KR20200087723A - DNA polymerase for detecting EGFR mutation and kit comprising the same - Google Patents

Abstract

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

Description

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

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

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 epithelial growth factor receptor (EGFR) mutations have high levels of EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib (Iressa, AstraZeneca), erlotinib (Tarceba, Roche), and osimmertinib (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 allows predicting drug response before treatment.

So far, one of the most used methods for the detection of EGFR mutations is direct sequencing, which directly identifies the nucleotide sequence of a gene corresponding to exons 18 to 21 of EGFR. This method has the disadvantage 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 it is possible to analyze the base of the target site as well as a specific base. It has the advantage of being possible. Accordingly, there is a continuous need for a kit for more accurately predicting drug reactivity of lung cancer patients by improving the sensitivity of detecting EGFR mutations.

On the other hand, Korean Patent Publication No. 10-2015-0102468 discloses a kit for diagnosing lung cancer, which includes a primer set for amplifying a lung cancer-related mutation and a probe complementary to the mutation, but a polymerase for detecting a mutation in the EGFR gene and There is no known reaction buffer to increase its activity.

Accordingly, the present inventors have developed a kit comprising a DNA polymerase capable of detecting 44 somatic mutations in exons 18, 19, 20 and 21 of the EGFR gene with high sensitivity and a reaction buffer for increasing its activity. The invention was completed.

An object of the present invention is to provide a DNA polymerase for detecting EGFR gene mutation.

Another object of the present invention is to provide a primer set for detecting EGFR gene mutations.

Another object of the present invention is to provide a probe for detecting an EGFR gene mutation.

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

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-described problem, the present invention is a 507th amino acid residue glutamic acid (E) in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine (K), the 536th amino acid residue arginine (R) is lysine (K) Provided is a DNA polymerase for detecting EGFR gene mutations, including Taq polymerase substituted with valine (V), wherein arginine (R), the 660th amino acid residue, is substituted with

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

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

According to one 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. Fluorophores 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, 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 Taq polymerase in which the amino acid residue arginine (R) is substituted with valine (V); And / or at least one primer selected from the group consisting of SEQ ID NO: 3 to 54; provides a kit for detecting EGFR gene mutation.

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

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 to 7 mM (NH ₄ ) ₂ SO ₄ ; And 5 to 50 mM of TMAC (Tetra methyl ammonium chloride), and may further include a PCR buffer composition having a final pH of 8.0 to 9.5.

The present invention also provides a method for detecting an EGFR gene mutation comprising the following steps: (a) extracting a nucleic acid from an isolated biological sample; (b) processing the kit of the present invention on the extracted nucleic acid to perform PCR (polymerase chain reaction); And (c) confirming the amplification result by the PCR with fluorescence.

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 amplification result by the PCR by measuring the Ct (cycle threshold) value.

According to a preferred embodiment of the present invention, the EGFR gene mutation includes one or more selected from the group consisting of deletion, substitution and insertion mutations in 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 719th amino acid glycine substitution in exon 18 of EGFR, a deletion in exon 19, a 768th amino acid in exon 20. Serine, 790th amino acid threonine, 797th amino acid cysteine substitution, exon 20 insertion, exon 21 858 amino acid leucine and 861 amino acid leucine substitution. It may include the above.

According to another preferred embodiment of the present invention, the EGFR gene mutation detection method may be for predicting responsiveness to drugs of lung cancer patients.

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 osimitinib.

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

The kit of the present invention shows high detection sensitivity (up to 0.01%, 3 mutant copies in 30,000 wild-type copies), high specificity and reproducibility, and is applicable to both liquid biopsy and tissue biopsy. In addition, by simultaneously detecting 44 mutations within exons 18 to 21 of the EGFR gene, it is possible to accurately predict drug reactivity to lung cancer patients against tyrosine kinase inhibitors.

