KR102370550B1 - DNA polymerase for detecting EGFR mutation and kit comprising the same - Google Patents

Abstract

The present invention relates to a DNA polymerase for detecting EGFR mutation and a kit comprising the same, and more particularly, a DNA polymerase capable of detecting 44 somatic mutations in exons 18 to 21 of the EGFR gene with high sensitivity, a primer set , probes, kits, and a method for detecting EGFR gene mutations using the kit.

Description

DNA polymerase for detecting EGFR mutation and kit comprising the same {DNA polymerase for detecting EGFR mutation and kit comprising the same}

The present invention relates to a DNA polymerase for detecting EGFR mutation and a kit comprising the same, and more particularly, a DNA polymerase capable of detecting 44 somatic mutations in exons 18 to 21 of the EGFR gene with high sensitivity, a primer set , probes, kits, and a method for detecting EGFR gene mutations using the kit.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for 80-85% of all lung cancer diagnoses. Patients with epidermal growth factor receptor (EGFR) mutations have elevated levels of EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib (Iressa, AstraZeneca), erlotinib (Tarceba, Roche) and osimertinib (Tagrisso, AstraZeneca). It is known to exhibit drug response or resistance. 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 can predict drug response before treatment.

One of the methods most frequently used for detection of EGFR mutations so far is direct sequencing, which directly identifies the nucleotide sequence of the gene corresponding to exons 18 to 21 of EGFR. This method has a disadvantage in that it takes a lot of time compared to real-time-polymerase chain reaction or DNA chip analysis, but it is the most accurate analysis in that not only specific bases but the entire target site can be analyzed. It has the advantage of being able to Therefore, the development of a kit for more accurately predicting drug reactivity in lung cancer patients by improving the detection sensitivity of EGFR mutations is continuously required.

On the other hand, Korean Patent Application Laid-Open No. 10-2015-0102468 discloses a kit for diagnosing lung cancer including a primer set for amplifying lung cancer-related mutations, and a probe complementary to the mutation, but a polymerase for detecting a mutation in the EGFR gene and It is not known about a reaction buffer for increasing its activity.

Accordingly, the present inventors 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 the activity thereof, and the present invention The invention was completed.

It is an object of the present invention to provide a DNA polymerase for detecting EGFR gene mutations.

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 EGFR gene mutation.

Another object of the present invention is to provide a kit for detecting EGFR gene mutations, including the above-described DNA polymerase and/or primer set.

Another object of the present invention is to provide a method for detecting EGFR gene mutations using the aforementioned kit.

In order to solve the above problems, in the present invention, in the amino acid sequence of SEQ ID NO: 1, glutamic acid (E), the 507th amino acid residue, is substituted with lysine (K), and the 536th amino acid residue, arginine (R), is lysine (K) It provides a DNA polymerase for detecting EGFR gene mutations, including a Taq polymerase in which arginine (R), which is the 660th amino acid residue, is substituted with valine (V),

The present invention also provides a primer set for detecting EGFR gene mutation, comprising one or more primers selected from the group consisting of SEQ ID NOs: 3 to 54.

The present invention also provides a probe for detecting EGFR gene mutation comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 55 to 63.

According to a preferred embodiment of the present invention, 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. A fluorophore of the species 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.

In the present invention, in the amino acid sequence of SEQ ID NO: 1, glutamic acid (E), which is the 507th amino acid residue, is substituted with lysine (K), arginine (R), which is the 536th amino acid residue, is substituted with lysine (K), and 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 NOs: 3 to 54; provides a kit for detecting EGFR gene mutations, including.

According to a preferred embodiment of the present invention, the kit may further include a probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 55 to 63.

According to another preferred embodiment of the present invention, 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 type of fluorophore may be labeled, and one type of quencher selected from the group consisting of BHQ-1 BHQ1 nova and BHQ-2 may be labeled at the 3'-end.

According to a preferred embodiment of the present invention, 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.

According to another preferred embodiment of the present invention, the kit comprises 25 to 100 mM KCl; 1-7 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 steps of: (a) extracting a nucleic acid from an isolated biological sample; (b) performing PCR (polymerase chain reaction) by treating the extracted nucleic acid with the kit of the present invention; and (c) confirming the results of the PCR amplification with fluorescence.

According to a preferred embodiment of the present invention, the PCR may be allele-specific PCR or real-time PCR.

According to another preferred embodiment of the present invention, (d) may further include the step of confirming the result of the PCR amplification by measuring a Ct (cycle threshold) value.

According to a preferred embodiment of the present invention, the EGFR gene mutation includes at least one selected from the group consisting of deletion, substitution and insertional mutations within exons 18, 19, 20 and 21 of the EGFR gene. can do.

According to another preferred embodiment of the present invention, the EGFR gene mutation is a substitution of glycine at the 719th amino acid within exon 18 of EGFR, a deletion within exon 19, and the 768th amino acid within exon 20. One selected from the group consisting of serine, threonine at amino acid 790, cysteine at amino acid 797, insertion within exon 20, leucine at amino acid 858 within exon 21, and leucine at amino acid 861 may include more than one.

According to another preferred embodiment of the present invention, the EGFR gene mutation detection method may be for predicting the reactivity of a lung cancer patient to a drug.

According to another preferred embodiment of the present invention, the lung cancer may be non-small cell lung cancer.

According to another preferred embodiment of the present invention, the drug may be a tyrosine kinase inhibitor.

According to another preferred embodiment of the present invention, the tyrosine kinase inhibitor may be gefitinib, erlotinib or osimertinib.

According to another preferred embodiment of the present invention, the nucleic acid of step (a) may be a formalin-fixed paraffin embedded sample of a tissue biopsy or extracted from a liquid biopsy.

The kit of the present invention exhibits 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. In addition, by simultaneously detecting 44 mutations within exons 18 to 21 of the EGFR gene, it is possible to accurately predict drug responsiveness to a tyrosine kinase inhibitor in lung cancer patients.

1 shows the production process of Taq DNA polymerase containing R536K, R660V and R536K / R660V mutations, respectively, (a) is a schematic representation of fragment PCR and overlap PCR, (b) is amplified in fragment PCR The result of confirming the product by electrophoresis is shown, and (c) shows the result of confirming the amplified product by electrophoresis by amplifying the full length by overlap PCR.
2 is a result of electrophoresis confirmation of the overlap PCR product of FIG. 1(c) purified with the pUC19 vector treated with SAP after digestion with restriction enzymes EcoRI/XbaI for gel extraction.
Figure 3 schematically shows 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 is a result of confirming by electrophoresis the overlap PCR product of FIG. 3 purified with the pUC19 vector treated with SAP after digestion with restriction enzymes EcoRI/XbaI for gel extraction.
5A to 5L show the results of detecting mutations by AS-qPCR in Ex19del C1 to Ex19del C12 mutant plasmids (3, 10 ² and 10 ⁴ copies) templates.
6A to 6L show the results of detecting mutations by AS-qPCR in Ex19del C13 to Ex19del C24 mutant plasmids (3, 10 ² and 10 ⁴ copies) templates.
7A to 7G show the results of detecting mutations by AS-qPCR in Ex19del C25 to Ex19del C31 mutant plasmids (3, 10 ² and 10 ⁴ copies) template, and 7h is S768I mutant plasmid (3, 10 ² and 10 ⁴ ) Copy) shows the results of detecting mutations by AS-qPCR in the template, and 7i shows the results of detecting mutations by AS-qPCR in WT.
8 shows the results of detecting mutations by AS-qPCR in the T790M mutant plasmid (0, 3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template.
9A shows the results of detecting mutations by AS-qPCR in the G719S mutant plasmid (3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template.
9B shows the results of detecting mutations by AS-qPCR in the G719C mutant plasmid (3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template.
Figure 9c shows the results of detecting mutations by AS-qPCR in the G719A mutant plasmid (3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template.
9d shows the results of detecting mutations in WT by AS-qPCR.
Figure 9e shows the results of detecting mutations by 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 by AS-qPCR in Ex20Ins C1 to Ex20Ins C5 mutant plasmids (3, 10 ² and 10 ⁴ copies) templates, and 10f is the result of detecting mutations by AS-qPCR in WT. is shown, and Figure 10g shows the result of detecting the mutation by AS-qPCR in the L858R mutant plasmid (3, 10 ² and 10 ⁴ copies) template, and 10h shows the result of detecting the mutation by AS-qPCR in the WT. will be.

As described above, the development of a kit for more accurately predicting drug reactivity in lung cancer patients by improving the detection sensitivity of EGFR mutations is continuously required. Accordingly, 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 for increasing the activity thereof. A solution to the above-mentioned 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. In addition, it features semi-quantitative analysis, and 4 channels allow analysis on any qPCR instrument.

Hereinafter, the terms used herein will be described.

"Amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. As used herein, the term "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 or V).

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 analogs of these groups. Exemplary side chains are, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, borate, boronate, phospho, phosphono, fosifine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups.

In the DNA polymerases of the present invention, the term "mutant" refers to a recombinant polypeptide comprising one or more amino acid substitutions relative to the corresponding naturally occurring or unmodified DNA polymerase.

The term "thermostable polymerase" (referring to an enzyme that is heat stable) is heat resistant, retains sufficient activity to achieve a subsequent polynucleotide extension reaction, and is subjected to elevated temperature for a period of time required to achieve denaturation of the double-stranded nucleic acid. It is not irreversibly denatured (inactivated) when As used herein, it is suitable for use at a temperature cycling reaction such as PCR. Irreversible denaturation herein refers to permanent and complete loss of enzymatic activity. For thermostable polymerases, enzymatic activity refers to catalyzing the combination of nucleotides in an appropriate manner to form a polynucleotide extension product complementary to the template nucleic acid strand. Thermostable DNA polymerases from thermophilic bacteria include, for example, Thermotoga maritima, Thermus aquaticus, Thermus thermophilus, Thermus flabus, Thermophylliformis, Thermus sp. DNA polymerases from Sps17, Thermos sp. Z05, Thermos caldophilus, Bacillus caldotenax, Thermotoga neopolitana, and Thermocypo africanus.

