WO2020145711A1 - Adn polymérase pour la détection de mutation d'egfr et trousse comprenant celle-ci - Google Patents

Adn polymérase pour la détection de mutation d'egfr et trousse comprenant celle-ci Download PDF

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WO2020145711A1
WO2020145711A1 PCT/KR2020/000448 KR2020000448W WO2020145711A1 WO 2020145711 A1 WO2020145711 A1 WO 2020145711A1 KR 2020000448 W KR2020000448 W KR 2020000448W WO 2020145711 A1 WO2020145711 A1 WO 2020145711A1
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egfr gene
pcr
amino acid
gene mutation
egfr
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이병철
박일현
최지현
박지은
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주식회사 진캐스트
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Definitions

  • the present invention relates to a DNA polymerase for detecting EGFR mutations and a kit comprising the same, and more specifically, a DNA polymerase and primer set capable of detecting 44 somatic mutations in exons 18 to 21 of the EGFR gene with high sensitivity.
  • Probes, kits and methods for detecting EGFR gene mutations using the kits are provided.
  • G719X, S768I, Ex19 deletion, L858R and L861Q mutations are associated with susceptibility to EGFR TKI, whereas T790M and most Ex20 insertion mutations are associated with reduced EGFR TKI response.
  • Early detection of EGFR gene mutations in lung cancer patients allows predicting drug response before treatment.
  • Korean Patent Publication No. 10-2015-0102468 discloses a kit for diagnosing lung cancer, which includes a primer set for amplifying a lung cancer-related mutation and a probe complementary to the mutation, but a polymerase for detecting a mutation in the EGFR gene and There is no known reaction buffer to increase its activity.
  • the present inventors have developed a kit comprising a DNA polymerase capable of detecting 44 somatic mutations in exons 18, 19, 20 and 21 of the EGFR gene with high sensitivity and a reaction buffer for increasing its activity.
  • the invention was completed.
  • An object of the present invention is to provide a DNA polymerase for detecting EGFR gene mutation.
  • Another object of the present invention is to provide a primer set for detecting EGFR gene mutations.
  • Another object of the present invention is to provide a probe for detecting an EGFR gene mutation.
  • 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)
  • a DNA polymerase for detecting EGFR gene mutations including 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 EGFR gene mutation detection, comprising at least one primer selected from the group consisting of SEQ ID NOs: 3 to 54.
  • the present invention also provides a probe for detecting an EGFR gene mutation, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 55 to 63.
  • the 507th amino acid residue glutamic acid (E) is substituted with lysine (K)
  • the 536th amino acid residue arginine (R) is substituted with lysine (K)
  • the 660th Taq polymerase in which the amino acid residue arginine (R) is substituted with valine (V);
  • And / or at least one primer selected from the group consisting of SEQ ID NO: 3 to 54; provides a kit for detecting EGFR gene mutation.
  • the nucleotide sequences of SEQ ID NOs: 55 to 63 are each selected from the group consisting of FAM, CAL Fluor Orange 560, VIC, HEX, JOE, Quasar 670 and CY5 at the 5'-end.
  • One fluorophore may be labeled, and one quencher selected from the group consisting of BHQ-1 BHQ1 nova 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 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 an EGFR gene mutation comprising the following steps: (a) extracting a nucleic acid from an isolated biological sample; (b) processing the kit of the present invention on the extracted nucleic acid to perform PCR (polymerase chain reaction); And (c) confirming the amplification result by the PCR with fluorescence.
  • 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 EGFR gene mutation includes at least one selected from the group consisting of deletion, substitution and insertion mutations in exons 18, 19, 20 and 21 of the EGFR gene. can do.
  • the EGFR gene mutation is a 719th amino acid glycine substitution in exon 18 of EGFR, a deletion in exon 19, a 768th amino acid in exon 20.
  • the EGFR gene mutation detection method may be for predicting responsiveness to drugs of lung cancer patients.
  • the lung cancer may be non-small cell lung cancer.
  • the drug may be a tyrosine kinase inhibitor.
  • the tyrosine kinase inhibitor may be gefitinib, erlotinib or osimitinib.
  • the nucleic acid of step (a) may be extracted from a formalin-fixed paraffin embedded sample or a liquid biopsy of a tissue 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.
  • 5A to 5L show the results of detecting mutations with AS-qPCR in the Ex19del C1 to Ex19del C12 mutant plasmid (3, 10 2 and 10 4 copies) template.
