KR20140033854A - Multiplex pcr primer set for detecting egfr mutant and kit comprising the same - Google Patents

Abstract

The present invention relates to a primer set for multiplex polymerase chain reaction to analyze mutations indicated in exon 18, 19, 20, and 21 of an EGFR gene at once with an aim of predicting responsiveness to drugs of lung cancer patients, an EGFR mutation detection kit comprising the same, and an EGFR mutation detection method.

Description

Multiplex PCR primer set for detecting EGFR mutant and kit comprising the same}

The present invention provides a primer set for multiple polymerase chain reaction for analyzing mutations in exons 18, 19, 20 and 21 of the EGFR gene at one time for the purpose of predicting the reactivity of the drug in lung cancer patients, comprising the same EGFR mutation detection kits, and EGFR mutation detection methods.

Lung cancer is one of the leading causes of death worldwide, accounting for 30% of cancer deaths worldwide. More than 75% of all lung cancers are non-small cell lung cancer (NSCLC), with an average 5-year survival rate of 15%.

Lung cancer is a type of epithelial cell cancer characterized by abnormally accelerated growth of epithelial cells. The epidermal growth factor receptor (EGFR) expressed on the surface of epithelial cells is a cancerous process. It is known to play an important role in. EGFR is a cell membrane glycoprotein belonging to the tyrosine kinase, an epithelial receptor in the human body, and has its own tyrosine kinase activity, which when activated, causes gene expression, cell proliferation, invasion of surrounding tissues, inhibition of cell death and angiogenesis. It causes such effects.

Thus, EGFR has been recognized as a target of lung cancer treatment, and a representative example of cancer therapy targeting EGFR is the development of EGFR tyrosine kinase inhibitor (EGFR-TKI). Currently, gefitinib (Iressa) and erlotinib (Tarceva) have been developed as tyrosine kinase inhibitors useful for the treatment of lung cancer, particularly non-small cell lung cancer.

By the way, the EGFR tyrosine kinase inhibitors are not tolerated or initially responded to many patients in clinical application, and only 10 to 15% of advanced lung cancer patients are known to respond to EGFR kinase inhibitors. Previous studies on this have found that the presence of somatic mutations in the kinase domain of EGFR significantly increases the sensitivity of EGFR kinase inhibitor drugs. The EGFR mutations are mainly observed in women, Asians, nonsmokers, and adenocarcinoma lung cancer patients, and the presence of these mutations is associated with drug reactivity and the patient's prognosis. Mutations of EGFR are mostly observed in exons 18 to exon 21, among which in-frame deletions in exon 19 or L858R point mutations in exon 21 are the most common mutations. Non-small cell lung cancer patients with EGFR mutations were significantly more responsive to EGFR kinase inhibitor drugs than non-small cell lung cancer patients without EGFR mutations, and a significant increase in survival was reported.

As such, the presence of an EGFR mutation acts as a strong predictor of drug sensitivity in response to EGFR kinase inhibitors such as zephytinib or erlotinib, thus requiring an effective and rapid method of detecting EGFR mutations for optimal therapeutic approaches. .

To date, one of the most commonly used methods for the detection of EGFR mutations is direct sequencing, which directly identifies the nucleotide sequences of genes corresponding to exons 18-21 of EGFR. This method has a disadvantage that it takes more time than real-time polymerase chain reaction or DNA chip analysis, but the most accurate analysis is possible in that it can analyze not only a specific base but the entire target site. It has the advantage of being possible.

For direct sequencing, polymerase chain reaction for amplification of specific sequences should be prioritized. The reaction is to amplify hundreds of thousands of times more genes in a short time using primer pairs of bases complementary to the ends of the sequence. However, the exons 18, 19, 20, and 21 of the EGFR gene have a total base length of 17,954 bp, which is impossible to amplify at a time through a single polymerase chain reaction. In addition, the sample used for the EGFR mutation test is fixed with paraffin as the biological tissue of the patient. The DNA extracted from the paraffin is characterized by more fragmentation than the DNA extracted from the blood. When less than 300bp DNA amplification occurs well. Therefore, four single polymerase chain reactions are required to amplify all exons 18, 19, 20, and 21. Since the tissues obtained from lung cancer patients are significantly smaller than the surgical tissues, There is a problem that it is not enough.