The term “thermoactive” refers to an enzyme that retains its catalytic properties at temperatures commonly used for reverse transcription or annealing/extension steps in RT-PCR and/or PCR reactions (ie, 45-80° C.). Thermostable enzymes are those that are irreversibly inactivated or not denatured when subjected to the elevated temperatures required for denaturation of nucleic acids. A thermoactive enzyme may or may not be thermostable. A thermoactive DNA polymerase may be DNA or RNA dependent from a thermophilic or mesophilic species, including but not limited to:

The term “nucleotide” is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that exists in single- or double-stranded form, unless specifically stated otherwise. It may contain analogs of natural nucleotides unless otherwise specified.

The term “nucleic acid” or “polynucleotide” refers to a polymer that may correspond to a DNA or RNA polymer, or an analog thereof. Nucleic acids are, for example, chromosomes or chromosomal segments, vectors (eg, expression vectors), expression cassettes, naked DNA or RNA polymers, products of polymerase chain reaction (PCR), oligonucleotides, probes, and primers. may or may include. A nucleic acid may be, for example, single-stranded, double-stranded, or triple-stranded, but is not limited to any particular length. Unless otherwise stated, a particular nucleic acid sequence comprises or encodes a complementary sequence in addition to any sequence specified.

The term “primer” refers to a polynucleotide capable of serving as an initiation point for nucleic acid synthesis in the template-direction when subjected to conditions in which polynucleotide extension is initiated. Primers can also be used in a variety of other oligonucleotide-mediated synthetic 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 a primer depends on the intended use, typically in the range of 6 to 40 nucleotides, more typically in the range of 15 to 35 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybridization complexes with the template. Primers are not required to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with the template to be extended. In certain embodiments, the term "primer pair" includes a 5'-sense primer that hybridizes complementary to the 5'-end of the nucleic acid sequence to be amplified, and 3'-antisense primers that hybridize to the 3'-end of the sequence to be amplified. It means a set of primers comprising Primers can be labeled, if desired, by incorporating a label that can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful labels include: ³² P, fluorescent dyes, electron-dense reagents, enzymes (commonly used in ELISA assays), biotin, or haptens and proteins for which antisera or monoclonal antibodies can be used. .

The term "5'-nuclease probe" refers to an oligonucleotide comprising one or more luminescent label moieties used in a 5'-nuclease reaction to target nucleic acid detection. In some embodiments, for example, a 5'-nuclease probe comprises only a single luminescent moiety (eg, a fluorescent dye, etc.). In certain embodiments, a 5'-nuclease probe comprises a self-complementary region such that the probe can form a hairpin structure under selective conditions. In some embodiments, the 5'-nuclease probe comprises two or more label moieties and is released with increased radiation intensity after one of the two labels is separated from or degraded from the oligonucleotide. In certain embodiments, a 5'-nuclease probe is labeled with two different fluorescent dyes, eg, a 5'-terminal reporter dye and a 3'-terminal quencher dye or moiety. In some embodiments, 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 fluorophores such that the fluorescence emission from the reporter dye is at least partially quenched. During the elongation step of the polymerase chain reaction, for example, a 5'-nuclease probe bound to the template nucleic acid has an activity such that the fluorescence emission of the reporter dye is no longer quenched, e.g. Taq polymerase or other It is degraded by the 5' to 3'-nuclease activity of the polymerase. In some embodiments, a 5'-nuclease probe may be labeled with two or more different reporter dyes and a 3'-terminal quencher dye or moiety.

The term "FRET" or "fluorescence resonance energy transfer" or "Forester resonance energy transfer" refers to the transfer of energy between two or more chromophores, a donor chromophore and an acceptor chromophore (referred to as a quencher). A donor typically transfers energy to an acceptor when the donor is excited by radiation of a suitable wavelength. The receptor re-radiates the displaced energy, typically in the form of light emitted at different wavelengths. When the receptor is a "cancer" quencher, it dissipates the energy transferred to a form other than light. Whether a particular fluorophore acts as a donor or acceptor depends on the properties of the other members of the FRET pair. A commonly used donor-acceptor pair includes the FAM-TAMRA pair. Commonly used matting agents are DABCYL and TAMRA. Commonly used cancer quenchers include: BlackHole Quenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry™ Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

The terms “conventional” or “native” when referring to a nucleic acid base, nucleoside triphosphate, or nucleotide refer to that which occurs naturally in the polynucleotide being described (i.e., for DNA they are dATP, dGTP, dCTP and dTTP). In addition, 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.

The term "unconventional" or "modified" when referring to a nucleic acid base, nucleoside, or nucleotide refers to a modification, derivative or include analogs. Certain unusual nucleotides are modified at the 2' position of the ribose sugar compared to conventional dNTPs. Thus, although for RNA a naturally occurring nucleotide is a ribonucleotide (i.e., ATP, GTP, CTP, UTP, collective rNTP), since the nucleotide has a hydroxyl group at the 2' position of the sugar, it is comparatively free of dNTPs, As used herein, ribonucleotides are unconventional nucleotides as substrates for DNA polymerases. As used herein, unconventional 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 triphosphates ddATP, ddTTP, ddCTP and ddGTP are collectively referred to as ddNTP. Additional examples of terminator compounds include 2′-PO ₄ analogs of ribonucleotides. Other uncommon nucleotides are phosphorothioate dNTPs ([[α]-S]dNTPs), 5′-[α]-borano-dNTPs, [α]-methyl-phosphonate dNTPs, and ribonucleosides. triphosphate (rNTP). Unconventional bases include radioactive isotopes such as ³² P, ³³ P, or ³⁵ S; fluorescent label; markers of chemiluminescence; markers of bioluminescence; hapten labels such as biotin; or with an enzymatic label such as streptavidin or avidin. The fluorescent label may comprise a negatively charged dye, such as a dye of the fluorescein family, or a neutrally charged dye, such as a dye of the rhodamine family, or a positively charged dye, such as a dye of the cyanine family. Dyes of the Fluoxane family include, for example, FAM, HEX, TET, JOE, NAN and ZOE. Dyes of the rhodamine family 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 were obtained from Perkin-Elmer (Boston, MA), Applied Biosystems (Foster City, CA), or Invitrogen /Molecular Probes (Eugene, OR). Dyes of the cyanine family include Cy2, Cy3, Cy5, and Cy7 and are marketed by GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Hereinafter, the present invention will be described in more detail.

In the present invention, in the amino acid sequence of SEQ ID NO: 1, 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), and the 660th amino acid residue Provided is a DNA polymerase for detecting EGFR gene mutation, comprising Taq polymerase in which phosphorus arginine (R) is substituted with valine (V).

The "Taq polymerase" is a thermostable DNA polymerase named after the thermophilic bacterium Thermos aquaticus and was first isolated from the bacterium. Thermos aquaticus is a bacterium that inhabits hot springs and hydrothermal vents, and Taq polymerase has been identified as an enzyme that can withstand the protein denaturation conditions (high temperature) required in the PCR process. The optimal activation temperature of Taq polymerase is 75-80 °C, and has a half-life of more than 2 hours at 92.5 °C, 40 minutes at 95 °C, and 9 minutes at 97.5 °C, and is capable of replicating 1000 base pairs of DNA within 10 seconds at 72 °C. can It lacks 3'→5' exonuclease-correcting activity, and the error rate is measured at about 1 out of 9,000 nucleotides. For example, using heat-resistant Taq, PCR can be performed at high temperatures (over 60 °C). The amino acid sequence shown in SEQ ID NO: 1 for Taq polymerase is used as a reference sequence.

In the present invention, in the amino acid sequence of SEQ ID NO: 1, the 507th amino acid residue is substituted from glutamic acid (E) to lysine (K), the 536th amino acid residue is substituted from arginine (R) to lysine (K), and the 660th amino acid is substituted The Taq polymerase in which the residue was substituted from arginine (R) to valine (V) was named "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 mutation comprising one or more primers selected from the group consisting of SEQ ID NOs: 3 to 54.

The primer set for detecting EGFR gene mutations of the present invention may be, for example, those described in Tables 16 to 19 of Example 3, but is not limited thereto.

Preferably, the polymerase according to the present invention has excellent sensitivity for detecting EGFR mutations, especially when used together with the primer sequences shown in Tables 16 to 19.

The present invention also provides a kit for detecting EGFR gene mutation comprising the above-described DNA polymerase and/or primer set.

The kit of the present invention can be used for research (Research Use Only, RUO) or in vitro diagnostic (In-Vitro Diagnostic, IVD).

The kit of the present invention may be a PCR kit, and may include any reagent or other element recognized by those skilled in the art to be used in the primer extension process.

For example, the PCR kit of the present invention comprises (a) nucleoside triphosphate; (b) reagents for quantification of binding to double-stranded DNA; (c) a polymerase blocking antibody; (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.

According to a preferred embodiment of the present invention, 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.

More preferably, 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.

According to another preferred embodiment of the present invention, the kit comprises 25 to 100 mM KCl; 1-7 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.

More preferably, the kit comprises 40 to 90 mM KCl; 1-5 mM (NH ₄ ) ₂ SO ₄ ; and 10 to 40 mM 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 of the present invention may further include Tris·Cl (pH 8.0 to 9.5), MaCl ₂ , Tween 20, and BSA (Bovine serum albumin) in addition to KCl, (NH ₄ ) ₂ SO ₄ , and TMAC, and these components The concentration of can be used by a person skilled in the art by adjusting it to an appropriate range.

The PCR buffer composition as described above enables reliable gene mutation-specific amplification by significantly improving the activity of the DNA polymerase of the present invention.

According to a preferred embodiment of the present invention, the PCR kit can be applied to general PCR (1st generation PCR), real-time PCR (2nd generation PCR), digital PCR (3rd generation PCR) or mass array (MassARRAY).

In the PCR kit of the present invention, 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 samples containing large amounts of normal wild-type gDNA. Allele-specific TaqMan ^® qPCR is allele-specific to suppress non-specific amplification from wild-type alleles. It can be combined with gene-specific MGB blockers to produce specificity that is superior to traditional allele-specific PCR.

The “Droplet Digital PCR” is a system that counts target DNA after dividing 20 μl of PCR reaction into 20,000 droplets and amplifying them. Positive droplets (1) and negative droplet (0) as a digital signal, counting, calculating the target DNA copy number through Poisson distribution, and finally confirming the result value in the number of copies per μl of sample It can be used for amplification and to simultaneously confirm the type of mutation.

