * 2011-04-22 2012-10-31 주식회사 파나진 Ｐｎａ 기반의 실시간 ｐｃｒ 클램핑을 이용한 ｊａｋ２ ｖ６１７ｆ 돌연변이 검출 방법 및 키트
KR20140066776A (ko) * 2011-09-30 2014-06-02 에피스템 리미티드 Ｊａｋ２의 돌연변이 분석

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118547058A (zh) * 2024-06-05 2024-08-27 广州凯普医学检验所有限公司 一种用于检测jak2 v617f突变的引物和探针组合及试剂盒

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