Figure 1 shows the production process of Taq DNA polymerase containing R536K, R660V and R536K/R660V mutations respectively, (a) schematically shows fragment PCR and overlap PCR, and (b) is amplified in fragment PCR The result of confirming the product by electrophoresis, and (c) shows the result of confirming the amplified product by electrophoresis by amplifying the entire length by overlap PCR.
FIG. 2 shows the results of confirming the overlap PCR product of FIG. 1 (c) purified by digestion with the restriction enzyme EcoRI/XbaI and then the SAP-treated pUC19 vector for gel extraction by electrophoresis.
Figure 3 is a schematic diagram showing fragment PCR and overlap PCR during the preparation of Taq DNA polymerase containing E507K, E507K/R536K, E507K/R660V and E507K/R536K/R660V mutations, respectively.
FIG. 4 shows the results of confirming the overlap PCR product of FIG. 3 purified by electrophoresis after digestion with the restriction enzyme EcoRI/XbaI and then treatment with SAP for gel extraction.
5A to 5L show the results of detecting mutations with AS-qPCR in the Ex19del C1 to Ex19del C12 mutant plasmid (3, 10 ² and 10 ⁴ copies) template.
6A to 6L show the results of detecting mutations with AS-qPCR in the Ex19del C13 to Ex19del C24 mutant plasmid (3, 10 ² and 10 ⁴ copies) template.
7A to 7G show the results of detecting mutations with AS-qPCR in the Ex19del C25 to Ex19del C31 mutant plasmid (3, 10 ² and 10 ⁴ copies) template, and 7h is S768I mutant plasmid (3, 10 ² and 10 ⁴ Copy) shows the result of detecting the mutation with AS-qPCR in the template, and 7i shows the result of detecting the mutation with AS-qPCR in WT.
8 shows the results of detecting mutations with AS-qPCR in the T790M mutant plasmid (0, 3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template.
Figure 9a shows the results of detecting the mutation in the G719S mutant plasmid (3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template with AS-qPCR.
Figure 9b shows the results of detecting the mutation in the G719C mutant plasmid (3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template with AS-qPCR.
Figure 9c shows the results of detecting the mutation in the G719A mutant plasmid (3, 10 ¹ , 30, 10 ² and 10 ⁴ copies) template with AS-qPCR.
Figure 9d shows the results of detecting the mutation in WT AS-qPCR.
9E shows the results of detecting mutations with AS-qPCR in the L861Q mutant plasmid (0, 3, 10 ² and 10 ⁴ copies) template.
10A to 10E show the results of detecting mutations with AS-qPCR in the Ex20Ins C1 to Ex20Ins C5 mutant plasmid (3, 10 ² and 10 ⁴ copies) template, and 10f is the result of detecting mutations from WT to AS-qPCR. 10g shows the results of detecting mutations with AS-qPCR in the L858R mutant plasmid (3, 10 ² and 10 ⁴ copies) template, and 10h shows the results of detecting mutations in WT with AS-qPCR. will be.

As described above, there is a continuous need to develop a kit for more accurately predicting drug reactivity of lung cancer patients by improving the sensitivity of detecting EGFR mutations. Thus, the present inventors have provided an optimized kit comprising a DNA polymerase capable of detecting 44 somatic mutations in exons 18, 19, 20 and 21 of the EGFR gene with high sensitivity and a reaction buffer to increase its activity. The solution to the above problem was sought. The kit of the present invention exhibits high detection sensitivity, high specificity and reproducibility, and is applicable to both liquid biopsy and tissue biopsy. In addition, it features a semi-quantitative analysis, and analysis is possible on all qPCR devices with 4 channels.

Hereinafter, terms used in the present application 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 analogue of these groups. Exemplary side chains include, for example, thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynyl, ether, Borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thio acid, hydroxylamine, or any combination of these groups.

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

The term “thermal stable polymerase” (referring to a heat stable enzyme) is heat resistant, retains sufficient activity to achieve subsequent polynucleotide elongation reactions and is treated with elevated temperature for the time required to achieve denaturation of the double-stranded nucleic acid Does not irreversibly denature (deactivate) when As used herein, it is suitable for use at temperatures cycling reactions such as PCR. Irreversible denaturation herein refers to permanent and complete loss of enzyme activity. For thermostable polymerases, enzymatic activity refers to catalyzing a combination of nucleotides in a suitable way to form a polynucleotide extension product complementary to the template nucleic acid strand. Thermostable DNA polymerases derived from thermophilic bacteria include, for example: Thermomoto maritima, thermos aquaticus, thermos thermophilus, thermos flavus, thermomod philipformis, thermos species DNA polymerase derived from Sps17, Thermos species Z05, Thermos Caldophyllus, Bacillus caldotenax, Thermomoto Neopolitana, and Thermosippo africanus.

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

The term “nucleotide” is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that exists in the form of a single strand or a double strand, and is not specifically mentioned otherwise. Unless it can contain analogs of natural nucleotides.

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

The term “primer” refers to a polynucleotide that can serve as a starting point for nucleic acid synthesis in the template-direction when placed under conditions where polynucleotide elongation is initiated. Primers can also be used in a variety of other oligonucleotide-mediated synthesis processes, including as initiators of de novo RNA synthesis and in vitro transcription-related processes. Primers are typically single-stranded oligonucleotides (eg, oligodeoxyribonucleotides). The appropriate length of the primer will typically depend on the intended use in the range of 6 to 40 nucleotides, more typically in the range of 15 to 35 nucleotides. Short primer molecules generally require a lower temperature to form a sufficiently stable hybridization complex 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 for elongation of the primer. In certain embodiments, the term “primer pair” includes a 5′-sense primer that hybridizes complementarily to the 5′-end of the amplified nucleic acid sequence, and a 3′-antisense primer that hybridizes to the 3′ end of the amplified sequence. Means a set of primers comprising Primers can be labeled, if necessary, by incorporating a label that can be detected by spectroscopic, photochemical, biochemical, immunochemical or chemical means. For example, useful labels include: ³² P, fluorescent dyes, electron-dense reagents, enzymes (usually used in ELISA assays), biotin, or haptens and proteins in which antiserum or monoclonal antibodies can be used. .