The “mass array” is a multiplexing analysis method that can be applied to various genome studies such as genotyping using MALDI-TOF mass spectrometer, and quickly analyzes multiple samples and targets at a low cost It can be used when wanting to, or when wanting to perform a customized analysis only for a specific target.

The kit for detecting EGFR 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 types of the probe sequence, the fluorophore, and the quencher may be, for example, as shown in Table 18 of Example 5, but are not limited thereto.

The kit of the present invention employs AS-PCR (Allele-specific PCR) and real-time PCR techniques, and includes a specific primer and a fluorescent probe for detecting EGFR mutations in cfDNA in human plasma samples. During nucleic acid amplification, the targeted mutant DNA is matched with a base at the 3' end of the primer, amplified selectively and efficiently, and then the mutant amplicon is detected by a fluorescent probe labeled with FAM or CAL Fluor Orange 560 (CFO560). Wild-type DNA cannot be matched with a specific primer and no amplification occurs. The kit of the present invention may consist of EGFR Master Mixture 1-4, ADPS ^TM smart DNA polymerase, EGFR positive control, and nuclease-free distilled water.

The kit for detecting EGFR gene mutations of the present invention, for example, may be configured as shown in Table 1, but is not limited thereto.

composition main ingredient sheep EGFR Master Mixture 1 Buffer containing dNTPs, primers/probes for EGFR target and IC 240 μL/tube Х1 EGFR Master Mixture 2 Buffer containing dNTPs, primers/probes for EGFR target and IC 240 μL/tube Х1 EGFR Master Mixture 3 Buffer containing dNTPs, primers/probes for EGFR target and IC 240 μL/tube Х1 EGFR Master Mixture 4 Buffer containing dNTPs, primers/probes for EGFR target and IC 240 μL/tube Х1 ADPS ^TM Smart DNA Polymerase ADPS ^TM Smart DNA Polymerase 60 μL/tube Х1 EGFR positive control Recombinant plasmid blends containing each EGFR mutation 160 μL/tube Х1 Nuclease-free distilled water PCR grade distilled water 1.0 mL/tube Х1

An exemplary configuration of each EGFR Master Mix of Table 1 is shown in Table 2.

final concentration MMX 1-4 ADPS buffer Tris Cl (pH 8.8) 50 mM MgCl ₂ 2.5 mM KCl 60 mM (NH ₄ ) ₂ SO ₄ 2.5 mM TMAC 25 mM Tween 20 0.1% BSA 0.01% dNTP 0.25 mM each Primer/Probe specific concentration ROX standard dye 1X

The detection information of EGFR Master Mix 1-4 is shown in Table 3.

reagent fluorescence signal FAM CAL Fluor Orange 560 ^* Quasar 670 ^* EGFR Master Mixture 1 S768I Ex19Del IC ^** EGFR Master Mixture 2 T790M - IC EGFR Master Mixture 3 L861Q G719X IC EGFR Master Mixture 4 Ex20Ins L858R IC

*Replaceable dyes: CAL Fluor Orange 560 (VIC/HEX/JOE), Quasar 670 (CY5)

**IC: internal control for PCR

The present invention also provides a method for detecting EGFR gene mutations comprising the steps of:

(a) extracting nucleic acids from the isolated biological sample;

(b) performing a polymerase chain reaction by treating the extracted nucleic acid with the kit of the present invention; and

(c) analyzing the size or nucleotide sequence of the amplification product as a result of the polymerase chain reaction.

According to a preferred embodiment of the present invention, 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 PCR amplification result by measuring a Ct (cycle threshold) value.

The cycle threshold (Ct) value means the number of cycles in which 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 in the reaction have accumulated. The Ct value is the cycle in which an increase in ΔRn was first detected. Rn denotes the magnitude of the fluorescence signal generated during PCR at each time point, and ΔRn denotes the fluorescence emission intensity of the reporter dye (normalized reporter signal) divided by the fluorescence emission intensity of the reference dye. The Ct value is also called Cp (crossing point) in LightCycler. The Ct value represents the point at which the system begins to detect an increase in fluorescence signal associated with exponential growth of the PCR product in a log-linear phase. This period provides the most useful information about the reaction. The slope of the log-linear step represents the amplification efficiency (Eff) (https://www.appliedbiosystems.co.kr/).

On the other hand, TaqMan probes typically contain a primer (eg, 20-) comprising a fluorophore at the 5'-end and a quencher (eg, TAMRA or non-fluorescence quencher (NFQ)) at the 3'-end. oligonucleotides longer than 30 nucleotides). The excited fluorescent material transfers energy to a nearby quenching material rather than fluorescing it (FRET = Frster or fluorescence resonance energy transfer; Chen, X., et al., Proc Natl Acad Sci USA, 94(20): 10756- 61 (1997)). Therefore, when the probe is normal, no fluorescence is generated. TaqMan probes are designed to anneal to the internal sites of PCR products.

The TaqMan probe specifically hybridizes to the template DNA during the annealing step, but fluorescence is suppressed by the quencher on the probe. During the extension reaction, the TaqMan probe hybridized to the template is decomposed by the 5' to 3' nuclease activity of Taq DNA polymerase, and the fluorescent dye is released from the probe, the inhibition by the quencher is released, and fluorescence is displayed. In this case, the 5'-end of the TaqMan probe should be located downstream of the 3'-end of the extension primer. That is, when the 3'-terminus of the extension primer is extended by a template-dependent nucleic acid polymerase, the 5'-end of the TaqMan probe is cleaved by the 5' to 3' nuclease activity of this polymerase to form a reporter molecule. A fluorescence signal is generated.

The reporter molecule and the quencher molecule bound to the TaqMan probe include a fluorescent material and a non-fluorescent material. Fluorescent reporter molecules and quencher molecules that can be used in the present invention can be any known in the art, examples of which 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 TMR (568), BODIPY558/568 (568) , BODIPY564/570 (570), Cy3 ^TM (570), Alexa ^TM 546 (570), TRITC (572), Magnesium Orange ^TM (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange ^TM (576) ), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red ^™ (590), Cy3.5 ^™ (596), ROX (608), Calcium Crimson ^™ (615), Alexa ^™ 594 ( 615), Texas Red (615), Nile Red (628), YO-PRO ^TM -3 (631), YOYO ^TM -3 (631), R-phycocyanin (642), CPhycocyanin (648), TO-PRO ^TM - 3 (660), TOTO3 (660), DiD DilC (5) (665), Cy5 ^TM (670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), VIC (546), BHQ-1 ( 534), BHQ-2 (579), BHQ-3 (672), Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705) and Quasar 705 (610). The number in parentheses is the maximum emission wavelength expressed in nanometers.

Suitable reporter-quencher pairs are disclosed in many literatures: Pesce et al., editors, FLUORESCENCE SPECTROSCOPY (Marcel Dekker, New York, 1971); White et al., FLUORESCENCE ANALYSIS: A PRACTICAL APPROACH (Marcel Dekker, New York, 1970); Berlman, HANDBOOK OF FLUORESCENCE SPECTRA OF AROMATIC MOLECULES, 2 ^nd EDITION (Academic Press, New York, 1971); Griffiths, COLOR AND CONSTITUTION OF ORGANIC MOLECULES (Academic Press, New York, 1976); Bishop, editor, INDICATORS (Pergamon Press, Oxford, 1972); Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS (Molecular Probes, Eugene, 1992); Pringsheim, FLUORESCENCE AND PHOSPHORESCENCE (Interscience Publishers, New York, 1949); Haugland, RP, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Sixth Edition, Molecular Probes, Eugene, Oreg., 1996; US Pat. Nos. 3,996,345 and 4,351,760.

In addition, the non-fluorescent material used for the reporter molecule and the quencher molecule bound to the TaqMan probe may include a minor groove binding (MGB) moiety. As used herein, the term "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. olivomycin, anthramycin, sibiromycin, pentamidine, stilbamidine, berenil, CC-1065, Hoechst 33258, DAPI (4-6 diamidino-2-phenylindole), dimers, trimers, tetramers and pentamers of CDPI, N-methylpyrrole-4-carbox-2-amide (MPC) and dimers, trimers, tetramers and pentamers thereof. it's not going to be

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 an increased melting temperature (Tm) of the hybrid duplex formed by the MGB-conjugated probe compared to the normal probe. Therefore, MGB stabilizes the van der Waals force to increase the melting temperature (Tm) of the MGB-conjugated probe without increasing the probe length, resulting in shorter probes (e.g., 21 nucleotides or less) in Taqman real-time PCR under more stringent conditions. makes the use of

In addition, the MGB-conjugated probe removes the background fluorescence more efficiently. Accordingly, the probe of the present invention may be in the form of a TaqMan MGB-conjugate, wherein the length of the probe includes, but is not limited to, 15-21 nucleotides.

According to a preferred embodiment of the present invention, the EGFR gene mutation is the EGFR gene mutation is selected from the group consisting of deletion, substitution and insertion mutations within exons 18, 19, 20 and 21 of the EGFR gene. It may include one or more types.

Specifically, the EGFR gene mutation is a substitution of glycine at the 719th amino acid within exon 18 of EGFR, a deletion within exon 19, serine at the 768th amino acid within exon 20, threonine at the 790th amino acid, It may include one or more selected from the group consisting of substitution of cysteine at the 797th amino acid, insertion within exon 20, leucine at the 858th amino acid in exon 21, and leucine at the 861th amino acid.

