Quantitative Cluster Analysis Method Of Target Protein By Using Next-Generation Sequencing And Use Thereof

Abstract

Disclosed is a method of quantitatively analyzing a target protein population in a sample to be analyzed, the method including (a) treating a sample to be analyzed with an aptamer library specific to a target protein population present in the sample so as to form complexes between target proteins and aptamers binding specifically thereto, thereby forming a target protein-aptamer complex population, (b) isolating the complex population from unbound aptamers, and (c) analyzing the sequence of each aptamer of the complex population through a next-generation sequencing process so as to quantify each aptamer of the complex population, thereby quantifying each target protein in the complex population. The method of the present invention can be very useful in collectively quantifying proteins in an analytical sample.

Claims

1. A method of quantitatively analyzing a target protein population in a sample to be analyzed, the method comprising: (a) treating a sample to be analyzed with an aptamer library specific to a target protein population present in the sample so as to form complexes between target proteins and aptamers binding specifically thereto, thereby forming a target protein-aptamer complex population, (b) isolating the complex population from unbound aptamers, and (c) analyzing a sequence of each aptamer of the complex population through a next-generation sequencing process so as to quantify each aptamer of the complex population, thereby quantifying each target protein in the complex population.

2. The method of claim 1, wherein the aptamer library is obtained by (i) preparing an aptamer pool having a random sequence to thus have potential binding capacity to various proteins, (ii) reacting the aptamer pool with the target protein population of the same sample as in step (a) so as to induce specific binding between aptamers and target proteins to thereby form a complex population, (iii) isolating the complex population by excluding unbound aptamers, and (iv) amplifying aptamers of the complex population.

3. The method of claim 1, wherein each aptamer of the aptamer library has 5 and 3 regions comprising conserved regions of known sequences and a middle region therebetween comprising a variable region of any random sequence.

4. The method of claim 1, wherein the sample to be analyzed is a processed sample obtained by removing a protein present in a large amount from the sample.

5. The method of claim 1, wherein step (c) is performed by preparing a double-stranded DNA population from aptamers of the complex population and analyzing the double-stranded DNA population through a next-generation sequencing process.

6. The method of claim 5, wherein each aptamer of the aptamer library has 5 and 3 regions comprising conserved regions of known sequences and a middle region therebetween comprising a variable region of any random sequence, whereby the double-stranded DNA population is prepared using a set of a forward primer and a reverse primer.

7. The method of claim 1, wherein the sample to be analyzed before treatment with the aptamer library in step (a) is added with two or more external standard proteins having different quantification values (i.e. concentrations) that are absent in the sample, and the aptamer library in step (a) uses an aptamer library further including aptamers for the external standard proteins, whereby, in step (c), results of quantifying the aptamers for the external standard proteins are obtained, in addition to results of quantifying the aptamers for the target proteins, and aptamer quantification results for the external standard proteins and aptamer quantification results for the target proteins are compared, thereby quantifying the target proteins.

8. The method of claim 1, wherein the aptamers are single-stranded DNA or single-stranded RNA.

9. The method of claim 1, wherein the target protein population is a population of unknown proteins, a population of known proteins, or a mixed population of unknown proteins and known proteins.

10. The method of claim 1, wherein when a predetermined protein of the target protein population is an unknown protein, isolating and identifying the unknown protein using an aptamer specific to the unknown protein that is contained in the aptamer library is further performed.

11. The method of claim 1, wherein the quantifying in step (c) is performed by counting a number of reads of the same sequence for the aptamers, counting a number of sequences considered to be the same as the reads taking into account an error frequency of a next-generation sequencing process, and summing the number of reads and the number of sequences so that the target proteins are quantified based on summed values.

12. The method of claim 1, wherein step (c) is performed by comparing a reference sequence, which is a known sequence for each aptamer obtained by analyzing a sequence of each aptamer of the aptamer library, with a sequence analysis result of each aptamer of the complex population.

13. A method of selecting a candidate protein as a biomarker, the method comprising: (a) treating a sample to be analyzed with an aptamer library specific to a target protein population present in the sample so as to form complexes between target proteins and aptamers binding specifically thereto, thereby forming a target protein-aptamer complex population, (b) isolating the complex population from unbound aptamers, and (c) analyzing a sequence of each aptamer of the complex population so as to quantify each aptamer of the complex population, thereby quantifying each target protein in the complex population, wherein the method further comprises: (i) performing steps (a) to (c) for an additional sample to be analyzed, which is different from the sample to be analyzed, and (ii) determining one or more target proteins having different quantification results by comparing target protein quantification results obtained through step (c) between the two samples to be analyzed.

14. The method of claim 13, wherein the aptamer library uses the same aptamer library for the two samples to be analyzed.

15. The method of claim 13, wherein the same aptamer library for the two samples to be analyzed is used, and the aptamer library is obtained by (i) preparing an aptamer pool having a random sequence to thus have potential binding capacity to various proteins, (ii) reacting the aptamer pool with the target protein population of any one of the two samples to be analyzed so as to induce specific binding between aptamers and target proteins to thereby form a complex population, (iii) isolating the complex population by excluding unbound aptamers, and (iv) amplifying aptamers of the complex population.

16. The method of claim 13, wherein each aptamer of the aptamer library has 5 and 3 regions comprising conserved regions of known sequences and a middle region therebetween comprising a variable region of any random sequence.

17. The method of claim 13, wherein each of the two samples to be analyzed is a processed sample obtained by removing a protein present in a large amount from the sample.

18. The method of claim 13, wherein step (c) is performed by preparing a double-stranded DNA population from aptamers of the complex population and analyzing the double-stranded DNA population through a next-generation sequencing process.

19. The method of claim 18, wherein each aptamer of the aptamer library has 5 and 3 regions comprising conserved regions of known sequences and a middle region therebetween comprising a variable region of any random sequence, whereby the double-stranded DNA population is prepared using a set of a forward primer and a reverse primer.

20. The method of claim 13, wherein the sample to be analyzed before treatment with the aptamer library in step (a) is added with two or more external standard proteins having different quantification values that are absent in the sample, and the aptamer library in step (a) uses an aptamer library further including aptamers for the external standard proteins, whereby, in step (c), results of quantifying the aptamers for the external standard proteins are obtained, in addition to results of quantifying the aptamers for the target proteins, and aptamer quantification results for the external standard proteins and aptamer quantification results for the target proteins are compared, thereby quantifying the target proteins, the additional sample to be analyzed before treatment with the aptamer library in step (i) is added with two or more external standard proteins having different quantification values that are absent in the sample, and the aptamer library uses an aptamer library further including aptamers for the external standard proteins, whereby results of quantifying the aptamers for the external standard proteins are obtained, in addition to results of quantifying the aptamers for the target proteins, and aptamer quantification results for the external standard proteins and aptamer quantification results for the target proteins are compared, thereby quantifying the target proteins, step (ii) is performed by comparing target protein quantification results of the two samples to be analyzed, and the external standard proteins added to the two samples to be analyzed are the same as each other.

21.-46. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0113] FIG. 1 shows the results of electrophoresis after removal of proteins present in large amounts from human serum protein samples;

[0114] FIGS. 2 and 3 are a schematic view and a flowchart showing a process of preparing a secondary library by performing SSH on a molecule-binding nucleic acid primary library;

[0115] FIG. 4 is a flowchart of a process of preparing a secondary library from a molecule-binding nucleic acid primary library using DSN;

[0116] FIG. 5 shows the electrophoresis results of the extent of subtraction of the secondary library obtained using DSN;

[0117] FIG. 6 shows the results of calculation of the appearance frequencies and the base sequences of the molecule-binding nucleic acids constituting the molecule-binding nucleic acid primary library in an analytical sample S and a comparative sample C;

[0118] FIG. 7 is a graph showing the distribution of 100 molecule-binding nucleic acids obtained by analyzing the myocardial infarction patient serum as an analytical sample and the unstable angina patient serum as a comparative sample with 1,149 molecule-binding nucleic acids of the molecule-binding nucleic acid secondary library;

[0119] FIG. 8 shows the results of clustering of samples using 100 molecule-binding nucleic acids obtained by analyzing the myocardial infarction patient serum as an analytical sample and the unstable angina patient serum as a comparative sample with 1,149 molecule-binding nucleic acids of the molecule-binding nucleic acid secondary library;

[0120] FIGS. 9A and 9B show the electrophoresis results 9A of certain molecule-binding nucleic acids contributing to the biological meaning analysis in the myocardial infarction patient serum as an analytical sample and the unstable angina patient serum as a comparative sample and the graph 9B of the electrophoretic band densities thereof;

[0121] FIG. 10 shows the results of observation of anticancer-drug-treated cells and untreated cells by labeling, with a fluorescent substance, molecule-binding nucleic acids selected by analyzing the extent of binding of anticancer-drug-treated cells and untreated cells to proteins;

[0122] FIG. 11 shows the results of evaluation of the selected liver-cancer-cell-line-specific molecule-binding nucleic acids specifically binding to a liver cancer cell line (Hep3B); and

[0123] FIG. 12 shows the analysis results of cdk2 mRNA after treatment of a liver cancer cell line with a Hep3B molecule-binding nucleic acid-siRNA cdk2 complex through various processes.