Accordingly, Korean Patent Publication No. 10-2011-0098440 discloses a technique for selectively detecting only mutations by inhibiting amplification of wild type using a PNA (Peptide Nucleic Acid) probe that specifically binds to the wild type corresponding to the EGFR mutation position. However, this method has a limitation in that it does not detect all mutations in exons 18, 19, 20, and 21. In particular, the amount of sample is required because it must be reacted with several PNA probes to determine whether the mutation is detected. This costs a lot of disadvantages.

Thus, the present inventors applied multiplex PCR (hereinafter referred to as "multiple PCR" for convenience) to detect EGFR mutations more effectively and quickly in order to assay drug susceptibility of lung cancer. As a result, we have devised a technique for amplifying 20, 21 at once. Multiple PCR is a method of amplifying several genes at the same time. Since the reactions are carried out at the same time in one reaction vessel, it is economical and can greatly reduce time and labor. When designing primers consisting of bases at the start of gene amplification for multiple PCR reactions, each primer should only amplify a specific gene fragment, and each amplification of each gene fragment should be sufficient for the next analysis. Conditions such as no interference between primers should be satisfied.

The inventors have developed a set of primers for multiple PCR that can detect mutations in exons 18, 19, 20 and 21 of the EGFR gene at once, and the detection rate of mutations is significantly increased than previously reported using the primer sets. The present invention was completed by confirming that the analysis sensitivity was improved.

One object of the present invention is to provide a primer set for detecting the EGFR gene mutation for predicting the reactivity to the drug of lung cancer patients.

It is another object of the present invention to provide a kit for detecting EGFR gene mutations comprising the primer set.

Another object of the present invention is to provide a method for detecting multiple PCR-based EGFR gene mutation using the primer set.

The present invention provides multiple polymerase chain reaction based assays capable of analyzing mutations in exons 18, 19, 20 and 21 of the EGFR gene at one time. The present invention enables simultaneous analysis of mutations in exons 18, 19, 20, and 21 of the EGFR gene, and the presence or absence of the EGFR mutation detected by the present invention can serve as an important indicator for the prescription of tyrosine kinase inhibitors in lung cancer patients. Efficient treatment of lung cancer patients can be enabled.

Thus, in one aspect, the present invention relates to a primer set for detecting EGFR gene mutations for predicting responsiveness to drugs of lung cancer patients.

In a preferred embodiment, the present invention

A primer pair consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2,

A primer pair consisting of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4,

A primer pair consisting of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6, and

Comprising a primer pair consisting of the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8,

The present invention relates to a primer set for detecting EGFR gene mutations for predicting responsiveness to drugs of lung cancer patients.

As another aspect, the present invention relates to a kit for detecting EGFR gene mutation for predicting the reactivity to the drug of a lung cancer patient comprising the primer set.

As another aspect, the present invention relates to a multiple PCR-based EGFR gene mutation detection method using the primer set.

In a preferred embodiment, the present invention

(a) extracting nucleic acid from a sample of lung cancer patient,

(b) subjecting the extracted nucleic acid to the primer set of claim 1 to perform multiple PCR, and

(c) analyzing the size or sequence of the amplified product of the multiple PCR result;

A method for detecting EGFR gene mutations for predicting responsiveness to drugs in lung cancer patients.

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

The present invention relates to multiple PCR primers and multiple PCR-based assays using the same for analyzing mutations shown in exons 18, 19, 20, and 21 of the EGFR gene at one time, preferably, EGFR detectable by the present invention. Gene mutations are deletion, substitution or insertion mutations in exons 18, 19, 20 and 21 of the EGFR gene.