For example, 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.

exon mutation EGFR nucleic acid sequence COSMIC ID exon 18 G719X 2155G>A 6252 2155G>T 6253 2156G>C 6239 exon 19 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 12369 2240_2257del18 12370 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>GCA 12419 2238_2248>GC 12422 2237_2251del15 12678 2236_2253del18 12728 2235_2248>AATTC 13550 2235_2252>AAT 13551 2235_2251>AATTC 13552 2253_2276del24 13556 2237_2257>TCT 18427 2238_2252del15 23571 2233_2247del15 26038 2232_2249del15 221565 2234_2248del15 1190791 exon 20 S768I 2303G>T 6241 T790M 2369C>T 6240 Ex20Ins 2307_2308ins9GCCACGTGG 12376 2319_2320insCAC 12377 2310_2311insGGT 12378 2311_2312ins9GCGTGGACA 13428 2309_2310AC>CCAGCGTGGAT 13558 exon 21 L858R 2573T>G 6224 2573_2574TG>GT 12429 L861Q 2582T>A 6213

In the EGFR gene mutation detection method of the present invention, 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 contained in an animal, preferably a vertebrate, more preferably a human subject. For example, the biological sample of step (a) may be sputum, blood, saliva or urine, and the nucleic acid of step (a) is a formalin-fixed paraffin embedded sample of a tissue biopsy or a liquid biopsy. can be extracted from

The EGFR gene mutation detection method of the present invention may include melting temperature analysis using a double-stranded specific dye.

Melting temperature curve analysis can be performed on a real-time PCR instrument such as an ABI 5700/7000 (96 well format) or ABI 7900 (384 well format) instrument with onboard software (SDS 2.1). Alternatively, melting temperature curve analysis can be performed as an endpoint analysis.

"Dye that binds to double-stranded DNA" or "double-strand-specific dye" can be used when it has higher fluorescence when bound to double-stranded DNA than in an unbound state. Examples of such dyes include SOYTO-9, SOYTO-13, SOYTO-16, SOYTO-60, SOYTO-64, SYTO-82, ethidium bromide (EtBr), SYTOX Orange, TO-PRO-1, SYBR Green I, TO-PRO-3 or EvaGreen. Except for EtBr and EvaGreen (Quiagen), these dyes have been tested for 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 an agarose gel after standard PCR, gene mutation-specific amplification or allele-specific amplification through real-time PCR, Tetra-primer amplification-refractory mutagenesis system 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 a thermostable DNA polymerase such as Taq polymerase or Klen Taq. DNA polymerase uses single-stranded DNA as a template and enzymatically assembles a new DNA strand from nucleotides by using oligonucleotides (primers). 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 at the end of the PCR. In real-time PCR, the reaction is characterized by the time point during the cycle when amplification is first detected rather than the amount of target accumulated after a fixed number of cycles. Two methods are mainly used to perform quantitative PCR: dye-based detection and probe-based detection.

The "Allele Specific Amplification (ASA)" is an amplification technique in which PCR primers are designed to discriminate between templates having different single nucleotide residues.

The "gene mutation-specific amplification or allele-specific amplification through real-time PCR" detects a gene mutation or SNP in a very efficient way. Unlike most other methods for detecting genetic mutations or SNPs, preliminary amplification of the target genetic material is not required. ASA combines amplification and detection in a single reaction based on discrimination of matched and mismatched primer/target sequence complexes. The increase in amplified DNA during the reaction can be monitored in real time as 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 via real-time PCR shows a delay or absence of a fluorescence signal for mismatched cases. In the detection of genetic mutations or SNPs, this provides information on the presence or absence of genetic mutations or SNPs.

The "tetra-primer amplification-refractory mutation system PCR" amplifies both wild-type and mutant alleles together with control fragments in a single tube PCR reaction. A non-allele-specific control amplicon is amplified by two common (outer) primers flanked by the mutant region. The two allele-specific (inside) primers are designed in the opposite direction to the common primer and together with the common primer can amplify both wild-type and mutant amplicons simultaneously. Consequently, the two allele-specific amplicons have different lengths because the mutations are located asymmetrically with respect to the common (outer) primer and can be easily separated by standard gel electrophoresis. This control amplicon provides an 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 mutagenesis system PCR.

The above "isothermal amplification" means that the amplification of a nucleic acid is carried out at a lower temperature without relying on a thermocycler, and preferably without having to change the temperature during amplification. The temperature used in the isothermal amplification may be between room temperature (22-24 °C) and about 65 °C, or between about 60-65 °C, 45-50 °C, 37-42 °C, or 22-24 °C. The product of the isothermal amplification result is gel electrophoresis, ELISA, ELOSA (Enzyme linked oligosorbent assay), real-time PCR, ECL (enhanced chemiluminescence), RNA, DNA, and a chip-based capillary electrophoresis device that analyzes proteins or turbidity. It can be detected with a bioanalyzer.

When the EGFR gene mutation detection method of the present invention is performed by 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 an EGFR gene mutation of the present invention may be used for predicting the reactivity of a lung cancer patient to a drug.

Accordingly, the present invention may provide a DNA polymerase, primer set, probe and/or kit for detecting EGFR gene mutation for predicting the reactivity of lung cancer patients to drugs.

The present invention may 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.

According to another preferred embodiment of the present invention, the cancer may be non-small cell lung cancer.

The EGFR gene mutation detection method of the present invention provides information for diagnosing cancer early and establishing a treatment strategy for each patient by detecting the known EGFR mutations in Table 4, thereby treating patients more effectively. contribute

The DNA polymerase, primer set and/or kit for detecting EGFR gene mutations of the present invention can be used for diagnosis, prognosis, and drug reactivity prediction methods for all applicable diseases by detecting the mutations described in Table 4 above.

In the present invention, the term “prognosis” refers to an act of predicting the course and outcome of a disease in advance. More specifically, prognosis prediction means that the course of disease after treatment may vary depending on the physiological or environmental condition of the patient, and it is interpreted to mean any act of predicting the course of the disease after treatment by comprehensively considering the condition of the patient. can be

The prognosis prediction may be interpreted as an act of predicting disease-free survival or survival rate of a patient by predicting in advance whether the disease will progress and be cured after treatment of a specific disease. For example, predicting that "the prognosis is good" indicates that the patient's disease-free survival rate or survival rate is high after treatment of the disease, meaning that the patient is more likely to be cured, and predicting that "the prognosis is poor" indicates that the disease has a high level of survival. After treatment, the disease-free survival rate or survival rate of the patient is at a low level, which means that the disease is highly likely to recur or to die due to the disease.

As used herein, the term “disease-free survival rate” refers to the possibility that a patient can survive without recurrence of a disease after treatment for a specific disease.

As used herein, the term “survival rate” refers to the possibility that a patient can survive regardless of whether the disease recurs after treatment of a specific disease.

When the EGFR gene mutation detection method of the present invention is used for predicting the reactivity of a lung cancer patient to a drug, the drug may be a tyrosine kinase inhibitor, and the type of tyrosine kinase inhibitor is, for example, , gefitinib, erlotinib or osimertinib.

Hereinafter, the present invention will be described in more detail by way of Examples. These examples are merely for illustrating the present invention in more detail, and thereby it will be apparent to those of ordinary skill in the art that the technical scope of the present invention is not limited to these examples.

Mutagenesis of Taq Polymerase

1-1. fragment PCR

In this example, Taq DNA polymerase in which the 536th amino acid residue in the amino acid sequence of SEQ ID NO: 1 was substituted from arginine to lysine (hereinafter referred to as "R536K"), Taq DNA polymerase in which the 660th amino acid residue was substituted from arginine to valine (hereinafter referred to as "R660V") and Taq DNA polymerase 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 (hereinafter referred to as "R536K/R660V") was prepared as follows did

First, Taq DNA polymerase fragments (F1 to F5) were amplified by PCR using the mutation-specific primers shown in Table 5 as shown in FIG. 1 (a). Reaction conditions are shown in Table 6.

primer sequence (5'→3') SEQ ID NO: Eco-F GG GGTACC TCA TCA CCC CGG 64 R536K-R CTT GGT GAG CTC CTT GTA CTG CAG GAT 65 R536K-F ATC CTG CAG TAC AAG GAG CTC ACC AAG 66 R660V-R GAT GGT CTT GGC CGC CAC GCG CAT CAG GGG 67 R660V-F CCC CTG ATG CGC GTG GCG GCC AAG ACC ATC 68 Xba-R GC TCTAGA CTA TCA CTC CTT GGC GGA GAG CCA 69

10xpfu buffer (Solgent) 2.5 μl dNTPs (10 mM each) 1 μl F primer 1 μl R primer 1 μl Distilled water 18 μl pUC19-Taq (10 ng/μl) 1 μl Pfu polymerase 0.5 μl 30 cycles (Ta=60℃) 25 μl

As a result of confirming the PCR product on electrophoresis, as shown in FIG.

1-2. overlap PCR

Using each of the fragments amplified in 1-1 above as a template, the full length was amplified using primers at both ends (Eco-F and Xba-R primers). Reaction conditions are shown in Tables 7 and 8.

R660V or R536K 10xpfu buffer (Solgent) 5 μl 5x Enhancer (Solgent) 10 μl dNTPs (10 mM each) 1 μl Eco-F primer (10 pmol/μl) 2 μl Xba-R primer (10 pmol/μl) 2 μl Distilled water 27 μl Fragment 1 (or Fragment 3) 1 μl Fragment 2 (or Fragment 4) 1 μl Pfu polymerase 1 μl 40 cycles (Ta=62℃) 50 μl

R536K/R660V 10xpfu buffer (Solgent) 5 μl 5x Enhancer (Solgent) 10 μl dNTPs (10 mM each) 1 μl Eco-F primer (10 pmol/μl) 2 μl Xba-R primer (10 pmol/μl) 2 μl Distilled water 26 μl Fragment 2 1 μl Fragment 3 1 μl Fragment 5 1 μl Pfu polymerase 1 μl 40 cycles (Ta=62℃) 50 μl

As a result, it was confirmed that the Taq polymerase of "R536K", "R660V" and "R536K/R660V" was amplified as shown in FIG.

1-3. ligation

pUC19 was digested with restriction enzymes 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 at 37°C for 1 hour under the conditions of Table 10 to prepare a vector. .

10xCutSmart Buffer (NEB) 2.5 μl pUC19 (500 ng/μl) 21.5 μl EcoRI-HF (NEB) 0.5 μl XbaI (NEB) 0.5 μl 25 μl

10xSAP Buffer (Roche) 2 μl purified DNA 17 μl SAP (Roche) 1 μl 20 μl

In the case of insert, the overlap PCR product of Example 1-2 was purified and digested with restriction enzymes EcoRI/XbaI for 3 hours at 37°C under the conditions shown in Table 11, followed by gel extraction with the prepared vector (FIG. 2). ).