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

The terms "FRET" or "fluorescence resonance energy transfer" or "poster resonance energy transfer" refers to the transfer of energy between two or more chromophores, donor chromophores and receptor chromophores (referred to as quenchers). The donor typically transfers energy to the receptor when the donor is excited by emitting light of a suitable wavelength. Receptors typically re-emit energy transferred in the form of light emitted at different wavelengths. When the receptor is a “cancer” matting agent, it disperses the energy transferred in a form other than light. Whether a particular fluorescent substance acts as a donor or a receptor depends on the properties of other members of the FRET pair. Commonly used donor-receptor pairs include FAM-TAMRA pairs. Commonly used matting agents are DABCYL and TAMRA. Commonly used cancer matting agents include: BlackHole 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 term “conventional” or “natural” when referring to a nucleic acid base, nucleoside triphosphate, or nucleotide, refers to naturally occurring polynucleotides described (ie, 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, nucleotide, or nucleotide, is a conventional base, nucleotide, or modification, derivative, or nucleotide that occurs naturally in a particular polynucleotide, or Analogs. Certain unusual nucleotides are modified at the 2'position of the ribose sugar compared to conventional dNTP. Thus, even though naturally occurring nucleotides for RNA are ribonucleotides (i.e., ATP, GTP, CTP, UTP, collective rNTP), since nucleotides have hydroxyl groups at the 2'position of the sugar, this is compared to the absence of dNTP, As used herein, ribonucleotides are unusual nucleotides as substrates for DNA polymerases. As used herein, unusual nucleotides include, but are not limited to, compounds used as terminators for nucleic acid sequencing. Exemplary terminator compounds include, but are not limited to, compounds having a 2',3'- dideoxy structure, referred to as dideoxynucleoside triphosphate. Dideoxynucleoside triphosphate ddATP, ddTTP, ddCTP and ddGTP are collectively referred to as ddNTP. Additional examples of terminator compounds include 2'-PO ₄ analogues of ribonucleotides. Other unusual nucleotides are phosphorothioate dNTP ([[α]-S]dNTP), 5'-[α]-borano-dNTP, [α]-methyl-phosphonate dNTP, and ribonucleosides Triphosphate (rNTP). Uncommon bases include radioactive isotopes such as ³² P, ³³ P, or ³⁵ S; Fluorescent labels; A label for chemiluminescence; Bioluminescent markers; Hapten labels such as biotin; Or it can be labeled with an enzyme label such as streptavidin or avidin. Fluorescent labels can include negatively charged dyes, such as the dyes of the fluorescein family, or neutrally charged dyes, such as the dyes of the rhodamine family, or positively charged dyes, such as the dyes of the cyanine family. Dyes of the fluorescein family include, for example, FAM, HEX, TET, JOE, NAN and ZOE. Rhodamine family dyes include Texas Red, ROX, R110, R6G, and TAMRA. Various dyes or nucleotides labeled FAM, HEX, TET, JOE, NAN, ZOE, ROX, R110, R6G, Texas Red and TAMRA are Perkin-Elmer (Boston, MA), Applied Biosystems (Foster City, CA), or Invitrogen /Molecular Probes (Eugene, OR). The cyanine family dyes include Cy2, Cy3, Cy5, and Cy7, and are marketed by GE Healthcare UK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

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 replaced with lysine (K), and the 660th amino acid residue. Provided is a DNA polymerase for detecting EGFR gene mutations, including Taq polymerase in which phosphorus arginine (R) is substituted with valine (V).

The "Taq polymerase" was a thermophilic DNA polymerase named after the thermophilic bacterium Thermus aquaticus and was first isolated from the bacteria. Thermos Aquaticus is a bacterium inhabiting hot springs and hot water jets, and Taq polymerase has been identified as an enzyme capable of withstanding the protein denaturation conditions (high temperature) required in PCR. The optimum activity temperature of Taq polymerase is 75-80 ℃, has a half-life of 9 hours at 22.5 hours at 92.5 ℃, 40 minutes at 95 ℃, 9 minutes at 97.5 ℃, and replicates 1000 base pair DNA within 10 seconds at 72 ℃ Can. It lacks 3'→5' nucleic acid endonuclease correcting activity, and the error rate is measured in about 1 out of 9,000 nucleotides. For example, using the heat-resistant Taq, PCR can be performed at high temperatures (above 60°C). The amino acid sequence shown in SEQ ID NO: 1 for Taq polymerase is used as a reference sequence.

In the present invention, in the amino acid sequence of SEQ ID NO: 1, the 507th amino acid residue is substituted with glutamic acid (E) to lysine (K), the 536th amino acid residue is replaced with arginine (R) to lysine (K), and the 660th amino acid Taq polymerase in which the residue is substituted with valine (V) in arginine (R) was designated as "E507K/R536K/R660V", and its amino acid sequence and nucleotide sequence are shown in SEQ ID NO: 2 and SEQ ID NO: 72, respectively.

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

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

Preferably, the polymerase according to the present invention is particularly excellent in EGFR mutation detection sensitivity when used with the primer sequences of Tables 16 to 19.

The present invention also provides a kit for detecting EGFR gene mutations, which includes the DNA polymerase and/or primer set described above.

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

The kit of the present invention may be a PCR kit, and may contain any reagents or other elements recognized by those skilled in the art as being used in the primer extension process.

For example, the PCR kit of the present invention includes (a) nucleoside triphosphate; (b) a reagent for quantification that binds double-stranded DNA; (c) polymerase blocking antibodies; (d) one or more control values or control sequences; And (e) one or more templates; may further include one or more selected from the group consisting of.