More preferably, the mutation at exon 18 includes a mutation (named G719S) in which the guanine of nucleotide 2155 is substituted with adenine (named 2155G> A), where the wild type glycine of codon 719 is substituted with serine.

Mutations in exon 19 also include the result of the deletion of 15mer of nucleotides between nucleotides 2236 and nucleotides 2250 (named 2236_2250del15) and the deleted mutations of glutamic acid of codon 746 to alanine of 750 (named E746_A750del).

The mutation at exon 20 also resulted in the insertion of 15mer of nucleotides between nucleotides 2307 and 2308 (named 2307_2308ins9), resulting in the insertion of alanine-serine-valine (ASV) between valine of codon 769 and aspartic acid of 770. Mutations (named V769_D770insASV).

In addition, mutations in exon 21 include mutations (named L858R) in which the wild type leucine of codon 858 is replaced with arginine as a result of the substitution of thymine at nucleotide 2573 with guanine (named 2573T> G).

As used herein, the term 'primer' refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming complementary templates and base pairs and for template strand copying. By a short nucleic acid sequence that functions as a starting point. Primers can initiate DNA synthesis in the presence of four different nucleoside triphospates (NTPs) and reagents for polymerisation (DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.

As used herein, the term 'EGFR gene mutation detection primer set' means a specific binding to each mutation site of exons 18 to 21 of EGFR in order to detect mutations present in exons 18 to 21 of EGFR, thereby inducing a renal response. Refers to a set of possible primer pairs, preferably each primer pair consists of a sense and antisense nucleic acid sequence having 7 to 50 oligo sequences. Each of these primer pairs produces a PCR product for mutations in the EGFR gene but not for the wild type EGFR gene.

More preferably, the primer set of the present invention comprises a primer pair consisting of SEQ ID NO: 1 and SEQ ID NO: 2 for detecting mutations in exon 18 of EGFR, SEQ ID NO: 3 for detecting mutations in exon 19 of EGFR And a primer pair consisting of a nucleotide sequence of SEQ ID NO: 4, a pair of primers consisting of a base number of SEQ ID NO: 5 and a base sequence of SEQ ID NO: 6 for detecting a mutation of exon 20 of EGFR, and a sequence number 7 for detecting a mutation of exon 21 of EGFR And a primer pair consisting of the nucleotide sequence of SEQ ID NO: 8. More preferably, the primer pairs detect G749S substitution mutations in exon 18 of EGFR, deletion mutations of E746-A750 in exon 19, ASV insertion mutations of V769-D770 of exon 20, and L858R substitution mutations of exon 21, respectively. Primer pairs.

Primer set of the present invention, the primer size, Tm value, GC content of the primer, preventing the formation of the primer (dimer) by the self-complementary sequence in the primer, repeating three or more times of the same base sequence It is carefully designed to maximize the sensitivity and specificity of EGFR mutation detection with due consideration of prohibition.

In addition, the four pairs of primer pairs constituting the primer set of the present invention each amplify only a specific gene fragment, the amplification of each gene fragment is sufficient for the analysis of the next step, without interference between primers in the PCR reaction Each gene region can be amplified simultaneously. In addition, the four pairs of primer pairs form PCR amplification products of different sizes, thereby making it easier to identify the presence or absence of each mutation corresponding to EGFR exons 18 to 21.

Primers of the invention may incorporate additional features that do not change the basic properties. That is, the nucleic acid sequence can be modified using many means known in the art. Examples of such modifications include methylation, capping, substitution of one or more homologs of nucleotides and uncharged linkages, such as phosphonates, phosphoresteres, phosphoramidates or carbamates, or phosphorothioates or phosphorodithioates. Modification of the nucleotides to charged linkers, etc. is possible. In addition, the primer nucleic acid sequence of the present invention can be modified using a label that can provide a detectable signal directly or indirectly.