10xCutSmart Buffer (NEB) 2 μl Purified PCR product 17 μl EcoRI-HF (NEB) 0.5 μl XbaI (NEB) 0.5 μl 20 μl

After ligation at room temperature (RT) for 2 hours under the conditions shown in Table 12, E. coli DH5α was transformed and selected in a medium containing ampicillin. The plasmids prepared from the obtained colonies were sequenced to obtain Taq DNA polymerase mutants ("R536K", "R660V" and "R536K/R660V") into which the desired mutation was introduced.

vector sole vector+insert 10x ligase buffer (Solgent) 1 μl 1 μl vector 1 μl 1 μl insert - 3 μl Distilled water 7 μl 4 μl T4 DNA ligase (Solgent) 1 μl 1 μl 10 μl 10 μl

Introduction of E507K mutation

2-1. fragment PCR

As a result of testing the Taq polymerase activity of "R536K", "R660V" and "R536K / R660V" prepared in Example 1, it was confirmed that the activity was lowered (data not shown), R536K, R660V, R536K / R660V, respectively In addition to the E507K mutation (substitution of the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 from glutamic acid to lysine) was introduced, and E507K mutation was also introduced in wild-type Taq DNA polymerase (WT) for use as a control. The preparation method of the Taq DNA polymerase into which the E507K mutation is introduced is the same as in Example 1.

Taq DNA polymerase fragments (F6 to F7) were amplified by PCR as shown in FIG. 3 using the mutation-specific primers shown in Table 13. Reaction conditions are shown in Table 14.

primer sequence (5'→3') SEQ ID NO: Eco-F GG GGTACC TCA TCA CCC CGG 64 E507K-R CTT GCC GGT CTT TTT CGT CTT GCC GAT 70 E507K-F ATC GGC AAG ACG AAA AAG ACC GGC AAG 71 Xba-R GC TCTAGA CTA TCA CTC CTT GGC GGA GAG CCA 69

10xpfu buffer (Solgent) 2.5 μl dNTPs (10 mM each) 1 μl F primer (10 pmol/μl) 1 μl R primer (10 pmol/μl) 1 μl Distilled water 18 μl Template plasmid (10 ng/μl) 1 μl Pfu polymerase 0.5 μl 30 cycles (Ta=60℃) 25 μl

*Template plasmid: pUC19-Taq (WT)pUC19-Taq (R536K)

pUC19-Taq (R660V)

pUC19-Taq (R536K/R660V)

2-2. overlap PCR

Using each of the fragments amplified in 2-1 above as a template, the full length was amplified using primers at both ends (Eco-F and Xba-R primers). Reaction conditions are shown in Table 15.

10xpfu buffer (Solgent) 5 μl 5x Enhancer (Solgent) 10 μl dNTPs (10 mM each) 1 μl Eco-F primer (10 pmol/μl) 2 μl Xba-R primer (10 pmol/μl) 2 μl Distilled water 27 μl Fragment 6 1 μl Fragment 7 1 μl Pfu polymerase 1 μl 40 cycles (Ta=62℃) 50 μl

2-3. ligation

pUC19 was digested with restriction enzymes EcoRI/XbaI for 4 hours at 37°C under the conditions of Table 9, and then the DNA was purified, and the purified DNA was treated with SAP under the conditions of Table 10 at 37°C for 1 hour to prepare a vector. .

In the case of insert, the overlap PCR product of Example 2-2 was purified, digested with restriction enzymes EcoRI/XbaI for 3 hours at 37°C under the conditions of Table 11, and then gel-extracted with the prepared vector (FIG. 4). ).

After ligation at room temperature (RT) for 2 hours under the conditions shown in Table 12, E. coli DH5α or DH10β was transformed and selected in a medium containing ampicillin. The plasmid prepared from the obtained colonies was sequenced to obtain a Taq DNA polymerase mutant into which E507K mutation was introduced ("E507K/R536K", "E507K/R660V" and "E507K/R536K/R660V").

Preparation of primer sets

To design primers that generate PCR products for mutations in the EGFR gene but not wild-type EGFR genes, use the OligoAnalyzer Tool (https://sg.idtdna.com/calc/analyzer) program to determine the primer size and Tm value. , primer GC content, and whether there is a self-complementary sequence in the primer to exclude the possibility of forming a secondary structure, and then design the primer as shown in Tables 16 to 19 did.

mutant group primer sequence (5'-3') SEQ ID NO: Final concentration (nM) S768I S768I Rmt14+C CGG GGT TGT CCA CGA 3 200 S768I SF2 ACA CTG ACG TGC CTC TCC C 4 200 Ex19del Ex19del C1 Rmt20 TGT TGG CTT TCG GAG ATG CC 5 50 Ex19del C2 Rmt21 TTG GCT TTC GGA GAT GAT TCC 6 50 Ex19del C3 Rmt20 GTT GGC TTT CGG AGA TTC CT 7 40 Ex19del C4 Rmt24 GCT TTC GGA GAT GTT GCT TCC TTG 8 150 Ex19del C5 Rmt21 CCT TGT TGG CTT TCT GTT CCT 9 40 Ex19del C6 Rmt21 CCT TGT TGG CTT TCG GAT CCT 10 40 Ex19del C7 Rmt21 TGG CTT TCG GAG ATG TTT TGA 11 50 Ex19del C8 Rmt20 TGG CTT TCG GAG ATG TCT TG 12 50 Ex19del C9 Rmt21 TCC TTG TTG GCT TTC GGT TCC 13 40 Ex19del C10 Rmt2 (20) TGT TGG CTT TCG GAG CCT TG 14 50 Ex19del C11 Rmt20 TTG TTG GCT TTC GGA GAT TC 15 50 Ex19del C12 Rmt21 TTC CTT GTT GGC TTT CGA TTC 16 50 Ex19del C13 Rmt21 GCT TTC GGA GAT GTT GGT TCC 17 100 Ex19del C14 Rmt20 GTT GGC TTT CGG AGA TGG TT 18 100 Ex19del C15 Rmt20 CTT GTT GGC TTT CGG AAC CT 19 50 Ex19del C16 Rmt23 TTC CTT GTT GGC TTT CGG AAT TT 20 75 Ex19del C17 Rmt20 GTT GGC TTT CGG AGA TAC CT 21 50 Ex19del C18 Rmt3 (21) TTT CCT TGT TGG CTT TCG GTT 22 75 Ex19del C19 Rmt21 TGT TGG CTT TCG GAG AAG CAA 23 75 Ex19del C20 Rmt21 TGT TGG CTT TCG GAG ATT GCT 24 50 Ex19del C21 Rmt21 TTG GCT TTC GGA GAT GTT GGC 25 75 Ex19del C22 Rmt20 TGT TGG CTT TCG GAG ACT TG 26 50 Ex19del C23 Rmt22 TGG CTT TCG GAG ATG TTG GAA T 27 100 Ex19del C24 Rmt22 GTT GGC TTT CGG AGA TAT TTT G 28 50 Ex19del C25 Rmt22 TGT TGG CTT TCG GAG ATG GAA T 29 100 Ex19del C26 Rmt19+FL AAT AAA TCA TAA AAA CTC ACA TCG AGG GTT G 30 100 Ex19del C27 Rmt20 TCC TTG TTG GCT TTC GAG AC 31 50 Ex19del C28 Rmt2 (20) TTG TTG GCT TTC GGA GAT TC 32 40 Ex19del C29 Rmt21 GCT TTC GGA GAT GTT GCG ATA 33 50 Ex19del C30 Rmt23 GTT GGC TTT CGG AGA TGT TAT AG 34 75 Ex19del C31 Rmt20 GCT TTC GGA GAT GTT GTG AT 35 100 19del SF2 TCC TTC TCT CTC TGT CAT AGG G 36 200 EGFR IC EGFR IC Ex2F CAG TTG GGC ACT TTT GAA GAT C 37 100 EGFR IC Ex2R TCC AAA TTC CCA AGG ACC AC 38 100

mutant group primer sequence (5'-3') SEQ ID NO: final concentration (nM) T790M T790M Rmt(15) AGG GCA TGA GCT GCA 39 240 T790M SF5 ACC CCC ACG TGT GCC G 40 150 EGFR IC EGFR IC Ex2F CAG TTG GGC ACT TTT GAA GAT C 37 100 EGFR IC Ex2R TCC AAA TTC CCA AGG ACC AC 38 100

mutant group primer sequence (5'-3') SEQ ID NO: final concentration (nM) G719X G719S Rmt17 CCG AAC GCA CCG GAG CT 41 200 G719C Rmt16 CGA ACG CAC CGG AGC A 42 200 G719A Rmt18(-3T) TGC CGA ACG CAC CGT AGG 43 200 G719X SF4 GCC TCT TAC ACC CAG TGG AGA A 44 200 L861Q L861Q Rmt17 CTC TTC CGC ACC CAG CT 45 200 L861Q SF1 CGT ACT GGT GAA AAC ACC G 46 200 EGFR IC EGFR IC Ex2F CAG TTG GGC ACT TTT GAA GAT C 37 100 EGFR IC Ex2R TCC AAA TTC CCA AGG ACC AC 38 100

mutant group primer sequence (5'-3') SEQ ID NO: final concentration (nM) Ex20Ins 20Ins C1 Rmt2(15)-4A CCA CGC TGG CAA CGC 47 400 20Ins C2 Rmt3(15) GGC ACA CGT GGT GGG 48 200 20Ins C3 Rmt2(16) CAC ACG TGG GGG TTA C 49 300 20Ins C4 Rmt2(16)-3T TGT CCA CGC TGT TCA C 50 200 20Ins C5 Rmt2(16)-3T ATC CAC GCT GGC TAC G 51 100 20Ins SF4 GAA GCC ACA CTG ACG TGC C 52 200 L858R L858R Fmt21-2A CAA GAT CAC AGA TTT TGG ACG 53 400 L858R OR2 TGA CCT AAA GCC ACC TCC TTA CT 54 400 EGFR IC EGFR IC Ex2F CAG TTG GGC ACT TTT GAA GAT C 37 100 EGFR IC Ex2R TCC AAA TTC CCA AGG ACC AC 38 100

preparation of the sample

Mutant plasmid preparation

Using the gDNA of the HEK293T cell line, which is the wild-type DNA of EGFR, a primer set capable of amplifying the peripheral region of the target mutation in Table 4 was applied to prepare wild-type clones of exons 18, 19, 20, and 21 regions. Using the prepared wild-type DNA, mutagenesis was performed on 44 target mutations, and E.Coli DH5α cells were transformed to obtain each mutant-type clone. Identification of wild-type clones and mutant clones was confirmed by direct sequencing. Wild-type DNA and mutant DNA for each exon extracted through clones were used as standards for evaluating the performance of the EGFR mutation detection kit.