DETAILED DESCRIPTION

[0124] A better understanding of the present invention will be given through the following examples. These examples are merely set forth to illustrate the present invention but are not to be construed as limiting the scope of the present invention.

<Example 1> Preparation of Single-Stranded Nucleic Acid Library

<Example 1-1> Oligonucleotide of Single-Stranded Nucleic Acid Library and Primer

[0125] In order to prepare a single-stranded nucleic acid library reacting with a biosample (an analytical sample and a comparative example) containing a target protein population, oligonucleotides of <General Formula I> below (random single-stranded nucleic acids) were prepared (Bionia, Korea).

[0126] The oligonucleotides constituting the single-stranded nucleic acid library, as represented in <General Formula I>, were composed of 5 conserved region-variable region-3 conserved region.

TABLE-US-00001 <GeneralFormulaI> Oligonucleotidestructure: 5-GGGAGAGCGGAAGCGTGCTGGGCCN.sub.50CATAACCCAGAGGTCGA TGGATCCCCCC-3

[0127] The base sequences underlined above are conserved regions, which are fixed portions comprising the known sequences of the single-stranded nucleic acid library, and 50 bases N.sub.50, corresponding to the variable region, include adenine (A), guanine (G), thymine (T), and cytosine (C) present at the same concentration at individual positions.

[0128] A double-stranded DNA library was prepared by performing PCR using the oligonucleotide of <General Formula I> as a template. Here, the primers used are a DS forward primer (SEQ ID NO: 1) and a DS reverse primer (SEQ ID NO: 2) as represented below.

TABLE-US-00002 DSforwardprimer: (SEQIDNO:1) 5-GGGGCTAATACGACTCACTATAGGGAGAGCGGAAGCGTGCTGGG-3 DSreverseprimer: (SEQIDNO:2) 5-GGGGCATCGACCTCTGGGTTATG-3

[0129] The DS forward primer (SEQ ID NO: 1) may complementarily bind to the 5-end underlined sequence of the single-stranded DNA oligonucleotide of <General Formula I>. Furthermore, the underlined portion of SEQ ID NO: 1 is a T7 promoter sequence for RNA polymerase of bacteriophage T7.

[0130] The DS reverse primer (SEQ ID NO: 2) used for PCR may complementarily bind to the 3-end underlined sequence of the single-stranded DNA oligonucleotide of <General Formula I>.

[0131] PCR (polymerase chain reaction) was performed using the DS forward primer (SEQ ID NO: 1) and the DS reverse primer (SEQ ID NO: 2) and using the single-stranded nucleic acid library of <General Formula I> as a template.

[0132] Particularly, 1,000 pmol single-stranded nucleic acid library and 2,600 pmol DS primer pair (a DS forward primer, a DS reverse primer) were mixed with 60 mM KCl, 10 mM Tris-Cl (pH 8.3), 3 mM MgCl.sub.2, 0.5 mM dNTP (dATP, dCTP, dGTP, and dTTP) and 0.1 U Taq DNA polymerase (Perkin-Elmer, Foster City Calif.), and PCR was performed, followed by purification with a QIAquick-spin PCR purification column (QIAGEN Inc., Chatsworth Calif.). Thereby, double-stranded DNA containing the T7 promoter was prepared.

[0133] The corresponding PCR product is double-stranded DNA containing the T7 promoter, and the general formula thereof is represented by <General Formula II> below.

TABLE-US-00003 <GeneralFormulaII> PCRproduct: 5-GGGGGCTAATACGACTCACTATAGGGAGAGCGGAAGCGTGCT GGGCCN.sub.50CATAACCCAGAGGTCGATCCCC-3

[0134] As will be described below, the primers for RT-PCR are an RS forward primer (SEQ ID NO: 3) and an RS reverse primer (SEQ ID NO: 4), and the base sequences thereof are as follows.

TABLE-US-00004 RSforwardprimer: (SEQIDNO:3) 5-CGGAAGCGTGCTGGGCC-3 RSreverseprimer: (SEQIDNO:4) 5-TCGACCTCTGGGTTATG-3

<Example 1-2> Preparation of Single-Stranded RNA Library

[0135] A single-stranded RNA library reacting with a biosample (an analytical sample and a comparative sample) was prepared. This single-stranded RNA library is an RNA library containing 2-F-substituted pyrimidine as the modified nucleotide, and single-stranded RNA containing 2-F-substituted pyrimidine was prepared through purification after synthesis through in-vitro transcription using the PCR product of <General Formula II> prepared in <Example 1-1> by means of a DuraScribe T7 Transcription Kit (EPICENTRE, USA).

[0136] Particularly, 300 pmol double-stranded DNA prepared as above, 50 mM Tris-Cl (pH 8.0), 12 mM MgCl.sub.2, 5 mM DTT, 1 mM spermidine, 0.002% Triton X-100, 4% PEG 8000, 5 U T7 RNA polymerase, 1 mM (ATP, GTP) and 3 mM (2F-CTP, 2F-UTP) were mixed, reacted at 37 C. for 6 to 12 hr, and purified with a Bio-Spin 6 chromatography column (Bio-Rad Laboratories, Hercules Calif.), after which the amount of the purified nucleic acids and the purity thereof were analyzed using a UV spectrometer.

<Example 1-3> Preparation of External Standard Material for Quality Control and Measurement

[0137] As external standard proteins, five kinds of plant (Arabidopsis)-specific proteins, namely A, B, C, D and E, analogues of which are not present in humans, secured from Plant Genomics of Michigan State University (genomics.msu.edu/plant_specific/), were prepared using an Escherichia coli expression system, as shown in [Table 1] below.

TABLE-US-00005 TABLE 1 Plant-specific protein Accession Kind Locus Description number A At1g65390.1 defense/immunity protein GO: 0003793 B At5g39310.1 cell elongation GO: 0009826 C At4g15910.1 Drought-Induced Protein (Di21) GO: 0009414 D At1g12860.1 Bhlh Protein GO: 0003677 E At4g02540.1 Chloroplast Thylakoid Lumen GO: 0009543 Protein

<Example 2> Preparation of Molecule-Binding Nucleic Acid Primary Library

<Example 2-1> Preparation of Molecule-Binding Nucleic Acid Primary Library

[0138] In order to prepare a molecule-binding nucleic acid primary library, a biosample containing a protein population, for example, the serum, was used as a sample, and the myocardial infarction patient serum was used as an analytical sample and the unstable angina patient serum was used as a comparative sample.

[0139] In order to increase the detection sensitivity of useful target proteins, a sample obtained by removing excess protein present in the patient serum using a MARC column (Agilent Technologies Inc. USA) in accordance with the protocol provided by the manufacturer was used for actual experiments. The electrophoresis results of the excess-protein-free analytical sample and comparative sample are shown in FIG. 1.

[0140] In order to prepare a molecule-binding nucleic acid primary library, a single-stranded RNA pool specific to the protein population of the analytical sample (obtained by removing excess protein present in the myocardial infarction patient serum) was made, and the specific single-stranded RNA pool was allowed to react with the excess-protein-free analytical sample and comparative sample, thus preparing a molecule-binding nucleic acid primary library for each sample, which was then used to construct a sequencing library for NGS.

[0141] For the preparation of the single-stranded nucleic acid pool specific to the sample, the single-stranded RNA library synthesized in <Example 1> was allowed to react with the analytical sample obtained by removing excess protein present in the myocardial infarction patient serum to give a protein-single-stranded RNA complex, followed by repeating a washing process using the same washing buffer, thereby removing unbound or nonspecific single-stranded RNA. Next, the complex pool was isolated, and a single-stranded RNA pool obtained by dissociating the single-stranded RNA from the complex was amplified through RT-PCR (reverse transcription-PCR) using the RS forward primer and the RS reverse primer of <Example 1>, after which the resulting amplification product was subjected to in-vitro transcription in the same manner as in Example 1, thereby obtaining a single-stranded RNA pool.