  • 7A to 7G show the results of detecting mutations with AS-qPCR in the Ex19del C25 to Ex19del C31 mutant plasmid (3, 10 2 and 10 4 copies) template
  • 7h is S768I mutant plasmid (3, 10 2 and 10 4 Copy) shows the result of detecting the mutation with AS-qPCR in the template
  • 7i shows the result of detecting the mutation with AS-qPCR in WT.
  • Figure 9a shows the results of detecting the mutation in the G719S mutant plasmid (3, 10 1 , 30, 10 2 and 10 4 copies) template with AS-qPCR.
  • Figure 9b shows the results of detecting the mutation in the G719C mutant plasmid (3, 10 1 , 30, 10 2 and 10 4 copies) template with AS-qPCR.
  • Figure 9c shows the results of detecting the mutation in the G719A mutant plasmid (3, 10 1 , 30, 10 2 and 10 4 copies) template with AS-qPCR.
  • Figure 9d shows the results of detecting the mutation in WT AS-qPCR.
  • 9E shows the results of detecting mutations with AS-qPCR in the L861Q mutant plasmid (0, 3, 10 2 and 10 4 copies) template.
  • 10A to 10E show the results of detecting mutations with AS-qPCR in the Ex20Ins C1 to Ex20Ins C5 mutant plasmid (3, 10 2 and 10 4 copies) template
  • 10f is the result of detecting mutations from WT to AS-qPCR
  • 10g shows the results of detecting mutations with AS-qPCR in the L858R mutant plasmid (3, 10 2 and 10 4 copies) template
  • 10h shows the results of detecting mutations in WT with AS-qPCR. will be.
  • kits for more accurately predicting drug reactivity of lung cancer patients by improving the sensitivity of detecting EGFR mutations there is a continuous need to develop a kit for more accurately predicting drug reactivity of lung cancer patients by improving the sensitivity of detecting EGFR mutations.
  • the present inventors provide an optimized kit comprising a DNA polymerase capable of detecting 44 somatic mutations in exons 18, 19, 20 and 21 of the EGFR gene with high sensitivity and a reaction buffer to increase its activity.
  • the solution to the above problem was sought.
  • the kit of the present invention exhibits high detection sensitivity, high specificity and reproducibility, and is applicable to both liquid biopsy and tissue biopsy.
  • 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 EGFR 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 The Taq polymerase in which the residue is substituted with arginine (R) to valine (V) was designated as “E507K/R536K/R660V”, and its amino acid sequence and nucleotide sequence are shown in SEQ ID NO: 2 and SEQ ID NO: 72, respectively.
  • the present invention also provides a primer set for detecting EGFR gene mutations comprising one or more primers selected from the group consisting of SEQ ID NOs: 3 to 54.
  • the primer set for detecting EGFR gene mutation of the present invention may be, for example, those described in Tables 16 to 19 of Example 3, but is not limited thereto.
  • the polymerase according to the present invention is particularly excellent in EGFR mutation detection sensitivity when used with the primer sequences of Tables 16 to 19.
  • the present invention also provides a kit for detecting EGFR 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 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 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 “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 EGFR 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 18 of Example 5, but is not limited thereto.
  • the kit of the present invention employs AS-PCR (Allele-specific PCR) and real-time PCR technology, and includes a specific primer and a fluorescent probe for detecting EGFR mutation of cfDNA in human plasma 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 or CAL Fluor Orange 560 (CFO560). Wild-type DNA cannot match specific primers, and no amplification occurs.
  • the kit of the present invention may be composed of EGFR Master Mixture 1-4, ADPS TM smart DNA polymerase, EGFR positive control and nuclease-free distilled water.
  • the EGFR gene mutation detection kit of the present invention may be configured as shown in Table 1, for example, but is not limited thereto.
  • Table 2 Exemplary configuration of each EGFR Master Mix in Table 1 is shown in Table 2.
  • Table 3 shows the detection information of EGFR Master Mix 1-4.
  • the present invention also provides a method for detecting an EGFR gene mutation comprising the following steps:
  • the PCR may be allele-specific PCR or real-time PCR.
  • the EGFR gene mutation detection method of the present invention may further include (d) confirming the amplification result by the PCR by measuring a Ct (cycle threshold) 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/).
  • FRET Frster or fluorescence resonance energy transfer
  • 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 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.
  • 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 EGFR gene mutation is selected from the group consisting of deletion, substitution and insertion mutations in exons 18, 19, 20 and 21 of the EGFR gene. It may contain one or more.
  • the EGFR gene mutation is the substitution of the 719th amino acid glycine in exon 18 of EGFR, the deletion in exon 19, the 768th amino acid serine in exon 20, the 790th amino acid threonine, It may include one or more selected from the group consisting of substitution of the cysteine, the 797th amino acid, insertion in exon 20, leucine, which is the 858th amino acid in exon 21, and the replacement of the 861th amino acid, leucine.