As shown in Table 20, samples were prepared by adding 10,000 copies, 100 copies, 30 copies, 10 copies, or 3 copies of each mutant plasmid listed in Table 4 per 30,000 copies of HEK293T cell genomic DNA, and the mutant plasmid was not added. The group was used as a control.

group sample WT 30,000 copies of HEK293T cell genomic DNA WT+mt 10 ⁴ 30,000 copies of HEK293T cell genomic DNA + 10,000 copies of the corresponding mutant plasmid WT+mt 10 ² 30,000 copies of HEK293T cell genomic DNA + 100 copies of the corresponding mutant plasmid WT+mt 30 30,000 copies of HEK293T cell genomic DNA + 30 copies of the corresponding mutant plasmid WT+mt 10 30,000 copies of HEK293T cell genomic DNA + 10 copies of the corresponding mutant plasmid WT+mt 3 30,000 copies of HEK293T cell genomic DNA + 3 copies of the corresponding mutant plasmid

EGFR mutation detection

EGFR gene mutations in Table 4 from each group in Table 20 using the primer set of Example 3 and Taq polymerase (SEQ ID NO: 2) each containing the "E507K / R536K / R660V" mutation obtained in Example 2 above detected. Table 21 shows mutation information targeted by EGFR Master Mixture 1-4, respectively.

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_2256del1236 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>GCA (12419), 2238_2248>GC (12422), 2237_2251del15 (12678), 2236_2253del18 (12728), 2235_2248>AATTC (13550), 2235_2252>AAT (13551), 2235_2251>AATTC (13552) , 2253_2276del24 (13556), 2237_2257>TCT (18427), 2238_2252del15 (23571), 2233_2247del15 (26038), 2232_2249del15 (221565), 2234_2248del15 (1190791) MMX2 FAM T790M 2369C>T (6240) MMX3 FAM L861Q 2582T>A (6213) CFO560 G719X 2155G>A (6252), 2155G>T (6253), 2156G>C (6239) MMX4 FAM Ex20Ins 2307_2308ins9GCCAGCGTG (12376), 2319_2320insCAC (12377), 2310_2311insGGT (12378), 2311_2312ins9GCGTGGACA (13428), 2309_2310AC>CCAGCGTGGAT (13558) CFO560 L858R 2573T>G (6224), 2573_2574TG>GT (12429)

Specific qPCR (Applied Biosystems 7500 Fast) experimental conditions are shown in Tables 22 to 24 below, and the probes were double-labeled as shown in Table 23.

2X EGFR MMX 10 μl sample 2 μl Nuclease-free distilled water 7.5 μl ADPS Smart DNA Polymerase (1 U/ul) 0.5 μl gun 20 μl

mutant group probe sequence (5'-3') SEQ ID NO: 5' fluorophore 3' matting material final concentration (nM) S768I S768I FAM_R TCA CGT AGG CTT CCT GGA GGG A 55 FAM BHQ1 400 Ex19del 19del CFO_R2 CTC TGG ATC CCA GAA GGT GAG AAA G 56 CAL Fluor Orange 560 BHQ1 400 T790M T790M IR4 GTG ATG AGC TGC ACG G 57 FAM BHQ1 125 T790M IR2(-8T) GTG ATG AGT TGC ACG GTG G 58 FAM BHQ1 130 G719X G719X CFO_R2_nova AAA CTG AAT TCA AAA AGA TCA AAG TGC TG 59 CAL Fluor Orange 560 BHQ1 nova 400 L861Q L858R FAM_R AAA CTG AAT TCA AAA AGA TCA AAG TGC TG 60 FAM BHQ1 50 Ex20Ins Ex20Ins FAM_R TCA CGT AGG CTT CCT GGA GGG A 61 FAM BHQ1 400 L858R L858R CFO560_F CCA AAC TGC TGG GTG CGG AAG AG 62 CAL Fluor Orange 560 BHQ1 400 EGFR IC EGFR IC Ex2 QS670 TCT CAG CCT CCA GAG GAT GTT CAA 63 Quasar 670 BHQ2 200

step cycle temperature hour data collection One One 95℃ 5 minutes - 95℃ 10 seconds - 2 50 60℃ 30 seconds FAM / CAL Fluor Orange 560 (VIC/HEX/JOE) / Quasar670 (CY5) 72℃ 10 seconds -

Sufficient EGFR reaction mixtures containing each of the components listed in Table 22 were prepared in separate sterile centrifuge tubes, respectively, and the reaction Master Mixture was vortexed for 3 seconds to mix thoroughly and centrifuged briefly. Two PCR tubes were prepared for each sample as follows: 10.0 µL of the EGFR reaction mixture was aliquoted for each PCR tube, 5.0 µL of each sample DNA was added to each sample tube, and then the PCR tube was capped. Nuclease-free distilled water was added up to 20.0 μL to all PCR tubes, and PCR strips were briefly centrifuged to collect all liquid at the bottom of each PCR tube. A PCR strip tube was placed in a real-time PCR (real-time PCR) machine, and PCR was performed after setting the PCR protocol using the cycling parameters in Table 24. After completion of PCR, the FAM/CAL Fluor Orange 560/Quasar 670 Ct values of each sample were recorded for data analysis, and the ΔCt values for each well were calculated as follows:

ΔCt value = Sample Ct value (FAM of each MMX) - Positive control Ct value (FAM of each MMX)

ΔCt value = sample Ct value (CFO560 for each MMX) - Ct value for positive control (CFO560 for each MMX)

The results for each tube were interpreted according to the cutoff ΔCt values in Tables 25 and 26. If the ΔCt value is < the cutoff ΔCt value, the sample is considered positive, and if there is no fluorescence signal and the Ct value of the internal control is 21.5 < Ct < 40, the sample is judged negative or below the limit of detection (LoD).

determine the outcome EGFR MMXs decision Internal Control Ct Values 21.5 ≤ Ct ≤ 40 available Ct < 21.5 or Ct > 40 invalidity Sample Ct value Ct < 23.5 invalidity Ct > 45 or no signal voice

Cutoff ΔCt value for each target Determining Valid Results Cutoff ΔCt value range decision ΔCt cutoff value S768I-FAM 10.0 ΔCt≤10.0 positivity ΔCt > 10.0 voice Ex19Del-CFO560 14.5 ΔCt≤14.5 positivity ΔCt > 14.5 voice T790M-FAM 14.5 ΔCt≤14.5 positivity ΔCt > 14.5 voice L861Q-FAM 13.0 ΔCt≤13.0 positivity ΔCt > 13.0 voice G719X-CFO560 8.5 ΔCt≤8.5 positivity ΔCt≤8.5 voice Ex20Ins-FAM 16.0 ΔCt≤16.0 positivity ΔCt > 16.0 voice L858R-CFO560 10.0 ΔCt≤10.0 positivity ΔCt > 10.0 voice

As a result of analyzing the data as described above, no fluorescence signal was detected in WT as shown in FIGS. 5a to 5l, 6a to 6l, 7a to 7i, 8, 9a to 9e, and 10a to 10h, but EGFR gene mutations in Table 4 Fluorescent signals were detected in the samples containing

exon mutation EGFR nucleic acid sequence COSMIC ID LoD (copy) Detection sensitivity (%) Exon 18 G719X 2155G>A 6252 15 0.05 2155G>T 6253 10 0.03 2156G>C 6239 3 0.01 Exon 19 Ex19Del 2240_2251del12 6210 3 0.01 2239_2247del9 6218 3 0.01 2238_2255del18 6220 3 0.01 2235_2249del15 6223 3 0.01 2236_2250del15 6225 3 0.01 2239_2253del15 6254 3 0.01 2239_2256del18 6255 3 0.01 2237_2254del18 12367 3 0.01 2240_2254del15 12369 3 0.01 2240_2257del18 12370 3 0.01 2239_2248TTAAGAGAAG>C 12382 3 0.01 2239_2251>C 12383 3 0.01 2237_2255>T 12384 3 0.01 2235_2255>AAT 12385 3 0.01 2237_2252>T 12386 3 0.01 2239_2258>CA 12387 3 0.01 2239_2256>CAA 12403 3 0.01 2237_2253>TTGCT 12416 3 0.01 2238_2252>GCA 12419 3 0.01 2238_2248>GC 12422 3 0.01 2237_2251del15 12678 3 0.01 2236_2253del18 12728 3 0.01 2235_2248>AATTC 13550 3 0.01 2235_2252>AAT 13551 3 0.01 2235_2251>AATTC 13552 3 0.01 2253_2276del24 13556 3 0.01 2237_2257>TCT 18427 3 0.01 2238_2252del15 23571 3 0.01 2233_2247del15 26038 3 0.01 2232_2249del15 221565 3 0.01 2234_2248del15 1190791 3 0.01 Exon 20 S768I 2303G>T 6241 3 0.01 T790M 2369C>T 6240 10 0.03 Ex20Ins 2307_2308ins9GCCACGTGG 12376 3 0.01 2319_2320insCAC 12377 3 0.01 2310_2311insGGT 12378 3 0.01 2311_2312ins9GCGTGGACA 13428 3 0.01 2309_2310AC>CCAGCGTGGAT 13558 3 0.01 Exon 21 L858R 2573T>G 6224 3 0.01 2573_2574TG>GT 12429 3 0.01 L861Q 2582T>A 6213 3 0.01