The term 'EGFR gene mutation detection kit' of the present invention includes a primer set consisting of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 8, the presence or absence of mutations on the exon 18 to 21 of the EGFR gene through multiple PCR By detecting, it refers to a kit that can be used to predict responsiveness to drugs in lung cancer patients.

The kit of the present invention may further include substances required for PCR reaction and sequencing in addition to the primer set. For example, DNA polymerases (eg, thermally stable DNA polymerases obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), dNTP (dATP, It may include a PCR reaction mixture consisting of dCTP, dGTP, dTTP), a reaction buffer, distilled water and the like, and may further include agarose and electrophoretic buffer that can determine whether the PCR product is detected. In addition, the kits of the present invention can be prepared in a number of separate packaging or compartments comprising the reagent components described above.

The term 'EGFR gene mutation detection method' of the present invention means by detecting the presence or absence of mutations on the exons 18-21 of the EGFR gene by a multiplex PCR method using a primer set consisting of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 8 Refers to the method used to predict reactivity to drugs in patients with lung cancer.

Preferably, the EGFR gene mutation detection method of the present invention, (a) extracting a nucleic acid from a sample of lung cancer patient, (b) performing the multiple PCR by processing the primer set of claim 1 to the extracted nucleic acid, and (c) analyzing the size or sequence of the amplification product of the multiple PCR result.

In the step (a), 'lung cancer patient sample' means all biologically-derived samples including genes and / or mitochondria derived from lung cancer patients, and samples of cells, tissues, organs, body fluids, blood, and the like. Can be. In addition, the nucleic acid extracted from a sample of the lung cancer patient, as a nucleic acid containing all or part of the EGFR gene, which is a target gene in which the gene mutation to be detected, may be DNA or RNA, and includes exon 18-21 sites. To about 5 to 1,000,000 bp, preferably about 5 to 100,000 bp, more preferably about 5 to 5000 bp.

In the step (b), multiple PCR refers to a process of amplifying a target nucleic acid by using the nucleic acid extracted above as a template and repeating the denaturation, annealing and synthesis reactions using the primer set of the present invention. Preferably, multiple PCR can be carried out by treating the PCR reaction mixture consisting of the primer set of the present invention, DNA polymerase, dNTP, reaction buffer, distilled water and the like as a template nucleic acid extracted from a lung cancer patient sample. The four pairs of primer pairs constituting the primer set of the present invention have a common PCR condition and can be simultaneously applied to one PCR reaction. Specific PCR conditions include an initial denaturation by heat treatment at 90 to 95 ° C. for 5 to 20 minutes, and a denaturation step at 10 to 2 minutes at 90 to 95 ° C. (denaturation); Annealing at 58 to 60 ° C. for 10 seconds to 2 minutes; PCR amplification may be performed by repeating the synthesis step (extension) for 10 to 40 minutes at 70 to 75 ° C for a total of 10 to 40 times. The reaction temperature and reaction time conditions may be appropriately modified by those skilled in the art. More preferably, after denaturing at 94 ° C. for 15 minutes, 30 seconds at 94 ° C., 60 seconds at 60 ° C., and 10 seconds at 72 ° C. may be repeated 30 times, followed by reaction at 72 ° C. for 10 minutes.

Step (c) is a step of detecting the presence or absence of EGFR mutation by analyzing the multiple PCR results, the mutation can be detected through the size or sequence analysis of the multiple PCR amplification products.

For the analysis of the size of multiple PCR amplification products, any method known in the art can be used. For example, the size of the amplification product can be determined by comparison with a target size marker by agarose or polyacrylamide gel electrophoresis. Since the four pairs of primer pairs constituting the primer set of the present invention form PCR amplification products of different sizes, the presence or absence of each mutation corresponding to EGFR exons 18-21 can be more easily identified.

