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WO2020145734A1 - Adn polymérase pour détecter des mutations braf et trousse comprenant cette derniere - Google Patents

Adn polymérase pour détecter des mutations braf et trousse comprenant cette derniere Download PDF

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WO2020145734A1
WO2020145734A1 PCT/KR2020/000489 KR2020000489W WO2020145734A1 WO 2020145734 A1 WO2020145734 A1 WO 2020145734A1 KR 2020000489 W KR2020000489 W KR 2020000489W WO 2020145734 A1 WO2020145734 A1 WO 2020145734A1
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pcr
braf gene
gene mutation
braf
kit
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이병철
박일현
박세호
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Genecast Co Ltd
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    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a DNA polymerase for detecting BRAF mutations and a kit comprising the same, and more specifically, a DNA polymerase, primer set, and probe capable of detecting four somatic mutations in codon 600 of BRAF gene with high sensitivity. , Kit and BRAF gene mutation detection method using the kit.
  • BRAF is a Ser/Thr that acts on the Ras-Raf-MEK-MAPK signaling pathway that normally regulates cell proliferation and survival under the control of growth factors and hormones, but the mutated BRAF gene causes cancer.
  • the most common mutation is V600E in the active segment. This mutation has been extensively observed in papillary thyroid cancer (PTC), colorectal cancer (CRC), melanoma and non-small cell lung cancer (NSCLC). BRAF mutations occur in about 50% melanoma, about 30% ovarian cancer, about 10% CRC and about 10% prostate cancer. In particular, the V600E mutation is detected in approximately 40% of PTC and is considered a diagnostic marker for thyroid cancer.
  • An object of the present invention is to provide a DNA polymerase for detecting BRAF gene mutations.
  • the present invention also provides a probe for detecting a BRAF 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 BRAF gene mutations, which includes the DNA polymerase and/or primer set described above.
  • 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 present invention also provides a method for detecting a BRAF 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 BRAF gene mutation may include one or more selected from the group consisting of deletion, substitution and insertion mutations in the codon 600 of the BRAF gene.
  • the BRAF gene mutation may include substitution of valine, an amino acid of codon 600 of BRAF.
  • the thyroid cancer may be papillary thyroid cancer or anaplastic thyroid cancer.
  • 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.
  • 5A-5C show the results of detecting mutations with AS-qPCR in the V600E1 mutant plasmid (30,000, 3 and 0 copies) template.
  • the presence of the BRAF mutation acts as a predictor of the diagnosis of thyroid cancer, and thus an effective and rapid detection method of the BRAF mutation is required for early diagnosis and optimal therapeutic approach of thyroid cancer. Accordingly, the present inventors sought solutions to the above-mentioned problems by providing a kit containing a DNA polymerase capable of detecting four somatic mutations in codon 600 of the BRAF gene with high sensitivity and a reaction buffer for increasing its activity. Did.
  • 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
  • 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 BRAF gene mutations 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 Taq polymerase in which the residue is substituted with valine (V) in arginine (R) was designated as “E507K/R536K/R660V”, and its amino acid sequence and nucleotide sequence are shown in SEQ ID NOs: 2 and 17, respectively.
  • the present invention also provides a primer set for detecting BRAF gene mutations comprising one or more primers selected from the group consisting of SEQ ID NOs: 3 to 6.
  • the primer set for detecting a BRAF gene mutation 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 is particularly excellent in BRAF mutation detection sensitivity when used together with the primer sequences in Table 16.
  • the present invention also provides a probe for detecting a BRAF 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 BRAF gene mutations, which includes 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 of 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 components
  • TrisCl pH 8.0 to 9.5
  • MaCl 2 MaCl 2
  • Tween 20 Bovine serum albumin
  • BSA Bovine serum albumin
  • concentration of can be used by a person skilled in the art to adjust to an appropriate range.
  • 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 “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 kit for detecting a BRAF gene mutation 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 20 of Example 5, but is not limited thereto.
  • BRAF gene mutation detection kit of the present invention may be configured as shown in Table 1, but is not limited thereto.
  • 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 BRAF gene mutation may include one or more selected from the group consisting of deletion, substitution and insertion mutations in the codon 600 of the BRAF gene.
  • the BRAF gene mutation may include substitution of valine, which is the amino acid of codon 600 of BRAF.
  • one or more of the four mutations listed in Table 4 below can be simultaneously detected in the codon 600 of BRAF.
  • 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
  • the nucleic acid in step (a) is a formalin-fixed paraffin embedded sample or a liquid biopsy of a tissue biopsy. Can be extracted from
  • the BRAF 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 "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 DNA polymerase, primer set, probe and/or kit for detecting BRAF gene mutation of the present invention can be used for the diagnosis of thyroid cancer.
  • BRAF gene mutation detection can also be used to diagnose malignant melanoma, colorectal cancer, or ovarian cancer.
  • 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.
  • 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. .
  • 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”).
  • 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 600 region.
  • mutagenesis was performed on four target mutants, and transformed into E.Coli DH5 ⁇ cells to obtain each mutant-type clone. Wild type clones and mutant clones were identified by direct sequencing. The wild-type DNA and mutant DNA of codon 600 extracted through clones were used as standards to evaluate the performance of the BRAF mutation detection kit.
  • MMX Neon Mutant group BRAF nucleic acid sequence (COSMIC ID) MMX FAM V600E1 1799T>A (476) V600E2 1799_1800TG>AA (475) V600D1 1799_1800TG>AT (477) V600D2 1799_1800TG>AC (308550)
  • 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.
  • thyroid cancer can be accurately diagnosed by simultaneously detecting four mutations in the codon 600 of the BRAF gene.

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Abstract

La présente invention concerne une ADN polymérase pour détecter des mutations de BRAF, et un kit comprenant celle-ci et, plus spécifiquement: une ADN polymérase, un ensemble d'amorces, une sonde et une trousse, qui permettent une détection très sensible de quatre mutations somatiques dans le codon 600 d'un gène BRAF; et un procédé de détection de mutations de gène BRAF à l'aide de la trousse.
PCT/KR2020/000489 2019-01-11 2020-01-10 Adn polymérase pour détecter des mutations braf et trousse comprenant cette derniere Ceased WO2020145734A1 (fr)

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