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 to 7 mM (NH ₄ ) ₂ SO ₄ ; And 5 to 50 mM of TMAC (Tetra methyl ammonium chloride), and may further include a PCR buffer composition having a final pH of 8.0 to 9.5.

More preferably, the kit comprises 40 to 90 mM KCl; 1 to 5 mM (NH ₄ ) ₂ SO ₄ ; And 10 to 40 mM of TMAC (Tetra methyl ammonium chloride), and may further include a PCR buffer composition having a final pH of 8.0 to 9.0.

The kit of the present invention may further include TrisCl (pH 8.0 to 9.5), MaCl ₂ , Tween 20, and Bovine serum albumin (BSA) in addition to KCl, (NH ₄ ) ₂ SO ₄ , and TMAC, and these components The concentration of can be used by a person skilled in the art to adjust to an appropriate range.

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

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

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 a sample containing a large amount of normal wild type gDNA, allele-specific TaqMan ^® qPCR to inhibit non-specific amplification from wild type alleles. Combination with gene-specific MGB blockers can produce specificity superior to traditional allele-specific PCR.

The “Droplet digital PCR” is a system for counting target DNA by splitting and amplifying a PCR reaction of 20 μl into 20,000 droplets, and depending on whether or not the target DNA is amplified in the droplet, positive droplets (1) It is counted as a digital signal with a negative drop (0), counts the copy number of the target DNA through Poisson distribution, and finally, the result can be confirmed by the number of copies per µl of the sample. Rare mutation detection, very small amount of gene It can be used when amplification, mutation type, etc. are to be simultaneously confirmed.

The “mass array” is a multiplexing analysis method applicable to various genomic studies such as genotyping using a MALDI-TOF mass spectrometer, and rapidly analyzes multiple samples and targets at a low cost. It can be used when you want to do it, or if you want to do customized analysis only for a specific target.

The EGFR gene mutation detection kit of the present invention may further include a probe, a fluorophore, and/or a quencher. The fluorophore may be VIC, HEX, JOE, FAM, CAL Flour Orange 560, Quasar 670, CY5 EverGreen dye, etc., but is not limited thereto. The probe sequence, the type of the fluorophore and the quencher may be the same as in Table 18 of Example 5, but is not limited thereto.

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

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

Configuration main ingredient amount EGFR Master Mixture 1 Primers/probes for buffers, EGFR targets and ICs containing dNTPs 240 μL/tube Х1 EGFR Master Mixture 2 Primers/probes for buffers, EGFR targets and ICs containing dNTPs 240 μL/tube Х1 EGFR Master Mixture 3 Primers/probes for buffers, EGFR targets and ICs containing dNTPs 240 μL/tube Х1 EGFR Master Mixture 4 Primers/probes for buffers, EGFR targets and ICs containing dNTPs 240 μL/tube Х1 ADPS ^TM Smart DNA Polymerase ADPS ^TM Smart DNA Polymerase 60 μL/tube Х1 EGFR positive control Recombinant plasmid blend containing each EGFR mutation 160 μL/tube Х1 Nuclease-free distilled water PCR grade distilled water 1.0 mL/tube Х1

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

Final concentration MMX 1-4 ADPS buffer TrisCl (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

Table 3 shows the detection information of EGFR Master Mix 1-4.

reagent Fluorescent 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

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

**IC: internal control for PCR

The present invention also provides a method for detecting an EGFR gene mutation comprising the following steps:

(A) extracting the nucleic acid from the isolated biological sample;

(B) processing the kit of the present invention to the extracted nucleic acid to perform a polymerase chain reaction; And

(c) analyzing the size or base 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 amplification result by the PCR by measuring a Ct (cycle threshold) value.

The cycle threshold (Ct) value means the number of cycles in which the fluorescence generated in the reaction exceeds a threshold, which is inversely proportional to the logarithm of the initial copy number. Therefore, the Ct value assigned to a particular well reflects the number of cycles in which a sufficient number of amplicons have accumulated in the reaction. The Ct value is the cycle in which the increase in ΔRn was first detected. Rn means the magnitude of the fluorescence signal generated during PCR at each time point, and ΔRn means the fluorescence emission intensity (standardized reporter signal) of the reporter dye divided by the fluorescence emission intensity of the reference dye. The Ct value is also referred to as a crossing point (Cp) in LightCycler. The Ct value represents the point at which the system begins to detect an increase in the fluorescence signal associated with the 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 phase represents the amplification efficiency (Eff) (http://www.appliedbiosystems.co.kr/).

On the other hand, TaqMan probes typically include a 5'-terminal fluorophore and a 3'-terminal quencher (e.g., TAMRA or non-fluorescent quencher (NFQ)) primers (e.g., 20- 30 nucleotides). Excited phosphors transfer energy to nearby quenchers rather than fluorescence (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 inner part of the PCR product.

The TaqMan probe specifically hybridizes to the template DNA in the annealing step, but fluorescence is suppressed by quenching on the probe. During the extension reaction, the TaqMan probe hybridized to the template is decomposed by the 5'to 3'nuclease activity of the Taq DNA polymerase, and the fluorescent dye is released from the probe. At this time, the 5'-end of the TaqMan probe should be located downstream of the 3'-end of the extension primer. That is, when the 3'-end of the extension primer is extended by a template-dependent nucleic acid polymerase, the 5'to 3'nuclease activity of this polymerase cuts the 5'-end of the TaqMan probe, thereby Fluorescence signal is generated.