As the sequencing method, a sequencing method known in the art may be used. For example, an automatic sequencing method, Sanger sequencing or pyro sequencing may be used as a method of directly determining the sequence portion. (pyrosequencing) and the like, but is not limited thereto.

The presence or absence of EGFR mutations identified in the present invention can provide useful information for predicting the prognosis of lung cancer, particularly non-small cell lung cancer treatment, for drug prescription. More preferably, the present invention is useful for predicting reactivity and prognosis for tyrosine kinase inhibitors such as zephytinib or erlotinib.

Preferably, when a PCR product using the primer set of the present invention does not generate any amplification products at all, it can be determined that the tyrosine kinase inhibitor drug does not react positively, and when one or more amplification products are produced, the tyrosine kinase inhibitor drug This positive reaction can be judged to be a good prognosis.

Single PCR and multiple PCR were performed on EGFR exons 18, 19, 20, and 21 using the primer set of the present invention, respectively, and compared by electrophoresis. It was confirmed that the size of the product was shown (Fig. 1), and the same result was confirmed through the sequencing of the single PCR and multiple PCR products.

In addition, when the EGFR mutation was detected from 162 clinical samples of 162 non-small cell lung cancer patients who received EGFR gene screening using the kit of the present invention, the mutation detection rate was 40.1%. In 2009, the Korean Association of Cardiopulmonary Pathology under the Korean Society of Pathology surveyed 1,753 non-small cell lung cancer patients from 15 hospitals nationwide who received EGFR gene screening for the expression rate of EGFR mutation in Korean lung cancer patients. It has been reported that EGFR mutations have been found, the present invention can be seen that can improve the detection rate of EGFR mutations by 11.69% compared to the previous report. This shows that the primer set of the present invention is superior in performance to other primers prepared previously.

Furthermore, as a result of evaluating the analytical sensitivity of the kit of the present invention by setting the ratio of the mutant DNA to the wild-type DNA as 1%, 5%, 10%, 20%, 40%, 50%, the ratio of the mutation was 10%. Mutations were detected (FIGS. 2 and 3). In this regard, previous studies have shown that sequencing can detect mutations when the concentration of mutagenic DNA is greater than 25% (J Thorac Oncol. 2012 Feb; 7 (2): 355-64. In comparison with this previous report, the present invention shows a very excellent sensitivity of the kit of the present invention because mutations can be detected even at a mutant DNA ratio of 10%, which is much lower than 25%.

As such, the primer set of the present invention and the multiple PCR-based EGFR mutation detection technique using the same are superior in specificity, accuracy, precision and sensitivity as compared to the conventional single PCR-based detection method, and many reactions are carried out in one reaction vessel. This is an economical and efficient advantage that can significantly reduce time and labor.

The multiplex PCR method of the present invention and the primers used in the PCR method are superior in specificity and sensitivity as compared to the conventional PCR methods and primers, and have an excellent effect of detecting EGFR mutations quickly and accurately. The presence or absence of EGFR mutations identified in the present invention provides useful information in predicting the susceptibility and prognosis of lung cancer treatment for drug prescription, and in particular, the methods of the present invention predict the prognosis of tyrosine kinase inhibitors such as zephytinib or erlotinib. Useful for

Figure 1 shows the results of electrophoresis comparing a single PCR product with multiple PCR products. Lane 1 shows exon 18 (150 bp), lane 2 shows exon 19 (204 bp), lane 3 shows exon 20 (262 bp), lane 4 shows single PCR amplification products of exon 21 (289 bp), and lane 5 shows exon 18, 19 Multiple PCR amplification products of 20, 21 are shown.
2 shows the results of evaluating analytical sensitivity by setting the ratio of mutant DNA in exons 18 and 19 to wild type DNA as 1%, 5%, 10%, 20%, 40%, 50%.
Figure 3 shows the results of evaluating analytical sensitivity by setting the ratio of mutant DNA in exons 20 and 21 to wild type DNA as 1%, 5%, 10%, 20%, 40%, 50%.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.