SEQUENCE LISTING <110> GeneCast Co., Ltd. <120> DNA polymerase for detecting EGFR mutation and kit comprising the same <130> 1066828 <150> KR 10-2019-0004216 <151> 2019-01-11 <160> 72 <170> PatentIn version 3.2 <210> 1 < 211> 832 <212> PRT <213> Artificial <220> <223> Thermus aquaticus DNA polymerase (Taq) <400> 1 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val 50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu 85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu 100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys 115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp 130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn 180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu 195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu 210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe 260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu 340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro 355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala 450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg 500 505 510 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515 520 525 Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr 530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala 595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro 645 650 655 Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His 770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 <210> 2 <211> 832 <212> PRT <213> Artificial <220> <223> Taq E507K/R536K/R660V <400> 2 Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu 1 5 10 15 Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly 20 25 30 Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala 35 40 45 Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val 50 55 60 Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly 65 70 75 80 Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu 85 90 95 Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu 100 105 110 Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys 115 120 125 Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp 130 135 140 Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Val Leu His Pro Glu Gly 145 150 155 160 Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro 165 170 175 Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn 180 185 190 Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu 195 200 205 Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu 210 215 220 Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys 225 230 235 240 Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe 260 265 270 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285 Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Glu Gly 290 295 300 Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp 305 310 315 320 Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro 325 330 335 Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu 340 345 350 Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro 355 360 365 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 370 375 380 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 385 390 395 400 Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu 405 410 415 Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu 420 425 430 Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala 450 455 460 Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His 465 470 475 480 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495 Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Lys Lys Thr Gly Lys Arg 500 505 510 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515 520 525 Val Glu Lys Ile Leu Gln Tyr Lys Glu Leu Thr Lys Leu Lys Ser Thr 530 535 540 Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu 545 550 555 560 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala 595 600 605 Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly 610 615 620 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr 625 630 635 640 Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro 645 650 655 Leu Met Arg Val Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly 660 665 670 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 690 695 700 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val 705 710 715 720 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg 725 730 735 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765 Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His 770 775 780 Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala 785 790 795 800 Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro 805 810 815 Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu 820 825 830 <210> 3 <211> 15 <212> DNA <213> Artificial <220> <223> S768I Rmt14+C <400> 3 cggggttgtc cacga 15 <210> 4 <211> 19 <212> DNA <213> Artificial <220> < 223> S768I SF2 <400> 4 acactgacgt gcctctccc 19 <210> 5 <211> 20 <212> DNA <213> Artificial <220> <223> Ex19del C1 Rmt20 <400> 5 tgttggcttt cggagatgcc 20 <210> 6 <211> 21 <212> DNA <213> Artificial <220> <223> Ex19del C2 Rmt21 <400> 6 ttggctttcg gagatgattc c 21 <210> 7 <211> 20 <212> DNA <213> Artificial <220> <223> Ex19del C3 Rmt20 <400> 7 gttggctttc ggagattcct 20 <210> 8 <211> 24 <212> DNA <213> Artificial <220> <223> Ex19del C4 Rmt24 <400> 8 gctttcggag atgttgcttc cttg 24 <210> 9 <211> 21 <212> DNA <213> Artificial <220> <223> Ex19del C5 Rmt21 <400> 9 ccttgttggc tttctgttcc t 21 <210> 10 <211> 21 <212> DNA < 213> Artificial <220> <223> Ex19del C6 Rmt21 <400> 10 ccttgttggc tttcggatcc t 21 <210> 11 <211> 21 <212> DNA <213> Artificial <220> <223> Ex19del C7 Rmt21 <400> 11 tggctttcgg agatgttttg a 21 <210> 12 <211> 20 <212> DNA <213> Artificial <220> <223> Ex19del C8 Rmt20 <400> 12 tggctttcgg agatgtcttg 20 <210> 13 <211> 21 <212> DNA <213> Artificial <220> <223> Ex19del C9 Rmt21 <400> 13 tccttgttgg ctttcggttc c 21 <210> 14 <211> 20 <212> DNA <213> Artificial <220> <223> Ex19del C10 Rmt2(20) <400> 14 tgttggcttt cggagccttg 20 <210> 15 <211> 20 <212> DNA <213> Artificial <220> <223> Ex19del C11 Rmt20 <400> 15 ttgttggctt tcggagattc 20 <210> 16 <211> 21 <212> DNA <213> Arti ficial <220> <223> Ex19del C12 Rmt21 <400> 16 ttccttgttg gctttcgatt c 21 <210> 17 <211> 21 <212> DNA <213> Artificial <220> <223> Ex19del C13 Rmt21 <400> 17 gctttcggag atgttggttc c 21 <210> 18 <211> 20 <212> DNA <213> Artificial <220> <223> Ex19del C14 Rmt20 <400> 18 gttggctttc ggagatggtt 20 <210> 19 <211> 20 <212> DNA <213> Artificial < 220> <223> Ex19del C15 Rmt20 <400> 19 cttgttggct ttcggaacct 20 <210> 20 <211> 23 <212> DNA <213> Artificial <220> <223> Ex19del C16 Rmt23 <400> 20 ttccttgttg gctttcggaa ttt 23 <210 > 21 <211> 20 <212> DNA <213> Artificial <220> <223> Ex19del C17 Rmt20 <400> 21 gttggctttc ggagatacct 20 <210> 22 <211> 21 <212> DNA <213> Artificial <220> < 223> Ex19del C18 Rmt3(21) <400> 22 tttccttgtt ggctttcggt t 21 <210> 23 <211> 21 <212> DNA <213> Artificial <220> <223> Ex19del C19 Rmt21 <400> 23 tgttggcttt cggagaagca a 21 < 210> 24 <211> 21 <212> DNA <213> Artificial <220> <223> Ex19del C20 Rmt21 <400> 24 tgttggcttt cggagattgc t 21 <210> 25 <211> 21 <212> DNA <213> Artifi cial <220> <223> Ex19del C21 Rmt21 <400> 25 ttggctttcg gagatgttgg c 21 <210> 26 <211> 20 <212> DNA <213> Artificial <220> <223> Ex19del C22 Rmt20 <400> 26 tgttggcttt cggagacttg 20 <210> 27 <211> 22 <212> DNA <213> Artificial <220> <223> Ex19del C23 Rmt22 <400> 27 tggctttcgg agatgttgga at 22 <210> 28 <211> 22 <212> DNA <213> Artificial < 220> <223> Ex19del C24 Rmt22 <400> 28 gttggctttc ggagatattt tg 22 <210> 29 <211> 22 <212> DNA <213> Artificial <220> <223> Ex19del C25 Rmt22 <400> 29 tgttggcttt cggagatgga at 22 < 210> 30 <211> 31 <212> DNA <213> Artificial <220> <223> Ex19del C26 Rmt19+FL <400> 30 aataaatcat aaaaactcac atcgagggtt g 31 <210> 31 <211> 20 <212> DNA <213> Artificial <220> <223> Ex19del C27 Rmt20 <400> 31 tccttgttgg ctttcgagac 20 <210> 32 <211> 20 <212> DNA <213> Artificial <220> <223> Ex19del C28 Rmt2(20) <400> 32 ttgttggctt tcggagattc 20 <210> 33 <211> 21 <212> DNA <213> Artificial <220> <223> Ex19del C29 Rmt21 <400> 33 gctttcggag atgttgcgat a 21 <210> 34 <211> 23 <212> DNA <213> Artificial <220> <223> Ex19del C30 Rmt23 <400> 34 gttggctttc ggagatgtta tag 23 <210> 35 <211> 20 <212> DNA <213> Artificial <220> <223> Ex19del C31 Rmt20 <400> 35 gctttcggag atgttgtgat 20 <210> 36 <211> 22 <212> DNA <213 > Artificial <220> <223> 19del SF2 <400> 36 tccttctctc tctgtcatag gg 22 <210> 37 <211> 22 <212> DNA <213> Artificial <220> <223> EGFR IC Ex2F <400> 37 cagttgggca cttttgaaga tc 22 <210> 38 <211> 20 <212> DNA <213> Artificial <220> <223> EGFR IC Ex2R <400> 38 tccaaattcc caaggaccac 20 <210> 39 <211> 15 <212> DNA <213> Artificial < 220> <223> T790M Rmt(15) <400> 39 agggcatgag ctgca 15 <210> 40 <211> 16 <212> DNA <213> Artificial <220> <223> T790M SF5 <400> 40 accccccacgt gtgccg 16 <210 > 41 <211> 17 <212> DNA <213> Artificial <220> <223> G719S Rmt17 <400> 41 ccgaacgcac cggagct 17 <210> 42 <211> 16 <212> DNA <213> Artificial <220> <223 > G719C Rmt16 <400> 42 cgaacgcacc ggagca 16 <210> 43 <211> 18 <212> DNA <213> Artificial <220> <223> G719A Rmt18(-3T) <400> 43 tgccgaacgc accgtagg 18 <210> 44 < 211> 22 <212> DNA <213> Artificial <220> <223> G719X SF4 <400> 44 gcctcttaca cccagtggag aa 22 <210> 45 <211> 17 <212> DNA <213> Artificial <220> <223> L861Q Rmt17 <400> 45 ctcttccgca cccagct 17 <210> 46 <211> 19 <212> DNA <213> Artificial <220> <223> L861Q SF1 <400> 46 cgtactggtg aaaacaccg 19 <210> 47 <211> 15 <212> DNA <213> Artificial <220> <223> 20Ins C1 Rmt2(15)-4A <400> 47 ccacgctggc aacgc 15 <210> 48 <211> 15 <212> DNA <213> Artificial <220> <223> 20Ins C2 Rmt3(15) <400> 48 ggcacacgtg gtggg 15 <210> 49 <211> 16 <212> DNA <213> Artificial <220> <223> 20Ins C3 Rmt2(16) <400> 49 cacacgtggg ggttac 16 <210> 50 <211> 16 <212> DNA <213> Artificial <220> <223> 20Ins C4 Rmt2(16)-3T <400> 50 tgtccacgct gttcac 16 <210> 51 <211> 16 <212> DNA <213> Artificial <220> <223> 20Ins C5 Rmt2 (16)-3T <400> 51 atccacgctg gctacg 16 <210> 52 <211> 19 <212> DNA <213> Artificial <220> <223> 20Ins SF4 <400> 52 gaagccacac tgacgtgcc 19 <210> 53 <211> 21 <212> DNA <213> Artificial <220> <223> L858R Fmt21-2A <400> 53 caagatcaca gattttggac g 21 <210> 54 <211> 23 <212> DNA <213> Artificial <220> <223> L858R OR2 <400> 54 tgacctaaag ccacctcctt act 23 <210> 55 <211> 22 <212> DNA <213> Artificial <220> <223 > S768I FAM_R <400> 55 tcacgtaggc ttcctggagg ga 22 <210> 56 <211> 25 <212> DNA <213> Artificial <220> <223> 19del CFO_R2 <400> 56 ctctggatcc cagaaggtga gaaag 25 <210> 57 <211> 24 <212> DNA <213> Artificial <220> <223> EGFR IC Ex2 QS670 <400> 57 tctcagcctc cagaggatgt tcaa 24 <210> 58 <211> 16 <212> DNA <213> Artificial <220> <223> T790M IR4 <400> 58 gtgatgagct gcacgg 16 <210> 59 <211> 19 <212> DNA <213> Artificial <220> <223> T790M IR2(-8T) <400> 59 gtgatgagtt gcacggtgg 19 <210> 60 <211> 29 <212> DNA <213> Artificial <220> <223> G719X CFO_R2_nova <400> 60 aaactgaatt caaaaagatc aaagtgctg 29 <210> 61 <211> 28 <212> DNA <213> Artificial <220> <223> L858R FAM_R < 400> 61 cagcatgtca agatcacaga ttttgggc 28 <210> 62 <211> 22 <212> DNA <213> Artificial <220> <223> Ex20Ins FAM_R <400> 62 tcacgtaggc ttcctggagg ga 22 <210> 63 <211> 23 <212> DNA <213> Artificial <220> <223> L858R CFO560_F <400> 63 ccaaactgct gggtgcggaa gag 23 <210> 64 <211> 20 <212> DNA <213> Artificial <220> <223> Eco-F <400> 64 gg ggtacctc atcaccccgg 20 <210> 65 <211> 27 <212> DNA <213> Artificial <220> <223> R536K-R <400> 65 cttggtgagc tccttgtact gcaggat 27 <210> 66 <211> 27 <212> DNA <213 > Artificial <220> <223> R536K-F <400> 66 atcctgcagt acaaggagct caccaag 27 <210> 67 <211> 30 <212> DNA <213> Artificial <220> <223> R660V-R <400> 67 gatggtcttg gccgccacgc gcatcagggg 30 <210> 68 <211> 30 <212> DNA <213> Artificial <220> <223> R660V-F <400> 68 cccctgatgc gcgtggcggc caagaccatc 30 <210> 69 <211> 32 <212> DNA <213> Artificial <220> <223> Xba-R <400> 69 gctctagact atcactcctt ggcggagagc ca 32 <210> 70 <211> 27 <212> DNA <213> Artificial <220> <223> E507K-R <400> 70 cttgccggtc tttttcgtct tgccgat 27 <210> 71 <211> 27 <212> DNA <213> Artificial <220> <223> E507K-F <400> 71 atcggcaaga cgaaaaagac cggcaag 27 <210> 72 <211> 2499 <212> DNA <213> Artificial <220> <223> Taq E507K/R536K/R660V < 400> 72 atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60 cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120 gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180 gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240 tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300 gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360 gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420 gccgacaaag acctttacca gctcctttcc gaccgcatcc acgtcctcca ccccgagggg 480 tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540 gactaccg ccctgaccgg gtcc gacaaccttc ccggggtcaa gggcatcggg 600 gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660 ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720 ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780 aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840 ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900 ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960 cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020 gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080 ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140 gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200 gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260 gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320 ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380 ctggaggtgg ccgaggagat cgcccgcctc gaggccga gg tcttccgcct ggccggccac 1440 cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500 cccgccatcg gcaagacgaa aaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560 gccctccgcg aggcccaccc catcgtggag aagatcctgc agtacaagga gctcaccaag 1620 ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680 cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740 ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800 gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860 cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920 gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgcgtg 1980 gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040 gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100 cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160 gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220 cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agg gcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460gtggggatag gggaggactg gctctccgcc aaggagtga 2499