The reporter molecule and quencher molecule bound to the TaqMan probe include fluorescent and non-fluorescent materials. Fluorescent reporter molecules and quencher molecules that can be used in the present invention can use any of those known in the art, and examples thereof are as follows (numbers in parentheses are the maximum emission wavelength in nanometers): Cy2 ^TM 506, YOPRO ^TM -1 (509), YOYO ^TM -1 (509), Calcein (517), FITC (518), FluorX ^TM (519), Alexa ^TM (520), Rhodamine 110 (520), 5-FAM (522), Oregon Green ^TM 500 (522), Oregon Green ^TM 488 (524), RiboGreen ^TM (525), Rhodamine Green ^TM (527), Rhodamine 123 (529), Magnesium Green ^TM (531), Calcium Green ^TM (533), TO-PRO ^TM -1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY 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 ^TM (590), Cy3.5 ^TM (596), ROX (608), Calcium Crimson ^TM (615), Alexa ^TM 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 wavelength of light emitted in nanometers.

Suitable reporter-quencher pairs have been described in many documents: 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, COLOUR 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 in the reporter molecule and the quencher molecule bound to the TaqMan probe may include a minor groove binding (MGB) moiety. The term "TaqMan MGB-conjugate probe" herein refers to a TaqMan probe conjugated with MGB at the 3'-end of the probe.

MGB is a substance that binds to the minor groove of DNA with high affinity, such as netropsin, distamicin, lexitropsin, mitramycin, chromomycin A3, and olibo. Olivomycin, anthramycin, sibiromycin, pentamidine, stilbamidine, berenil, CC-1065, Hoechst 33258, DAPI (4-6- diamidino-2-phenylindole), CDPI dimers, trimers, tetramers and pentamers, MPC (N-methylpyrrole-4-carbox-2-amide) and dimers thereof, trimers, tetramers and pentamers It does not work.

Conjugation of the probe and MGB significantly increases the stability of the hybrid formed between the probe and its target. More specifically, increased stability (ie, increased degree of hybridization) results in increased melting temperature (Tm) of the hybrid duplex formed by MGB-conjugated probes compared to normal probes. Thus, MGB stabilizes the van der Waals force, thereby increasing the melting temperature (Tm) of the MGB-conjugate probe without increasing the probe length, resulting in shorter probes (e.g. no more than 21 nucleotides) in Taqman real-time PCR under more stringent conditions. Enables the use of.

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

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

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

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.

Exxon 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_2308ins9GCCAGCGTG 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 included in an animal, preferably a vertebrate, more preferably a human subject. For example, the biological sample in step (a) may be sputum, blood, saliva, or urine, and the nucleic acid in step (a) is a formalin-fixed paraffin embedded sample or a liquid biopsy of a tissue biopsy. It can be extracted from.

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

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

“Dye binding to double-stranded DNA” or “double-strand specific dye” can be used when it has a higher fluorescence when bound to double-stranded DNA than to the unbound state. Examples of such dyes are SOYTO-9, SOYTO-13, SOYTO-16, SOYTO-60, SOYTO-64, SYTO-82, Etidium Bromide (EtBr), SYTOX Orange, TO-PRO-1, SYBR Green I, TO-PRO-3 or EvaGreen. These dyes, except EtBr and EvaGreen (Quiagen), have been tested in real-time applications.

The EGFR gene mutation detection method of the present invention includes real-time PCR (RT-PCR) or quantitative PCR (qPCR), analysis on agarose gel after standard PCR, gene mutation specific amplification or allele-specific amplification through real-time PCR, Tetra-primer amplification-refractory mutant systems can be performed by PCR or isothermal amplification.

The "standard PCR" is a technique for amplifying single or several copies of DNA or cDNA known to those skilled in the art. Almost all PCR uses thermostable DNA polymerases such as Taq polymerase or Klen Taq. DNA polymerases use single-stranded DNA as a template and enzymatically assemble new DNA strands from nucleotides by using oligonucleotides (primers). The amplicon generated by PCR can be analyzed, for example, on an agarose gel.

The "real-time PCR" can monitor the process in real time when performing PCR. Therefore, data is collected throughout the PCR process, not when the PCR is finished. In real-time PCR, the reaction is characterized by a time point during the cycle when amplification is first detected, rather than the target amount accumulated after a fixed number of cycles. Two methods, mainly dye-based detection and probe-based detection, are used to perform quantitative PCR.

The “allele specific amplification (ASA)” is an amplification technique designed to distinguish PCR primers from different templates with a single nucleotide residue.

The "gene mutation specific amplification or allele-specific amplification through real-time PCR" detects gene mutation or SNP in a very efficient manner. Unlike most other methods for detecting gene mutations or SNPs, preliminary amplification of the target genetic material is not required. ASA combines amplification and detection in a single reaction based on the distinction of matched and mismatched primer/target sequence complexes. The increase in DNA amplified during the reaction can be monitored in real time with an increase in the fluorescence signal caused by dyes such as SYBR Green I, which emit light upon binding to double-stranded DNA. Gene mutation-specific amplification or allele-specific amplification through real-time PCR shows delay or absence of a fluorescent signal for mismatched cases. In detecting genetic variation or SNP, it provides information on the presence or absence of genetic variation or SNP.