Example  One. primer  Production of sets

To design primers that produce PCR products for mutations in the EGFR gene but not for wild-type EGFR genes, the Primer3Plus program is used to determine primer size, Tm value, GC content of the primer, and self-complementary sequences within the primer ( Eight primers were designed after eliminating the possibility of forming a secondary structure by checking whether there is a self-complementary sequence. At this time, the size of the PCR amplification products of exon 18, 19, 20, 21 by each primer pair is designed to be different from each other to facilitate identification during electrophoresis. The base sequences of the designed primers are shown in Table 1.

Exon Base sequence Size of amplified product (bp) 18 Forward 5'-CCCCAGCTTGTGGAGCCTCT-3 '(SEQ ID NO: 1) 150 Reverse 5'-GGCCTGTGCCAGGGACCTTA-3 '(SEQ ID NO: 2) 19 Forward 5'-TGCCAGTTAACGTCTTCCTT-3 '(SEQ ID NO: 3) 204 Reverse 5'-GCCAGACATGAGAAAAGGTG-3 '(SEQ ID NO: 4) 20 Forward 5'-ACCATGCGAAGCCACACT-3 '(SEQ ID NO: 5) 262 Reverse 5'-TATCTCCCCTCCCCGTATCT-3 '(SEQ ID NO: 6) 21 Forward 5'-ATGATCTGTCCCTCACAGCAG-3 '(SEQ ID NO: 7) 289 Reverse 5'-CCTCCCCTGCATGTGTTAAA-3 '(SEQ ID NO: 8)

Example  2. Preparation of Standards

Gene sets of EGFR exons 18, 19, 20, and 21 were amplified by applying primer sets prepared for DNA extracted from tissues identified as having mutations. Amplified nucleic acids of exons 18, 19, 20, 21 were transformed into E. Coli DH5α cells using T-Blunt vector (Solgent, KR) to obtain wild-type and mutant DNA of each exon. The exon 21 amplified nucleic acid was transformed into E. coli DH5α cells using pTOP TA2 vector (Enzynomics, KR) to obtain wild type clones and mutant clones. Identification of wild type and mutant clones was confirmed by direct sequencing. The wild type DNA and mutant DNA of exons 18, 19, 20, and 21 extracted through the clones were used as a standard for evaluating the performance of the EGFR mutation detection kit.

The types of mutations used as standards are shown in Table 2.

number Exon Amino acid change Change of base One 18 p.G719S c.2155G> A 2 19 p.E746_A750del c.2236_2250del15 3 20 p.V769_D770insASV c.2307_2308ins9 4 21 p.L858R c.2573T> G

Example  3. Multiple Polymerase chain reaction

10ul of template DNA solution (0.1ng / ul) prepared as a standard, 5ul of primer mixture (2 pmol/ul) shown in Table 1, 25ul of 2X Multiplex PCR Master Mix (Qiagen), and 10ul of distilled water were mixed in one tube. . After denaturing at 94 degrees for 15 minutes using a DNA amplifier (PCR machine), repeating 30 times at 94 degrees, 60 seconds at 60 degrees, 10 seconds at 72 degrees, and reacting for 10 minutes at 72 degrees, 4 degrees Stored at. DNA amplification was confirmed after electrophoresis on 2% agarose gel.

After the single polymerase chain reaction and the multiple polymerase chain reaction of exon 18, 19, 20, 21 and electrophoresis, the comparison is shown in FIG. Lanes 1, 2, 3, and 4 are electrophoretic results of the products of the single polymerase chain reaction, and lanes 5 are the electrophoretic results of the multiple polymerase chain reaction products. As a result of the multiple polymerase chain reaction, it can be seen that the same amplification products are shown in comparison with the single polymerase chain reaction. In addition, it was confirmed that the single polymerase chain reaction and the multiple polymerase chain reaction product showed the same results through sequencing.