[0142] The process of preparing the single-stranded RNA pool is described in detail below.

[0143] A reaction solution (50 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KCl, 5 mM NgCl.sub.2, 1 mM EDTA, and 0.05% TWEEN-30) containing 300 L of a 10.sup.14 base sequence/mL solution of the 300 pmol RNA library prepared in <Example 1-2> was heated at 80 C. for 10 min and then allowed to stand in ice for 10 min.

[0144] This reaction solution was added with yeast tRNA (Life Technologies) in an amount five times the amount of the single-stranded nucleic acids used above and 0.2% BSA (bovine serum albumin, Merck), thus affording a nonspecific-reaction-blocking buffer solution.

[0145] Before immobilization of the serum protein on an NC (nitrocellulose) membrane piece (0.30.3 mm.sup.2), the reaction solution containing the RNA library was added with 60 L of 100 mM DTT and the nonspecific-reaction-blocking buffer solution was added with 400 L of 100 mM DTT and 10% BSA.

[0146] 60 L of the reaction solution (50 mM HEPES, pH 7.5, 125 mM NaCl, 5 mM KCl, 5 mM NgCl.sub.2, 1 mM EDTA, 0.05% TWEEN-30) and 600 g of the prepared serum protein were mixed in a 1.5 mL binding reaction tube. Tapping and then quick-spinning were performed so that the NC membrane was completely immersed in the reaction solution. Mixing was performed using a stirrer for 40 min at 100 rpm.

[0147] The NC membrane was dried at room temperature for 10 min and then placed in a new 1.5 mL tube. 300 L of the nonspecific-reaction-blocking buffer solution was added thereto and mixed using a stirrer at 100 rpm for 40 min and the NC membrane was then transferred into a new 1.5 mL tube. 500 L of the reaction solution was added thereto and mixed using a stirrer at 100 rpm for 10 min and the NC membrane was then transferred into a new 1.5 mL tube. Further, 500 L of the reaction solution was added thereto and mixed using a stirrer at 100 rpm for 10 min and the NC membrane was then transferred into a new 1.5 mL tube. Thereafter, a washing process for removing the unbound serum protein was performed using the reaction solution.

[0148] 5 L of the reaction solution (100 ng/L) containing the single-stranded RNA pool and 195 L of a binding buffer solution were added thereto and mixed using a stirrer at 300 rpm for 40 min and the NC membrane was then transferred into a new 1.5 mL tube. Further, 500 L of the reaction solution was added thereto and mixed using a stirrer at 100 rpm for 10 min and the NC membrane was then transferred into a new 1.5 mL tube. Furthermore, 500 L of the reaction solution was added thereto and mixed using a stirrer at 100 rpm for 10 min and the NC membrane was then transferred into a new 1.5 mL tube.

[0149] The NC membrane was placed on a Whatman filter paper and dried at room temperature for 10 min. The dried NC membrane was placed in a new 1.5 mL tube, added with 50 L of DEPC sterile purified water, and allowed to stand in a heat block at 95 C. for 10 min, and thus RNA was eluted. RNA attached to the membrane was eluted through tapping, subjected to quick-spinning and then allowed to stand in ice. The RNA thus obtained was placed in a PCR tube and subjected to reverse transcription and PCR at 36 C. using the RS forward primer and the RS forward primer of <Example 1> and then to in-vitro transcription in the same manner as in <Example 1>, thereby preparing a single-stranded RNA pool. The molecule-binding nucleic acid primary library binding to the human serum sample protein was prepared in a manner in which procedures of reacting the proteins of a human serum sample and the RNA library and performing washing using various washing buffers (0 to 1 reaction solution, 0 to 5% Tween-30 or 0 to 600 mM EDTA solution) were repeated once.

[0150] A molecule-binding primary single-stranded nucleic acid library was prepared in a manner in which the single-stranded RNA pool obtained above was allowed to react with each of the analytical sample and the comparative sample to thus remove unbound or nonspecific single-stranded RNA, thereby separating the protein-complex pool, after which the single-stranded RNA was dissociated from the complex pool. Here, the washing process was performed only once.

[0151] Next, the molecule-binding single-stranded RNA was subjected to reverse transcription to afford cDNA, which was then subjected to one-way PCR once, thus obtaining double-stranded DNA fragments, which were then subjected to NGS analysis as below.

<Example 2-2> Preparation of Molecule-Binding Nucleic Acid Primary Library for Sample Containing External Standard Protein

[0152] In order to use the external standard proteins of Example 1 to quantify the target proteins of the analytical sample or the comparative sample, single-stranded RNA specifically binding to each external standard protein was attained using the single-stranded RNA library synthesized in <Example 1> through a standard SELEX method (Ellington, A. D. and J. W. Szostak. 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346: 818-822; Gold, L., P. Allen, J. Binkley, D. Brown, D. Schneider, S. R. Eddy, C. Tuerk, L. Green, S. Macdougal, and D. Tasset. 1993. RNA: the shape of things to come, pp. 497-510. In: R. F. Gestelend and J. F. Atkins (eds.). The RNA World, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.), followed by a sequencing process through cloning based on a known BAC library construction (Genome Res. 2001 March; 11(3):483-496), thereby predetermining the sequences thereof.

[0153] Five kinds of external standard proteins prepared in <Example 1>, A, B, C, D and E were added at concentrations of 0.01 pg/mL, 1.0 pg/mL, 100.0 pg/mL, 10.0 ng/mL, and 1.0 g/mL, respectively, to each of the analytical sample and the comparative sample to afford samples to be analyzed for the present example. Also, the single-stranded RNA pool reacting with these samples was added with single-stranded RNA specifically binding to each external standard protein, thus preparing a single-stranded RNA pool reacting with the sample. Each of the samples added with the five kinds of external standard proteins at different concentrations was reacted with the single-stranded RNA pool containing the single-stranded RNA specific to the external standard proteins to afford a molecule-binding nucleic acid primary library in the same manner as in <Example 2-1>, which was then subjected to reverse transcription and one-way PCR once, and the resulting product was subjected to NGS analysis, as will be described below.

<Example 3> Preparation of Molecule-Binding Nucleic Acid Secondary Library

<Example 3-1> Preparation of Molecule-Binding Nucleic Acid Secondary Library Using SSH

[0154] The preparation of the SSH molecule-binding nucleic acid secondary library was performed in the steps shown in [FIG. 2] and [FIG. 3].

[0155] RT-PCR was performed using, as a template, the molecule-binding primary RNA library for the proteins of the analytical sample prepared in <Example 2> and the molecule-binding primary RNA library for the proteins of the comparative sample, thereby preparing a DNA pool. Here, RT-PCR was conducted through a standard method.

[0156] Particularly, in order to carry out reverse transcription, 36 L of a reaction solution containing the primary library was placed in a PCR tube, and 10 L of a 5 reverse transcription buffer solution and 1 L of an RS reverse primer (25 pmol/L) solution were added thereto and mixed together. The resulting mixed solution was reacted at 65 C. for 5 min and then at 25 C. for 10 min using a PCR device. A mixed solution comprising 1.5 L of DEPC sterile purified water, 1 L of 30 mM dNTP, and 0.5 L of AMV RTase (10 /L BEAMS, Cat. No. 4001L) was prepared, and 3 L thereof was then added to the PCR tube in which the reaction was completed, so that the final volume was 60 L. The reaction in the PCR tube was carried out using a PCR device at 37 C. for 40 min and at 95 C. for 5 min.

[0157] Subsequently, PCR was performed as follows. In order to amplify the product cDNA, PCR amplification was carried out using a PCR mixed solution prepared in a manner in which 60 L of the RT reaction product was added with 10 L of a 10PCR buffer solution, 1 L of 30 mM dNTP, 1 L of a DS forward primer (25 pmol/L), 1 L of a DS reverse primer (25 pmol/L), 0.5 L of Taq polymerase (5 WA) and 36.5 L of tertiary sterile purified water so that the final volume was 100 L. A series of procedures of preliminary denaturation at 95 C. for 5 min, denaturation at 95 C. for 40 sec, binding at 55 C. for 40 sec, and extension at 72 C. for 40 sec was repeated 25 times using a PCR device, and final extension at 72 C. for 5 min was performed.