  • the EGFR gene mutation detection method of the present invention can simultaneously detect one or more of the 44 mutations listed in Table 4 below in exons 18, 19, 20 and 21 of EGFR.
  • 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.
  • the EGFR 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 EGFR gene mutation detection method of the present invention includes real-time PCR (RT-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.
  • RT-PCR real-time PCR
  • qPCR quantitative PCR
  • 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 of 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 EGFR gene mutation detection method of the present invention is performed with qPCR, for example, it may be performed under the conditions of Tables 22 to 24 below.
  • the DNA polymerase, primer set, probe, and/or kit for detecting EGFR gene mutations of the present invention can be used for predicting reactivity to drugs of lung cancer patients.
  • the present invention can provide a DNA polymerase, primer set, probe, and/or kit for detecting EGFR gene mutation to predict responsiveness to drugs of lung cancer patients.
  • the present invention can also provide a method for predicting the reactivity of a lung cancer patient to a drug using the aforementioned DNA polymerase, primer set, probe, and/or kit.
  • the cancer may be non-small cell lung cancer.
  • the EGFR gene mutation detection method of the present invention provides information to diagnose cancer early and establish a treatment strategy for each patient by detecting a known EGFR mutation of Table 4 above, thereby more effectively treating patients Contribute.
  • 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 and Tm value using the OligoAnalyzer Tool https://sg.idtdna.com/calc/analyzer) program to design primers that generate PCR products for mutations in the EGFR gene but not for the wild-type EGFR gene
  • a primer set capable of amplifying the periphery of the target mutation in Table 4 was applied to prepare wild-type clones at exon 18, 19, 20, and 21 sites. Mutagenesis was performed on 44 target mutations using the prepared wild-type DNA, and transformed into E.Coli DH5 ⁇ cells to obtain each mutant clone. Wild type clones and mutant clones were identified by direct sequencing. Wild-type DNA and mutant DNA for each exon extracted through clones were used as standards to evaluate the performance of the EGFR mutation detection kit.
  • samples were prepared by adding 10,000 copies, 100 copies, 30 copies, 10 copies, or 3 copies of each mutant plasmid described in Table 4 per 30,000 copies of HEK293T cell genomic DNA. The group was used as a control.
  • MMX Neon Mutant group EGFR nucleic acid sequence (COSMIC ID) MMX1 FAM S768I 2303G>T (6241) CFO560 Ex19Del 2240_2251del12 (6210), 2239_2247del9 (6218), 2238_2255del18 (6220), 2235_2249del15 (6223), 2236_2250del15 (6225), 2239_2253del15 (6254), 2239_2256del18 (6255), 2237_2254del18 (12367), 2240_2254del15 (370) 2239_2248TTAAGAGAAG>C (12382), 2239_2251>C (12383), 2237_2255>T (12384), 2235_2255>AAT (12385), 2237_2252>T (12386), 2239_2258>CA (12387), 2239_2256>CAA (12403), 2237_2253 >TTGCT (12416), 2238_2252
  • EGFR reaction mixtures containing each of the components listed in Table 22 were each prepared in separate sterile centrifuge tubes, and the reaction Master Mixture was vortexed for 3 seconds to thoroughly mix and centrifuge briefly.
  • Two PCR tubes were prepared for each sample as follows: 10.0 ⁇ L of EGFR reaction mixture was divided for each PCR tube, and 5.0 ⁇ L of each sample DNA was added to each sample tube, and then the PCR tube was covered. 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.
  • 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 24. After the PCR was completed, FAM/CAL Fluor Orange 560/Quasar 670 Ct values of each sample were recorded to analyze the data, and ⁇ Ct values per well were calculated as follows:
  • ⁇ Ct value sample Ct value (FAM of each MMX)-positive control Ct value (FAM of each MMX)
  • ⁇ Ct value sample Ct value (each MMX CFO560)-positive control Ct value (CFO560 of each MMX)
  • 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 D'EGFR et une trousse comprenant celle-ci et, plus spécifiquement, une ADN polymérase capable de détecter 44 mutations somatiques dans les exons 18 à 21 du gène EGFR avec une sensibilité élevée, un ensemble d'amorces, une sonde, une trousse et un procédé de détection d'une mutation du gène EGFR au moyen de la trousse.
PCT/KR2020/000448 2019-01-11 2020-01-10 Adn polymérase pour la détection de mutation d'egfr et trousse comprenant celle-ci WO2020145711A1 (fr)

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