In addition, when the EGFR mutation was detected from 162 clinical samples of 162 non-small cell lung cancer patients who received EGFR gene screening using the kit of the present invention, the mutation detection rate was 40.1% (Table 3).

EGFR mutation Wild Detection rate 62 cases (40.1%) 97 cases (59.9%)

In 2009, the Korean Association of Cardiopulmonary Pathology under the Korean Society of Pathology surveyed 1,753 non-small cell lung cancer patients in 15 hospitals nationwide who received EGFR gene screening. It has been reported that EGFR mutations have been found, the present invention can be seen that the detection rate can be improved by 11.69% compared to this previous report. This shows that the primer set of the present invention is superior in performance to other primers prepared previously.

Furthermore, as a result of evaluating the analytical sensitivity of the kit of the present invention by setting the ratio of the mutant DNA to the wild-type DNA as 1%, 5%, 10%, 20%, 40%, 50%, the ratio of the mutation was 10%. Mutations were detected (FIGS. 2 and 3). In this regard, previous studies have shown that sequencing can detect mutations when the concentration of mutagenic DNA is greater than 25% (J Thorac Oncol. 2012 Feb; 7 (2): 355-64. In comparison with this previous report, the present invention shows a very excellent sensitivity of the kit of the present invention because mutations can be detected even at a mutant DNA ratio of 10%, which is much lower than 25%.

&Lt; 110 > D & P Biotech, Ltd. <120> Multiplex PCR primer set for detecting EGFR mutant and kit          comprising the same <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification of EGFR exon 18 <400> 1 ccccagcttg tggagcctct 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification of EGFR exon 18 <400> 2 ggcctgtgcc agggacctta 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification of EGFR exon 19 <400> 3 tgccagttaa cgtcttcctt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification of EGFR exon 19 <400> 4 gccagacatg agaaaaggtg 20 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification of EGFR exon 20 <400> 5 accatgcgaa gccacact 18 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification of EGFR exon 20 <400> 6 tatctcccct ccccgtatct 20 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for amplification of EGFR exon 21 <400> 7 atgatctgtc cctcacagca g 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for amplification of EGFR exon 21 <400> 8 cctcccctgc atgtgttaaa 20

Claims (9)

A primer pair consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2,
A primer pair consisting of the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4,
A primer pair consisting of the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 6, and
Comprising a primer pair consisting of the nucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8,
A set of primers for detecting EGFR gene mutations for predicting responsiveness to drugs in lung cancer patients.
Kit for detecting the EGFR gene mutation for predicting the reactivity to the drug of the lung cancer patient, comprising the primer set of claim 1.
(a) extracting nucleic acid from a sample of lung cancer patient,
(b) treating the extracted nucleic acid with the primer set of claim 1 to perform a multiple polymerase chain reaction, and
(c) analyzing the size or sequence of the amplification product resulting from the multiplex polymerase chain reaction,
EGFR gene mutation detection method for predicting responsiveness to drugs in lung cancer patients.
The method of claim 3, wherein the EGFR gene mutation comprises a deletion, substitution, or insertion mutation within exons 18, 19, 20, and 21 of the EGFR gene.
The method of claim 4, wherein the EGFR gene mutation is a substitution of glycine which is an amino acid of codon 749 within exon 18 of EGFR, glutamic acid, leucine that is an amino acid of codons 746, 747, 748, 749 and 750 within exon 19 , Deletion of arginine, glutamic acid and alanine, amino acid insertion between valine, codon 769, and aspartic acid, 770, in exon 20, and substitution of leucine, amino acid of codon 858, in exon 21 How to do.
The method of claim 3, wherein the lung cancer is non-small cell lung cancer.
The method of claim 3, wherein the drug is a tyrosine kinase inhibitor.
The method of claim 3, wherein the drug is gefitinib or erlotinib.
The method of claim 3, wherein the nucleic acid is DNA or RNA.

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