[0158] The PCR product was transferred into a 1.5 mL tube, 100 L of deionized water was added so that the volume was increased to 300 L, and phenol, chloroform and isoamyl alcohol were added in 300 L of the same volume. After vortex mixing and then centrifugation at 13,000 rpm for 1 min, the supernatant was transferred into a new 1.5 mL tube. A two-fold volume of 100% ethanol and 0.1-fold volume of 3 M sodium acetate (pH 5.2) were added thereto and mixed, and the tube was stored at 30 C. for at least 60 min or at 80 C. for at least 10 min. The tube thus stored was centrifuged at 4 C. and at 13,000 rpm for 10 min, and the supernatant was removed. 600 L of 70% ethanol was added thereto, followed by centrifugation at 4 C. and at 13,000 rpm for 5 min, after which the supernatant was removed.

[0159] The remaining content of the tube was dried and dissolved in 40 L of tertiary sterile purified water. Then, 5 L thereof was loaded on a 2.5% agarose gel and 3 L of a 60 bp ladder DNA marker was then loaded thereon, followed by quantification in a 100 bp band using an Alphaimager program (BIO-RAD, USA).

[0160] The PCR product of the molecule-binding nucleic acid primary library binding to the proteins of the analytical sample was used as a pretester, and a tester for use in SSH was prepared from the pretester, and the PCR product of the molecule-binding nucleic acid primary library binding to the proteins of the comparative sample was used as a driver.

[0161] Tester 1 and Tester 2 were prepared from the pretester through PCR using a standard method. Here, Tester 1 was prepared using an SSH forward primer (SSH forward primer_Tester 1, SEQ ID NO: 5) comprising a newly designed adapter 1 and an RS forward primer (underlined portion of SEQ ID NO: 5) and an RS reverse primer, and Tester 2 was prepared using an RS forward primer and an SSH reverse primer (SSH reverse primer_Tester 2, SEQ ID NO: 6) comprising an adapter 2 and an RS reverse primer (underlined portion).

TABLE-US-00006 SSHforwardprimer_Tester1: (SEQIDNO:5) 5-TCGAGCGGCCGCCCGGGCAGGTCGGAAGCGTGCTGCC-3 SSHreverseprimer_Tester2: (SEQIDNO:6) 5-CAGCGTGGTCGCGGCCGAGGTTCGACCTCTGGGTTATG-3

[0162] 1.5 L of a 30 ng Tester 1, 1.5 L of a 600 ng driver PCR product, and 1.0 L of a 4 hybridization solution (300 m MHEPES pH 7.5, 2 M NaCl, 0.8 m MEDTA) were mixed and primary hybridization was performed at a reaction volume of 4.0 L at 60 C. for about 12 hr, and 1.5 L of a 30 ng Tester 2 (SEQ ID NO: 6), 1.5 L of a 600 ng driver PCR product, and 1.0 L of a 4 hybridization solution were mixed, followed by treatment at a reaction volume of 4.0 L at 95 C. for 5 min and then primary hybridization at 60 C. for about 12 hr. For the sake of convenience, the former is called Tester 1 hybrid solution, and the latter is called Tester 2 hybrid solution.

[0163] The Tester 2 hybrid solution was cautiously added to the driver hybrid solution and mixed therewith, and the resulting hybrid solution (a mixed hybrid solution of the Tester 2 hybrid solution and the driver hybrid solution) was then added to the Tester 1 hybrid solution and mixed well using a pipette, followed by secondary hybridization at 60 C. for 12 hr. The driver solution was prepared by mixing 1.0 L of a 130 g driver, 1.0 L of a 4 hybridization solution, and 2 L of water, adding a small amount of mineral oil and performing treatment using a PCR device at 98 C. for 90 sec. After termination of the secondary hybridization, the resulting solution was added with Taq polymerase and then subjected to extension at 75 C. for 5 min, whereby the cohesive ends of 5 and 3 ends of the double-stranded nucleic acids of the secondary hybridization product were added with bases and thus converted to blunt ends.

[0164] After termination of the extension reaction, the resulting solution was subjected to PCR through a standard method using an adapter 1 primer (SEQ ID NO: 7) and an adapter 2 primer (SEQ ID NO: 8) designed to have the sequence shown below.

TABLE-US-00007 Adapter1primer: (SEQIDNO:7) 5-TCGAGCGGCCGCCCGGGCAGGT-3 Adapter2primer: (SEQIDNO:8) 5-CAGCGTGGTCGCGGCCGAGGT-3

[0165] The PCR product thus obtained was subjected to nested PCR through a standard method using an RS forward primer (SEQ ID NO: 3) and an RS reverse primer (SEQ ID NO: 4).

[0166] The PCR amplification product was subjected to NGS analysis.

<Example 3-2> Preparation of Molecule-Binding Nucleic Acid Secondary Library Using DSN

[0167] A molecule-binding nucleic acid secondary library was prepared through subtraction using DSN (duplex-specific nuclease) in the steps shown in [FIG. 4].

[0168] In order to prepare the molecule-binding nucleic acid secondary library using DSN, the RT-PCR product of the molecule-binding RNA library for the proteins of the analytical sample prepared in <Example 2> was used as a tester, and the RT-PCR product of the molecule-binding RNA library for the proteins of the comparative sample was used as a driver.

[0169] PCR was performed using the SSH forward primer_Tester 1 and the SSH reverse primer_Tester 2 as primers from the above tester, after which PCR was performed using the SSH forward primer_Tester 1, thus obtaining tester double-stranded DNA. For the driver, double-stranded DNA was obtained through PCR using the RS forward primer and the RS reverse primer.

[0170] The double-stranded DNA thus obtained was subjected to hybridization and then DSN treatment. Particularly, 100 ng/L double-stranded DNA was prepared, 1.5 L each of which was then placed in PCR tubes, after which 1 L of a 4 hybridization solution and 1.5 L of distilled water were added thereto and mineral oil was overlaid thereon. Then, heating at 98 C. for 3 min and then hybridization at 60 C. for 4 hr were performed. After termination of the hybridization, 5 L of 2DSN buffer preheated to 60 C. (100 mM Tris-HCl pH 8.0, 10 mM MgCl.sub.2, 2 mM dithiothreitol) was added to the reaction mixture. Subsequently, a DSN enzyme (Wako, Japan) at 0.25 Kunitz units was added thereto and the reaction was then carried out. After the reaction for 30 min, 10 L of 5 mM EDTA was added thereto, thereby terminating the reaction.

[0171] 60 L of the resulting reaction product was added with 10 L of 10PCR buffer solution, 1 L of 30 mM dNTP, 1 L of SSH forward primer_Tester 1 (25 pmol/L), 1 L of SSH reverse primer_Tester 2 (25 pmol/L), 0.5 L of Taq polymerase (5 /L) and 36.5 L of tertiary sterile purified water so that the final volume was 100 L, thus preparing a PCR mixed solution. Using a PCR device, a series of procedures of preliminary denaturation at 95 C. for 5 min, denaturation at 95 C. for 40 sec, binding at 55 C. for 40 sec, and extension at 72 C. for 40 sec was repeated 25 times, followed by final extension at 72 C. for 5 min. Subsequently, 1 L of exonuclease I was added thereto and the reaction was carried out for 40 min, thus removing the remaining single-stranded nucleic acids.

[0172] The amplification product thus obtained was subjected to nested PCR through a standard method using an RS forward primer (SEQ ID NO: 3) and an RS reverse primer (SEQ ID NO: 4).

[0173] The PCR amplification product was subjected to NGS analysis.

<Example 3-4> Evaluation of Subtraction

[0174] In order to evaluate the extent of subtraction of the molecule-binding nucleic acid primary library, the serum sample was immobilized on an NC membrane and then reacted with the molecule-binding nucleic acid secondary library using the subtracted DSN. The molecule-binding nucleic acid binding to the serum protein was separated from the NC membrane and used as a template for RT-PCR, followed by RT-PCR and electrophoresis. The results thereof were confirmed.

[0175] In the present example, the myocardial infarction patient serum was used as an analytical sample and the unstable angina patient serum was used as a comparative sample. The results of evaluation of the molecule-binding nucleic acid library obtained through subtraction are shown in [FIG. 5]. The strong band was formed in the analytical sample and the weak band was formed in the comparative sample, from which the analytical sample was confirmed to be subtracted by the comparative sample.