The "tetra-primer amplification-refractory mutation system PCR" amplifies both wild type and mutant alleles with control fragments in a single tube PCR reaction. Non-allele specific control amplicons are amplified by two common (outer) primers flanking the mutation region. The two allele specific (inner) primers are designed in the opposite direction to the common primer, and can be amplified both wild-type and mutant amplicons simultaneously with the common primer. Consequently, the two allele-specific amplicons have different lengths and can be easily separated by standard gel electrophoresis because the mutations are located asymmetrically with respect to the common (outer) primer. The control amplicon provides internal control for false negatives as well as amplification failures, and at least one of the two allele-specific amplicons is always present in the tetra-primer amplification-refractory mutation system PCR.

The "isothermal amplification" does not depend on the thermocycler, and preferably means that the amplification of the nucleic acid takes place at a lower temperature without the need to change the temperature during amplification. The temperature used in isothermal amplification can be between room temperature (22-24 °C) to about 65 °C, or at room temperature of about 60-65 °C, 45-50 °C, 37-42 °C or 22-24 °C. The products of the isothermal amplification results are gel electrophoresis, ELISA, ELOSA (Enzyme linked oligosorbent assay), real-time PCR, ECL (improved chemiluminescence), RNA, DNA, and chip-based capillary electrophoresis devices that analyze protein or turbidity It can be detected with an analyzer (bioanalyzer).

When the EGFR gene mutation detection method of the present invention is performed with qPCR, for example, it may be performed under the conditions of Tables 22 to 24 below.

The DNA polymerase, primer set, probe, and/or kit for detecting EGFR gene mutations of the present invention can be used for predicting reactivity to drugs of lung cancer patients.

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

The present invention can also provide a method for predicting reactivity to drugs of lung cancer patients 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 to diagnose cancer early and establish a treatment strategy for each patient by detecting a known EGFR mutation of Table 4 above, thereby more effectively treating patients Contribute.

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

In the present invention, the term "prognosis" refers to the act of predicting the course and outcome of a disease in advance. More specifically, the prognosis prediction can be interpreted to mean any action that predicts the course of the disease after treatment by comprehensively taking into account the patient's physiological or environmental conditions. Can be.

The prognosis prediction may be interpreted as an action of predicting the disease-free survival rate or survival rate of a patient by predicting the progress and complete cure of the disease after treatment of a specific disease. For example, predicting that "the prognosis is good" means that the patient has a high probability of being treated without disease or having a high survival rate after treatment of the disease, and predicting that the "prognosis is bad" is a disease After treatment, the patient's disease-free survival rate or survival rate is low, indicating that the disease is likely to recur or die from the disease.

The term "no disease survival rate" of the present invention means the possibility that a patient can survive without recurrence of the disease after treatment of the specific disease.

The term "survival rate" of the present invention means 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 the purpose of predicting the reactivity to a drug in a lung cancer patient, the drug may be a tyrosine kinase inhibitor, and the type of the tyrosine kinase inhibitor is, for example , Gefitinib, erlotinib or osimmertinib.

Hereinafter, the present invention will be described in more detail by examples. These examples are only for explaining the present invention in more detail, whereby it will be apparent to those skilled 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 embodiment, Taq DNA polymerase in which the 536th amino acid residue in the amino acid sequence of SEQ ID NO: 1 is substituted with arginine to lysine (hereinafter referred to as "R536K"), Taq DNA polymerase in which the 660th amino acid residue is substituted with valine in arginine. (Hereinafter referred to as “R660V”) and Taq DNA polymerase (hereinafter referred to as “R536K/R660V”) in which the 536th amino acid residue is substituted from arginine to lysine and the 660th amino acid residue is substituted from arginine to valine is prepared as follows. Did.

First, Taq DNA polymerase fragments (F1 to F5) were amplified by PCR using the mutant specific primers listed in Table 5, as shown in Figure 1(a). The reaction conditions are shown in Table 6.

primer Sequence (5'→3') Sequence number 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 (solvent) 2.5 μl dNTP (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 the electrophoresis, as shown in Fig. 1 (b), it was confirmed that the band for each fragment was confirmed and the desired fragment was amplified.

1-2. Overlap PCR

Each fragment amplified in 1-1 was used as a template to amplify the full length using primers at both ends (Eco-F and Xba-R primers). The reaction conditions are shown in Tables 7 and 8.

R660V or R536K 10ⅹpfu buffer (solvent) 5 μl 5ⅹ Enhancer (Solgent) 10 μl dNTP (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 Short 2 (or Short 4) 1 μl Pfu polymerase 1 μl 40 cycles (Ta=62℃) 50 μl

R536K/R660V 10ⅹpfu buffer (solvent) 5 μl 5ⅹ Enhancer (Solgent) 10 μl dNTP (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 Short 2 1 μl Short 3 1 μl Short 5 1 μl Pfu polymerase 1 μl 40 cycles (Ta=62℃) 50 μl

As a result, it was confirmed that Taq polymerases of "R536K", "R660V" and "R536K/R660V" were amplified as shown in FIG. 1(c).