Claims (19)

delete delete delete delete In the amino acid sequence of SEQ ID NO: 1, 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), and the 660th amino acid residue, arginine ( R) Taq polymerase in which valine (V) is substituted;
a primer set comprising a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4;
a primer set comprising a forward primer of SEQ ID NO: 5 and a reverse primer of SEQ ID NO: 6;
a primer set comprising a forward primer of SEQ ID NO: 7 and a reverse primer of SEQ ID NO: 8;
a primer set comprising a forward primer of SEQ ID NO: 9 and a reverse primer of SEQ ID NO: 10;
a primer set comprising a forward primer of SEQ ID NO: 11 and a reverse primer of SEQ ID NO: 12;
a primer set comprising a forward primer of SEQ ID NO: 13 and a reverse primer of SEQ ID NO: 14;
a primer set comprising a forward primer of SEQ ID NO: 15 and a reverse primer of SEQ ID NO: 16;
a primer set comprising a forward primer of SEQ ID NO: 17 and a reverse primer of SEQ ID NO: 18;
a primer set comprising a forward primer of SEQ ID NO: 19 and a reverse primer of SEQ ID NO: 20;
a primer set comprising a forward primer of SEQ ID NO: 21 and a reverse primer of SEQ ID NO: 22;
a primer set comprising a forward primer of SEQ ID NO: 23 and a reverse primer of SEQ ID NO: 24;
a primer set comprising a forward primer of SEQ ID NO: 25 and a reverse primer of SEQ ID NO: 26;
a primer set comprising a forward primer of SEQ ID NO: 27 and a reverse primer of SEQ ID NO: 28;
a primer set comprising a forward primer of SEQ ID NO: 29 and a reverse primer of SEQ ID NO: 30;
a primer set comprising a forward primer of SEQ ID NO: 31 and a reverse primer of SEQ ID NO: 32;
a primer set comprising a forward primer of SEQ ID NO: 33 and a reverse primer of SEQ ID NO: 34;
a primer set comprising a forward primer of SEQ ID NO: 35 and a reverse primer of SEQ ID NO: 36;
a primer set comprising a forward primer of SEQ ID NO: 39 and a reverse primer of SEQ ID NO: 40;
a primer set comprising a forward primer of SEQ ID NO: 41 and a reverse primer of SEQ ID NO: 42;
a primer set comprising a forward primer of SEQ ID NO: 43 and a reverse primer of SEQ ID NO: 44;
a primer set comprising a forward primer of SEQ ID NO: 45 and a reverse primer of SEQ ID NO: 46;
a primer set comprising a forward primer of SEQ ID NO: 47 and a reverse primer of SEQ ID NO: 48;
a primer set comprising a forward primer of SEQ ID NO: 49 and a reverse primer of SEQ ID NO: 50;
a primer set comprising a forward primer of SEQ ID NO: 51 and a reverse primer of SEQ ID NO: 52; and
a primer set comprising a forward primer of SEQ ID NO: 53 and a reverse primer of SEQ ID NO: 54;
A kit for detecting EGFR gene mutations, including. The kit for detecting EGFR gene mutations according to claim 5, further comprising a primer set comprising a forward primer of SEQ ID NO: 37 and a reverse primer of SEQ ID NO: 38 for an internal control. 7. The method of claim 5 or 6, further comprising one or more probes selected from the group consisting of SEQ ID NOs: 55 to 63, wherein the nucleotide sequences of SEQ ID NOs: 55 to 63 are respectively FAM and CAL at the 5'-end One fluorophore selected from the group consisting of Fluor Orange 560, VIC, HEX, JOE, Quasar 670 and CY5 is labeled, and at the 3'-end, BHQ-1 from the group consisting of BHQ1 nova and BHQ-2 A kit for detecting EGFR gene mutations, in which one selected quencher is labeled. 6. The method of claim 5,
25-100 mM KCl; and
1-7 mM (NH ₄ ) ₂ SO ₄ ;
And, a kit for detecting EGFR gene mutations further comprising a PCR buffer composition having a final pH of 8.0 to 9.5. 6. The method of claim 5,
25-100 mM KCl;
1-7 mM (NH ₄ ) ₂ SO ₄ ; and
5 to 50 mM of TMAC (Tetra methyl ammonium chloride), and further comprising a PCR buffer composition having a final pH of 8.0 to 9.5, a kit for detecting EGFR gene mutations. A method for detecting EGFR gene mutations comprising the steps of:
(a) extracting nucleic acids from the isolated biological sample;
(b) performing PCR (polymerase chain reaction) by treating the kit of claim 5 or claim 6 on the extracted nucleic acid; and
(c) confirming the result of the PCR amplification with fluorescence. The method of claim 10, wherein the PCR is allele-specific PCR or real-time PCR. 11. The method of claim 10, (d) further comprising the step of determining the result of the PCR amplification by measuring a Ct (cycle threshold) value, EGFR gene mutation detection method. The EGFR gene according to claim 10, wherein the EGFR gene mutation comprises at least one selected from the group consisting of deletion, substitution and insertional mutations within exons 18, 19, 20 and 21 of the EGFR gene. Mutation detection method. 14. The method of claim 13, wherein the EGFR gene mutation is a substitution of glycine at amino acid 719 within exon 18 of EGFR, a deletion within exon 19, serine at amino acid 768 within exon 20, amino acid 790 In threonine, substitution of cysteine at the 797th amino acid, insertion within exon 20, leucine at the 858th amino acid within exon 21, and substitution of leucine at the 861th amino acid, including at least one selected from the group consisting of, EGFR gene mutation detection method. 11. The method of claim 10, EGFR gene mutation detection method is for predicting the responsiveness of lung cancer patients to drugs, EGFR gene mutation detection method. 16. The method of claim 15, wherein the lung cancer is non-small cell lung cancer, EGFR gene mutation detection method. The method of claim 15, wherein the drug is a tyrosine kinase inhibitor. 18. The method of claim 17, wherein the tyrosine kinase inhibitor is gefitinib, erlotinib or osimertinib, EGFR gene mutation detection method. The method of claim 10, wherein the nucleic acid of step (a) is extracted from a formalin-fixed paraffin embedded sample of a tissue biopsy or a liquid biopsy.

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