<Example 4> Determination of Base Sequence of Molecule-Binding Nucleic Acid Library and Appearance Frequency Thereof

[0176] The double-stranded DNA pool that was the RT-PCR product of the molecule-binding nucleic acid primary library prepared above and the double-stranded DNA pool that was the RT-PCR product of the molecule-binding nucleic acid secondary library were subjected to NGS (next-generation sequencing), and thus the base sequences of the molecule-binding nucleic acids and the appearance frequencies thereof were determined.

[0177] NGS analysis was performed for the DNA pool using a HiSeq 3000 (Illumina).

[0178] A sequencing library was prepared using the DNA pool prepared above and a TruSeq DNA sample preparation kit v.2 (IIlumina). Particularly, 136 ng DNA was subjected to end repair, adenosine addition, adapter addition and PCR in accordance with the manufacturer's protocol. Washing was performed using Agencourt AMPure XP beads (Beckman-Coulter, USA) included in the TruSeq kit at every step except for the adenosine base addition step. Then, a sequencing library was prepared by performing PCR through 15-cycle reactions using the PCR primers included in the kit in accordance with the manufacturer's protocol.

[0179] As the PCR product, the sequencing library was quantified using a Qubit fluorometer (Invitrogen). A 3 ng PCR product per sample was subjected to hybridization at a concentration of 5 pM in a flow cell of a cBOT cluster station of a HiSeq 3000. Bridge amplification on cBOT was performed 28 times per single DNA in a molecule-binding nucleic acid library to form a cluster. Each cluster was linearized and then hybridized with sequencing primers. Such a flow cell was loaded on a HiSeq 3000 and analyzed with a HiSeq 3000 Single-Read 80 Base Pair Recipe capable of sequencing 73 single-read bases and 7 multiplexed bases. Image production and analysis were conducted through Illumina Real-Time Analysis (RTA) and thus base-call files and quality scores were assayed in real time.

[0180] After termination of the sequencing of forward and reverse strands, quality analysis was performed using Casava software made by Illumina, and also, downstream analysis was performed using a proprietary software (AptaCDSS, BioEZ) analysis system.

[0181] The matching step of the quality analysis was performed for the pattern of 74 base pairs (bp) of the sequence comprising the 5 conserved region (17 bp), variable region (50 bp) and 3 conserved region (17 bp) in <General Formula I> of the oligonucleotides constituting the single-stranded nucleic acid library. The filtering step excludes any sequence that does not match the above pattern. One mismatch was allowed in each conserved region during pattern matching. The conserved region sequence was excluded after filtering, leaving only a 50 bp variable region sequence for downstream analysis. The reverse supplemental tag was coupled with the forward sequence tag. The counting of the molecule-binding nucleic acid reads for each round was performed through round enrichment analysis. The quality analysis and count round results were used to determine the base sequences of certain molecule-binding nucleic acids constituting the library and to determine the appearance frequency thereof.

[0182] The sequence analysis was performed on 15,000,000 to 18,000,000 single DNA molecules per DNA pool of molecule-binding nucleic acids.

[0183] The base sequences and appearance frequencies of 5,395 molecule-binding nucleic acids constituting each of the molecule-binding nucleic acid primary library prepared from the analytical sample S and the molecule-binding nucleic acid primary library prepared from the comparative sample C were compared with each other to confirm that they were different from each other [FIG. 6).

[0184] The base sequences of the molecule-binding nucleic acids constituting the molecule-binding nucleic acid secondary library prepared through SSH were subjected to clustering analysis using reference sequences constructed with the molecule-binding nucleic acid sequences determined in the molecule-binding nucleic acid primary library and using Omega Cluster of EBI website (www.ebi.ac.uk/Tools/msa/clustalo/).

[0185] Through comparison and analysis of these analytical and comparative samples, 1,149 molecule-binding nucleic acids representing the analytical sample were determined.

<Example 4-2> Determination of Biological Meaning of Molecule-Binding Nucleic Acid

[0186] Analysis was performed using the myocardial infarction patient serum (10 cases) as an analytical sample and the unstable angina patient serum (21 cases) as a comparative sample [Table 2]. Reference sequences were constructed with base sequences of 1,149 molecule-binding nucleic acids determined above. Double-stranded DNA, which is the RT-PCR product of the molecule-binding nucleic acid pool separated from the protein-molecule-binding nucleic acid complex pool formed by reacting the sample with a library of 1,149 molecule-binding nucleic acids, was used as a nucleic acid sample to prepare a sequencing library. Then, reads were produced through NGS and compared with the reference sequences, and thus the appearance frequencies of the molecule-binding nucleic acids were analyzed. The distribution of molecule-binding nucleic acids able to distinguish myocardial infarction and unstable angina statistically based on the appearance frequencies of 1,149 molecule-binding nucleic acids using a biological meaning determination system is shown in [FIG. 7].

[0187] Based on the analysis results of each of the analytical sample (10 cases) and the comparative sample (10 cases), a database for the analytical sample and the comparative sample was constructed, and molecule-binding nucleic acids that can distinguish the two groups were determined by one-way ANOVA, after which the appearance frequency of the determined molecule-binding nucleic acids was determined by 4*[(appearance frequency-minimum)/(maximum-minimum)]2. The results thereof are shown in [FIG. 7].

[0188] The results of class clustering with the appearance frequency of the selected molecule-binding nucleic acids are shown in [FIG. 8]. As shown in [FIG. 8], the myocardial infarction patient serum and the unstable angina patient serum were able to form independent clusters, from which the patients can be found to be distinguished based on the appearance frequencies of molecule-binding nucleic acids binding to the human serum proteins.

TABLE-US-00008 TABLE 2 Clinical information of cardiovascular patients Myocardial infarction Unstable angina Gender Male 10 21 Female 0 1 Age 50-49 1 0 60-59 5 9 60-69 4 11 70-79 0 1 Total 10 21

[0189] As is apparent from the above results, protein profiles in a certain biosample were searched for and analyzed using molecule-binding nucleic acids binding to multiple proteins of the biosample and the NGS technique, from which molecule-binding nucleic acids specifically binding to the myocardial infarction patient serum protein as the analytical sample and having biological meaning can be selected. Among these, based on the results of evaluation of the extent of binding of serial No. 768 (number assigned to the molecule-binding nucleic acid) molecule-binding nucleic acid to the myocardial infarction patient serum as the analytical sample and to the unstable angina patient serum as the comparative sample, it can be found to specifically respond only to the myocardial infarction patient serum [FIG. 9].

<Example 4-4> Selection and Use of Other Molecule-Binding Nucleic Acids

[0190] <4-4-1> Molecule-Binding Nucleic Acid for Protein in Response to Anticancer Drug

[0191] Selection of Molecule-Binding Nucleic Acid

[0192] In order to investigate molecule-binding nucleic acids and proteins capable of drug reaction monitoring, total protein of cells treated with the anticancer substance doxorubicin and total protein of untreated cells were prepared as biosamples. The target cancer cell line was liver cancer cells Hep3B, and a liver cancer cell line culture broth was treated with doxorubicin so that the final concentration thereof was 5 g/mL. 4 hr after treatment, it was confirmed that doxorubicin was absorbed into the cells.

[0193] The cells, which were treated with doxorubicin and then cultured for 6 hr (analytical sample), and the untreated cells (comparative sample) were collected and total protein was isolated therefrom. A molecule-binding nucleic acid primary library binding to each cell line was prepared using the single-stranded nucleic acid library prepared in <Example 1>. The library thus prepared was subjected to SSH as in <Example 3> to thus select and attain the liver cancer cell line SSH molecule-binding nucleic acid library as the analytical sample.

[0194] Confirmation of Binding Capacity of Molecule-Binding Nucleic Acid

[0195] The molecule-binding nucleic acid pool thus selected and attained was reacted with the cancer cell line Hep3B analytical sample and comparative sample, thus preparing an analytical sample-binding molecule-binding nucleic acid pool and a comparative sample-binding molecule-binding nucleic acid pool, the sequences of which were then determined through NGS and protein profiles were produced.

[0196] Based on the protein profiles produced and accumulated for the analytical sample and the comparative sample, molecule-binding nucleic acids specifically binding to the analytical sample were selected through ANOVA, and the binding capacity thereof was observed at the cellular level. The selected molecule-binding nucleic acids were labeled with a Rhodamine staining reagent and thus the treated cells and the untreated cells were stained and observed with a fluorescence microscope. The results are shown in [FIG. 10], in which the specific binding of the molecule-binding nucleic acids to the treated cells was confirmed.