1-3. Ligation

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

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 inserts, the overlap PCR product of Example 1-2 was purified, digested with restriction enzyme EcoRI/XbaI at 37°C for 3 hours under the conditions of Table 11, and then gel extracted with the prepared vector (FIG. 2 ). ).

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 of Table 12, E. coli DH5α was transformed to select from the medium containing ampicillin. The plasmid prepared from the obtained colonies was sequenced to obtain Taq DNA polymerase mutants ("R536K", "R660V" and "R536K/R660V") into which the desired mutation was introduced.

Vector sole Vector + insert 10ⅹ ligase buffer (solvent) 1 μl 1 μl vector 1 μl 1 μl insert - 3 μl Distilled water 7 μl 4 μl T4 DNA ligase (solvent) 1 μl 1 μl 10 μl 10 μl

Introduction of E507K mutation

2-1. Fragment PCR

Taq polymerase activity of "R536K", "R660V" and "R536K/R660V" prepared in Example 1 was tested to confirm that the activity was poor (data not shown), R536K, R660V, R536K/R660V, respectively In addition, an E507K mutation (substituting the 507th amino acid residue in the amino acid sequence of SEQ ID NO: 1 with glutamic acid for lysine) was introduced, and an E507K mutation was also introduced into the wild-type Taq DNA polymerase (WT) for use as a control. The production method of Taq DNA polymerase introduced with the E507K mutation is the same as in Example 1.

Taq DNA polymerase fragments (F6 to F7) were amplified by PCR using the mutant specific primers listed in Table 13. The reaction conditions are shown in Table 14.

primer Sequence (5'→3') Sequence number 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 (solvent) 2.5 μl dNTP (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

Each fragment amplified in 2-1 was used as a template, and the full length was amplified using primers (Eco-F and Xba-R primers) at both ends. The reaction conditions are shown in Table 15.

10ⅹpfu buffer (solvent) 5 μl 5ⅹ Enhancer (Solgent) 10 μl dNTP (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 Short 6 1 μl Short 7 1 μl Pfu polymerase 1 μl 40 cycles (Ta=62℃) 50 μl

2-3. Ligation

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

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

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

Production of primer sets

Primer size and Tm value using the OligoAnalyzer Tool (https://sg.idtdna.com/calc/analyzer) program to design primers that generate PCR products for mutations in the EGFR gene but not for the wild-type EGFR gene After designing primers as shown in Tables 16 to 19 after excluding the possibility of formation of a secondary structure by checking whether the primer GC content, whether there is a self-complementary sequence in the primer, etc. Did.

Mutant group primer Sequence (5'-3') Sequence number 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') Sequence number 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') Sequence number 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') Sequence number 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

Sample preparation

Mutant plasmid preparation

Using the gDNA of the HEK293T cell line, the wild-type DNA of EGFR, a primer set capable of amplifying the periphery of the target mutation in Table 4 was applied to prepare wild-type clones at exon 18, 19, 20, and 21 sites. Mutagenesis was performed on 44 target mutations using the prepared wild-type DNA, and transformed into E.Coli DH5α cells to obtain each mutant clone. Wild type clones and mutant clones were identified by direct sequencing. Wild-type DNA and mutant DNA for each exon extracted through clones were used as standards to evaluate the performance of the EGFR mutation detection kit.

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 described in Table 4 per 30,000 copies of HEK293T cell genomic DNA. The group was used as a control.

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

EGFR mutation detection

Using the Taq polymerase (SEQ ID NO: 2) and the primer set of Example 3, each containing the "E507K/R536K/R660V" mutation obtained in Example 2 above, the EGFR gene mutations of Table 4 from each group of Table 20 were obtained. Detected. Table 21 shows mutant 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_2256del18 (6255), 2237_2254del18 (12367), 2240_2254del15 (370) 2239_2248TTAAGAGAAG>C (12382), 2239_2251>C (12383), 2237_2255>T (12384), 2235_2255>AAT (12385), 2237_2252>T (12386), 2239_2258>CA (12387), 2239_2256>CAA (12403), 2237_2253 >TTGCT (12416), 2238_2252>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, and the probes were double-labeled as 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') Sequence number 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 time 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 each prepared in separate sterile centrifuge tubes, and the reaction Master Mixture was vortexed for 3 seconds to thoroughly mix and centrifuge briefly. Two PCR tubes were prepared for each sample as follows: 10.0 μL of EGFR reaction mixture was divided for each PCR tube, and 5.0 μL of each sample DNA was added to each sample tube, and then the PCR tube was covered. Nuclease-free distilled water was added to all PCR tubes to 20.0 μL, and PCR strips were briefly centrifuged to collect all liquids at the bottom of each PCR tube. The PCR strip tube was placed in a real-time PCR (real-time PCR) instrument, and PCR was performed after setting the PCR protocol using the cycling parameters in Table 24. After the PCR was completed, FAM/CAL Fluor Orange 560/Quasar 670 Ct values of each sample were recorded to analyze the data, and ΔCt values per 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 (each MMX CFO560)-positive control Ct value (CFO560 of each MMX)

The results for each tube were analyzed according to the cutoff ΔCt values in Tables 25 and 26. If the ΔCt value is <cutoff ΔCt value, the sample is judged to be 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 to be negative or below the detection limit (LoD).