[0197] <4-4-2> Liver-Cancer-Cell-Specific Molecule-Binding Nucleic Acid Selection of Molecule-Binding Nucleic Acid

[0198] A liver cancer cell line Hep3B sample and a GIBCO hepatocyte sample were treated with the single-stranded nucleic acid library prepared in <Example 1> to thus prepare a molecule-binding nucleic acid primary library binding to each cell line. The library thus prepared was subjected to SSH to thus select and attain a liver cancer cell line SSH molecule-binding nucleic acid library.

[0199] The selected molecule-binding nucleic acid pool was reacted with the cancer cell line Hep3B analytical sample and the GIBCO hepatocyte comparative sample, thus preparing a liver cancer cell line Hep3B-binding molecule-binding nucleic acid pool and a GIBCO hepatocyte-binding molecule-binding nucleic acid pool, the cell surface profiles of which were then produced through NGS.

[0200] Confirmation of Binding Capacity of Molecule-Binding Nucleic Acid

[0201] Based on the cell surface profiles produced and accumulated for the cancer cell line Hep3B analytical sample and the GIBCO hepatocyte comparative sample, molecule-binding nucleic acids specifically binding to the liver cancer cell line Hep3B were selected through ANOVA, and the binding capacity thereof was observed at the cellular level [FIG. 11]. The binding capacity of the selected molecule-binding nucleic acids to the liver cancer cell line Hep3B and to the GIBCO hepatocytes was determined by binding certain molecule-binding nucleic acids to the above cells, amplifying the bound molecule-binding nucleic acids and performing electrophoresis, resulting in the finding that they were nucleic acids specifically binding to the liver cancer cell line.

[0202] The selected molecule-binding nucleic acids were labeled with a Rhodamine staining reagent, and thus the liver cancer cell line Hep3B and the GIBCO hepatocytes were stained and observed with an optical microscope. The results are shown in [FIG. 11], in which the molecule-binding nucleic acids were specifically bound to the liver cancer cell line Hep3B but scarcely bound to the GIBCO hepatocytes.

[0203] Use of Molecule-Binding Nucleic Acid

[0204] Next, cdk2 siRNA, inhibiting the function of the cdk2 gene, which is mainly expressed in cancer cells and is thus known as a tumor gene, was prepared. Then, a complex, specifically acting on a liver cancer cell line by binding to a liver-cancer-cell-line-specific molecule-binding nucleic acid, was developed, and the function thereof was evaluated.

[0205] A Hep3B molecule-binding nucleic acid-cdk2 siRNA complex was prepared and then introduced into the cells through transfection or direct treatment. The Hep3B molecule-binding nucleic acid-cdk2 siRNA complex was treated at a concentration of 100 nM or 260 nM, or only cdk2 siRNA was treated for comparison. The liver cancer cell line Hep3B was treated for 3 days, and total RNA was separated from each sample, and thus the expression of cdk2 mRNA was compared and analyzed. The results thereof are shown in [FIG. 12].

[0206] The first lane of [FIG. 12] shows the results of treatment of the Hep3B molecule-binding nucleic acid-cdk2 siRNA complex at 100 nM and the second lane shows the results of treatment at 260 nM. The third lane shows the results of treatment of 100 nM cdk2 siRNA and the fourth lane shows the results of the untreated group.

[0207] Upon transfection, the amount of cdk2 mRNA was reduced in both the Hep3B molecule-binding nucleic acid-cdk2 siRNA complex and the cdk2 siRNA compared to the untreated group. However, upon direct treatment, the amount of cdk2 mRNA was reduced in the group treated with the Hep3B molecule-binding nucleic acid-cdk2 siRNA complex compared to the untreated group and the group treated with siRNA. In the case of the Hep3B molecule-binding nucleic acid-cdk2 siRNA complex, it can be confirmed that the analytical molecule-binding nucleic acid of the complex was bound to the protein of the liver cancer cell line Hep3B and migrated into the cells and thus acted as siRNA, thereby reducing the amount of cdk2 mRNA.

<Example 5> Isolation and Identification of Protein Binding to Molecule-Binding Nucleic Acid

[0208] A database of molecular-binding nucleic acids constructed by disease groups was compared and analyzed to determine useful spots that could contribute to biological meaning analysis, and the corresponding molecule-binding nucleic acids were prepared. Proteins to which the molecule-binding nucleic acids were specifically bound were isolated and identified using the molecule-binding nucleic acids.

[0209] Particularly, biotin was attached to one side of the selected molecule-binding nucleic acid (NABM), reacted with streptavidin, and reacted with the serum sample, thus obtaining a serum protein-molecule-binding nucleic acid complex, and the corresponding complex was separated from the sample using the biotin-immobilized support. The band formed through electrophoresis was isolated and the serum protein was identified. Through MALDI-TOF/TOF, the amino acid sequence of the protein binding to the single-stranded nucleic acid was determined. The dissociation constant Kd of the protein identified after isolation from the molecule-binding nucleic acid was measured to be less than 3010.sup.9.

<Example 7> Analysis of BiosampleSimultaneous Analysis of Nucleic Acid and Protein

[0210] NGS (next-generation sequencing) is a chip-based and PCR-based paired end method in which a whole genome is fragmented and the fragments are hybridized and sequenced at a very high speed. A lot of information about the genome can be produced using NGS.

[0211] Using NGS technology, single-nucleotide polymorphism (SNP), which is an allele coding for a genetic trait that appears in 2-5% of the human population, and amino acid mutation resulting from SNP, in which wild-type amino acid is mutated, can be analyzed quickly. WGS (whole genome sequencing) is a method of reading the whole human genome sequence by NGS at several magnifications, such as 10, 30, and 50, WES (whole exome sequencing) is a method of determining the base sequence of only the gene region involved in protein production in the above WGS, and TS (target sequencing) is a technique for sequencing only the gene region involved in target molecule production in the above WGS. Therefore, the data size is generated in descending order of WGS>WES>TS. However, it is advantageous to sequencing a large number of samples when a small region is analyzed. METseq is a sequencing technique for DNA methylation measurement of genes, and RNAseq is a sequencing technique for the expression of genes, namely DNA transcriptome. SV (structural variation) is variation in a large unit (DNA segment) of a chromosome caused by insertion, inversion, translocation, etc. in the mutation, and information thereon can also be produced by NGS.

[0212] The process of simultaneously producing protein and nucleic acid information using NGS technology is as follows.

[0213] Construction of Reference Sequence

[0214] First, reference sequences should be constructed with base sequences of nucleic acids to be analyzed and base sequences of molecule-binding nucleic acids including aptamers of proteins to be analyzed. In this example, reference sequences were constructed with base sequences of 1,149 molecule-binding nucleic acids and base sequences of the following nucleic acids to be analyzed.

<Example 7-1> Protein Analysis

[0215] A sample for protein analysis was prepared from a biosample. The prepared protein sample was brought into contact with a molecule-binding RNA pool, and the formed protein-molecule-binding nucleic acid complex pool was isolated. Particularly, the protein sample was attached to an NC disk and was brought into contact with a molecule-binding nucleic acid pool including aptamers in a reaction solution, and the formed protein-molecule-binding nucleic acid complex pool was washed with a washing solution. Thereby, unbound or nonspecifically bound molecule-binding nucleic acids were removed and the disk was separated. Reverse transcription and PCR were performed on the molecule-binding RNA obtained from the molecule-binding nucleic acid pool binding to multiple proteins attached to the disk, to thus afford a DNA pool. The 136 ng DNA pool thus obtained was subjected to end repair, adenosine addition, adapter addition and PCR using a TruSeq DNA sample preparation kit v.2 (IIlumina). Washing was performed with Agencourt AMPure XP beads (Beckman-Coulter) included in the TruSeq kit at every step except for the adenosine base addition step. A sequencing library was constructed by performing PCR through 15-cycle reactions using the PCR primers included in the kit in accordance with the manufacturer's protocol.

<Example 7-2> DNA Analysis

[0216] In order to analyze nucleic acid information, a special oligonucleotide design is required to amplify a target gene. The oligonucleotide has the purpose of simultaneously amplifying many targets, and a target-specific sequence (a target hybridization nucleotide sequence) and a 5-flanking assembly spacer sequence (an overlapping sequence) may be used.

[0217] In order to produce multiple target loci assembly sequencing (mTAS) oligonucleotides having optimal lengths that can be annealed at a predetermined temperature, the present inventors used the computer program PrimerPlex2.75 software (PREMIER Biosoft, USA). Oligonucleotide probes were prepared from the target-specific sequence (target hybridization nucleotide sequence) and the 5-flanking assembly spacer sequence (overlapping sequence). The probes are about 25 bp long and can be annealed at Tm 60 C.

[0218] Each target genomic locus is designed to have a gap of 7 bp including the SNP position (i.e. the left of the SNP position: 3 bp; SNP position: 1 bp; and the right of the SNP position: 3 bp). The spacing of the gaps was adjusted to 0-3 bp to facilitate design. Although the assembly spacer sequence was manufactured arbitrarily, the annealing sites present on the assembly sequence were determined based on nearest-neighbor methods (BMC Genomics. 2016; 17: 486.) to calculate temperature values for overlapping regions between oligonucleotides.

[0219] A nucleic acid sample was isolated from the biosample using a known method. DNA (gDNA) was extracted from the human serum sample using a QIAamp DNA extraction kit (Qiagen, Germany).

[0220] Particularly, for the isolation of gDNA, a serum sample was placed in a 1.5 mL microcentrifuge tube, mixed with 30 L of protease K and 180 L of a buffer solution, and reacted at 56 C. for 1 hr. The reaction solution thus obtained was purified using a QIAamp spin column and then washed two times with a buffer solution. Next, gDNA was extracted after dissolution in 60 L of a buffer solution preheated at 70 C., and was then stored at 30 C. Each gDNA extracted above was quantified using a Qubit dsDNA HS Assay Kit and a Qubit 2.0 (Life Technologies, USA).

[0221] 10 ng gDNA stored at 30 C. was used to manufacture a library for NGS using panel primers of 17 target genes. The corresponding panel primers are shown in [Table 3] below.

[0222] The library for NGS was prepared using an Ion AmpliSeq Library Kit 2.0 (Life technologies).

[0223] Particularly, in order to obtain only the mutation site of the target gene in the DNA extracted from the sample, amplification was performed using the above panel primer pool. 4 L of 5HiFi Master Mix, 10 L of the panel primer pool and 10 ng DNA were mixed and sterile distilled water was added thereto so that the total volume of the reaction solution was 30 L, thus obtaining a mixed reaction solution. The mixed reaction solution thus prepared was amplified by repeating the procedures at 99 C. for 2 min, 99 C. for 15 sec and 60 C. for 4 min 21 times using a PCR device. The resulting amplification product was added with 2 L of a Fupa reagent (Thermo Fisher Scientific, USA; used for partial cleavage and phosphorylation), and reacted at 60 C. for 10 min, 55 C. for 10 min, and 60 C. for 30 min, whereby both ends of the amplification product were partially cleaved. The partially cleaved amplification product was added with 2 L of Ion P1 adapter and 2 L of Ion Xpress Barcode and reacted at 22 C. for 40 min and at 72 C. for 10 min, and a sequencing adapter and a barcode capable of distinguishing the samples were bound to the amplification product. The amplification product having the sequencing adapter and the barcode bound thereto was washed with 45 L of an AMPure XP solution and quantified using an Agilent DNA 1000 chip, thus preparing a sequencing library.

<Example 7-3> RNA Analysis

[0224] Serum samples were sampled in an amount of 1.5 ml each and centrifuged at 10,000 g for 5 min at room temperature to recover the cells. Then, total RNA of the recovered cells was extracted using an RNeasy Plus Mini Kit (Qiagen). The concentration and quality of the extracted RNA were measured at 260 nm and 280 nm using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del., USA).

[0225] The sequence of the cDNA library prepared from the RNA sample was analyzed using a simultaneous mass technique. For example, a cDNA library was synthesized in accordance with the manufacturer's protocol or through slight modification using a product such as mRNA-Seq Sample Preparation Kit (Illumina). 5-fold diluted Klenow DNA polymerase was used in the end-repair step of plasma cDNA. A PCR purification kit (QIAquick MinElute Kit (Qiagen, USA)) was used to purify the end-repaired and adenylated product. A 10-fold diluted paired end adapter was coupled with the plasma cDNA sample, and the adapter-coupled product was purified two times using AMPure XP beads (Agencourt, USA), followed by quantification using an Agilent DNA 1000 chip, thereby preparing a sequencing library.

<Example 7-4> miRNA Analysis

[0226] Whole-genome micro RNA (genome-wide miRNA) was analyzed and compared using small ribonucleic acid (small RNA) sequencing in human serum samples. Total RNA rich in small ribonucleic acids was extracted from the serum samples using a mirVAna miRNA isolation kit (Ambion, Austin, Tex.), and a miRNA library was prepared using an Illumina library preparation protocol (Illumina, San Diego, Calif., USA). Each library was indexed with an Illumina adapter (6-base barcode). The small ribonucleic acid (small RNA) library was subjected to size fractionation using a 6% TBE urea polyacrylamide gel, and 150 to 160 base-pair fractions were obtained from the gel and purified. The purified miRNA library was quantified using an Agilent DNA 1000 chip to give a sequencing library.

[0227] Although a sequencing library may be prepared by mixing the nucleic acid sample isolated from the fractionated biosample and the protein-molecule-binding nucleic acid complex pool isolated as described above, in this example, sequencing libraries prepared separately were mixed to determine the base sequences. The base sequences of the nucleic acids constituting the prepared sequencing library were compared with the reference sequences to determine the appearance frequency.

[0228] In order to determine whether the sequencing library for NGS may be used for sequencing, the length and the amount of the library constructed were measured using an Agilent Bioanalyzer 2100 (Agilent, USA) and a high-sensitivity chip, and libraries were used for sequencing, with lengths ranging from 100 to 400 bp and amounts of 100 pmol/L or more. Moreover, the libraries were subjected to quality control using a high-sensitivity chip.

[0229] As shown above, the PCR product, which was mixed for each sample and to which the barcode sequence of the corresponding sample was attached, was quantified using a Qubit fluorometer (Invitrogen). A 3 ng PCR product per sample was hybridized at a concentration of 5 pM in a flow cell on a cBOT cluster station of a HiSeq 3000. Bridge amplification using cBOT was performed 28 times per single DNA protein to form a cluster. Each cluster was linearized and hybridized with sequencing primers. The corresponding flow cell was loaded on a HiSeq 3000. This flow cell was analyzed with a HiSeq 3000 Single Read 80 Base Pair Recipe capable of sequencing 73 single-read bases and 7 multiplexed bases. Image production and analysis were performed through Illumina Real-Time Analysis (RTA), and base-call files and quality scores were confirmed in real time.

[0230] After termination of the sequencing of forward and reverse strands, quality analysis was performed using Casava software of Illumina, and moreover, downstream analysis was performed using a proprietary software analysis system and reference sequences.

[0231] The appearance frequencies of the nucleic acids constituting the determined sequencing library reflect information on nucleic acids and proteins as target molecules, and the biological meaning of the biosample can be determined based on the above analysis results with the biological meaning determination system. The biological meaning of the biological meaning determination system indicates physiological changes of the sample or the person from whom the sample was taken, and also indicates information necessary for the process of making a decision for the purpose of health care such as prevention, diagnosis, treatment, amelioration and therapy based on the clinical test values.