Result determination EGFR MMXs decision Internal control Ct value 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, as shown in FIGS. 5A to 5L, 6A to 6L, 7A to 7I, 8, 9A to 9E, and 10A to 10H, fluorescence signals were not detected in WT, but the EGFR gene mutation of Table 4 Fluorescence signal was detected in the sample containing, and it was confirmed that EGFR gene mutation can be detected with a high sensitivity of up to 0.01% (3 mutant copies in 30,000 wild-type copies) as shown in Table 27.

Exxon 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_2308ins9GCCAGCGTG 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 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 Glu 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> Artificial <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> Artificial <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 acccccacgt 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 ggggtacctc 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 gactaccggg ccctgaccgg ggacgagtcc 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 gaggccgagg 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 agggcaccgc cgccgacctc 2280 atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340 cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400 cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460 gtggggatag gggaggactg gctctccgcc aaggagtga 2499

Claims (19)

In the amino acid sequence of SEQ ID NO: 1, the 507th amino acid residue glutamic acid (E) is replaced with lysine (K), the 536th amino acid residue arginine (R) is replaced with lysine (K), and the 660th amino acid residue arginine ( R) DNA polymerase for detecting EGFR gene mutations, including Taq polymerase substituted with valine (V). A primer set for EGFR gene mutation detection, comprising at least one primer selected from the group consisting of SEQ ID NOs: 3 to 54. A probe for detecting an EGFR gene mutation, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 55 to 63. The method of claim 3, wherein the nucleotide sequence of SEQ ID NO: 55 to 63, each of the 5'- terminal FAM, CAL Fluor Orange 560, VIC, HEX, JOE, Quasar 670 and CY5 is selected from the group consisting of one fluorescent material (fluorophore) is labeled, a probe for detecting the EGFR gene mutation, which is labeled with one quencher selected from the group consisting of BHQ-1 BHQ1 nova and BHQ-2 at the 3'-end. In the amino acid sequence of SEQ ID NO: 1, the 507th amino acid residue glutamic acid (E) is replaced with lysine (K), the 536th amino acid residue arginine (R) is replaced with lysine (K), and the 660th amino acid residue arginine ( R) Taq polymerase substituted with valine (V); And/or
At least one primer selected from the group consisting of SEQ ID NOs: 3 to 54;
Containing, EGFR gene mutation detection kit. The kit for detecting an EGFR gene mutation according to claim 5, further comprising a probe comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 55 to 63. The method of claim 6, wherein the nucleotide sequence of SEQ ID NO: 55 to 63 is a fluorescent substance of one selected from the group consisting of FAM, CAL Fluor Orange 560, VIC, HEX, JOE, Quasar 670 and CY5 at the 5'-terminal, respectively. A kit for detecting EGFR gene mutation, wherein (fluorophore) is labeled, and one quencher selected from the group consisting of BHQ-1 BHQ1 nova and BHQ-2 is labeled at the 3'-end. The method of claim 5,
25 to 100 mM KCl; And
1 to 7 mM (NH ₄ ) ₂ SO ₄ ;
And, the final pH is 8.0 to 9.5, further comprising a PCR buffer composition, EGFR gene mutation detection kit. The method of claim 5,
25 to 100 mM KCl;
1 to 7 mM (NH ₄ ) ₂ SO ₄ ; And
A kit for detecting EGFR gene mutations, comprising 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. EGFR gene mutation detection method comprising the following steps:
(A) extracting the nucleic acid from the isolated biological sample;
(b) performing a polymerase chain reaction (PCR) by treating the kit of any one of claims 5 to 9 with the extracted nucleic acid; And
(c) confirming the amplification result by PCR with fluorescence. The method according to claim 10, wherein the PCR is allele-specific PCR or real-time PCR. 11. The method of claim 10, (d) EGFR gene mutation detection method further comprising the step of confirming the amplification result by the PCR by measuring the Ct (cycle threshold) value. The method of claim 10, wherein the EGFR gene mutation is EGFR gene mutation is at least one selected from the group consisting of deletion, substitution and insertion mutations in exons 18, 19, 20 and 21 of the EGFR gene. Including, EGFR gene mutation detection method. 14. The method of claim 13, wherein the EGFR gene mutation is a substitution of the 719th amino acid glycine in exon 18 of EGFR, a deletion in exon 19, the 768th amino acid serine in exon 20, the 790th amino acid. An threonine, a substitution of cysteine at the 797th amino acid, insertion in exon 20, leucine at 858th amino acid in exon 21, and leucine at 861th amino acid; EGFR gene mutation detection method. The method of claim 10, wherein the EGFR gene mutation detection method is for predicting responsiveness to drugs of lung cancer patients. 16. The method of claim 15, wherein the lung cancer is non-small cell lung cancer. 16. 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 osimmertinib. The method of claim 10, wherein the nucleic acid of step (a) is extracted from a formalin-fixed paraffin embedded sample or a liquid biopsy of a tissue biopsy.

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