TABLE-US-00009 TABLE3 Targetgeneandprimer Target Forwardprimer ReversePrimer gene TCCTCATGTACTGGTCCCTCAT GGTGCACTGTAATAATCCAGACTGT KRAS CATACGCAGCCTGTACCCA GTGGATGCAGAAGGCAGACAG RET TGTCCTCTTCTCCTTCATCGTCT AGGAGTAGCTGACCGGGAA RET CCATCTCCTCAGCTGAGATGAC GGACCCTCACCAGGATCTTG RET CCTATTATGACTTGTCACAATGTCACCA TAGACGGGACTCGAGTGATGATT BRAF CACAGCAGGGTCTTCTCTGTTT CCTTCTGCATGGTATTCTTTCTCTTCC EGFR TGTCAAGATCACAGATTTTGGGCT ATGTGTTAAACAATACAGCTAGTGGGAA EGFR GGTGACCCTTGTCTCTGTGTTC AGGGACCTTACCTTATACACCGT EGFR GGAAACTGAATTCAAAAAGATCAAAGTGCT GGAAATATACAGCTTGCAAGGACTCT EGFR cttACCAGCTTGTTCATGTCTGGA AGAGGACTTCGCTGAATTGACC MET GCAGCGCGTTGACTTATTCATG CACAGCTACTCTCAGAAAGCACT MET AACTGAGCTTGTTGGAATAAGGATGTTAT CCATTTTGGTTTAATGTATGCTCCACAATC MET CCCATCCAGTGTCTCCAGAAGT CAAGTGACACTGGTTGTAAATATGCATTT MET GTTATGACAGGATTTGCACACATAGTT CCCAAGCCATTCAATGGGATCT MET CCCTTCTCTTCACAGATCACGA CAGACAGATCTGTTGAGTCCATGT MET GCTTGGGCTGCAGACATTTC GCCAGCTGTTAGAGATTCCTACC MET TGGTCCTGCACCAGTAATATGC ATTATAAGGCCTGCTGAAAATGACTGA KRAS CCATCCACAAAATGGATCCAGACA GCTCTGATAGGAAAATGAGATCTACTGTTT BRAF AGGAAATTCCCACTTAGGAACCATTG AGCAAACTCAGTTGAAATGGTTTGG MET GTTCAGTGTGTCAAACAGTATTCTTGAATG GTTGGATGAATTTCATAGACAATGGGATC MET ACAAGCATCTTCAGTTACCGTGAA AAAACTGCAATTCCTCTTGACTATTCTACA MET GCTATGGATGTTGCCAAGCTGT TAACATGAAAAAGGCTTTGGTTTTCAGG MET GCAACAGCTGAATCTGCAACTC ATTTTCATTGCCCATTGAGATCATCAC MET CTGTGTTTAAGCCTTTTGAAAAGCCA CCAAGTACAACAATTGTATTCACATAGCT MET CATAATTAAATGTTACGCAGTGCTAACCA GCAAACCACAAAAGTATACTCCATGGT MET CAGTCAAACCCTCAGGACAAGA CCCTCGGTCAGAAATTGGGAAA MET TCTCAGGAATCACTGACATAGGAGAAG CGAATGAAATTTCGAAGATCTCCATGTTT MET GCTGGTGGTCCTACCATACATG TTTTTAAAGACTCAGAGCAGGCCTATT MET GAGGCCAGATGAAATACTTCCTTCA AAAATCAGCAACCTTGACTGTGAATTTT MET TGTCCTTTCTGTAGGCTGGATGA GGTGGTAAACTTTTGAGTTTGCAGA MET GTGAAGTGGATGGCTTTGGAAAG AAACTGGAATTGGTGGTGTTGAATTTTT MET CGTCTCCTGGAGATGGATACTCT CTGTGGAGGAACTTTTCAAGCTG FGFR1 GCAGTTACTGGGCTTGTCCAT GAGGCGGAGAAGCTCTAACAC FGFR1 GCATGGACAGGTCCAGGTAC GGAAGACCTGGACCGCATC FGFR1 CCTTACCTGGTTGGAGGTCAA GGAGACGTCCCTGACCTTACA FGFR1 GAGTTCTTTGCTCCACTTGGGA CCACACTCTGCACCGCTAG FGFR1 GCACCTTACCTTGTTCAGGCAA TGGAGTATCCATGGAGATGTGGA FGFR1 AGGAATGCCTTCAAAAAGTTGGGA TCCAGTGCATCCATGAACTCTG FGFR1 GTGATGGCCGAACCAGAAGAAC CATGTGCCTCTGCCATTGTTG FGFR1 GAGAGAGGCCTTGGGACTGATA TCAGAAATGGAGATGATGAAGATGATCG FGFR1 AGCAGGTTGATGATATTCTTATGCTTCC CCACTCCCTTAGCCTTTATCCTG FGFR1 TTAAACCCAATGCCCAGACCCAAA CCCTTCTTCTTCCCATAGATGCTCT FGFR1 TCTCCTCTGAAGAGGAGtcatcatc GGTGTCCGTGTTCATCTGGAAC FGFR1 GGCAGAAAGAGGACTCCTCAGT CGACTGCCTGTGAAGTGGATG FGFR1 TGTAGATCCGGTCAAATAATGCCTC GGTTTCATCTGAGAAGCAAGGAGT FGFR1 GCATTAGAGGCCCAGAGAGAGA TCATCGTCTACAAGATGAAGAGTGGTA FGFR1 GCTGTGGAAGTCACTCTTCTTGG CTAACACCCTGTTCGCACTGA FGFR1 TTGGAATGGGACAAGATTTTCTTTGC CGAAAGACTGGTCTTAGGCAAAC FGFR1 AGGACAGAAGCATCACTTACACTTC CAACTTATGCCACTCTCTGTTTCC FGFR1 AAATGAAAAGCATGTAATCAGGACTTCCTA ACACTGCGCTGGTTGAAAAATG FGFR1 TGTGGTCAGGTTTGAATTCTTTGC GCCGTAGCTCCATATTGGACATC FGFR1 CATGCAATTTCTTTTCCATCTTTTCTGG CTCACAGGTGTTGGGCAGAT FGFR1 CTTTGTCATTTACAAGTACTTTGCAAACAC TCCCTAAAGCTGGAGTCCCAAATA ROS1 TTCTAGTAATTTGGGAATGCCTGGTT cgcctcTGAATATTTCTTTAATGTTGTCTT ROS1 GCCTAGGTGCTCCATAATGATGG AGTCTGGCATAGAAGATTAAAGAATCAAAA ROS1 ACCAATCATGATGCCGGAGAAAG ACCTGGTGTGGTTGTCAATACC ALK TCACCGAATGAGGGTGATGTTTT GCAGAGCCCTGAGTACAAGC ALK GGTTGTAGTCGGTCATGATGGT TGGCCTGTGTAGTGCTTCAAG ALK ccacttcacctagccAGAATTTTTC CTGACAGGCGATCTTGAACATCA EML4 AGATGATAGTATTTCTGCTGCAAGTACTTC CACATGATCTTCAGAGATTGCAAGAC EML4 CAAGAAGATGAAATCACTGTGCTAAAGG CAGCTTCAACTTTCAAAGAAAATATTGCAA EML4 CTCTGTCGGTCCGCTGAAT GCTGTGTGCGGAAGGAAAAA EML4 GTTCTAGTTCAGTCTTCTTCATTGTACCTT CGGAAGAAGGTAAACATTAGCTCTACAG EML4 TCTGTTAAGATATGGTTATCGAGGAAAGGA CAACCATTTCACACAGTCTGTATGG EML4 AGAGAACTCAGCGACACTACCT TCTGAGCTTTCCACAAGAAATCACTT EML4 GCTTCCAAATAGAAGTACAGGTAAGCT CTGAGCACATGTCAAGAGCAAATC EML4 TCTTGCCACACATCCCTTCAAA GCCAGTGTGAGGAGTTTCTGTG EML4 ATGGTATTTCTTTCTTAGAATGTTAGCCCTATC AAATTTACCTTTATTTCTGCTCCTTTTGCTTT EML4 GAGCATATGCTTACTGTATGGGACT GCTTTTGCGACTTACGAATAGATAGTTCTA EML4 TTTTTCATTTGTTTAAATGTGTATTGTTCCATG GGAGAAGGTGATGCTCGAATTTG EML4 gaggaaTCTCATTCTAATGATCAAAGTCCA CCATTCCCTAGCTCTGTACTTGG EML4 TGTCAGTTACATGTCTTTGATACTCAGAA GGGTGTTGAAGGTATTTTCGACAATTTAT EML4 CTTGGGAAAATTCAGATGATAGCCGTA CCAGACATACATAACTGTACATGCAAACAT EML4 CCATAGGGAGACTTTCTCATGTACTC TAGCATTTAAACATCCCACCACTTCA EML4 CCCTCCTTCCAAATGGACTTAATTTTAAA TGCACCACTTCCATTGGTTATACAG EML4 CCTCGAGCAGTTATTCCCATGTC AGACAATTCTGCAATGTTAGTTTTTCCC EML4 aacttttacagtttcttgaggtgattttaatgg TTTCAATTAGCAAAAATTAAACTAAGAAGGGCA EML4 ACAAAGGTATTCTGTTGTTTCATGTTTCC CAATGTCTCTAGGTACAGAAGGCAT EML4 GTTCTACTGAAGTTTTCTTCCAAATAGACACT CTCCAGTTAATAACATCCCATTTCTCATCT EML4 GGCAGTGTGTTCACACTTTGTC GTGGGACAAAATACCTGAGTTTAGAGTATT EML4 GACTAAAACTTTGAAGTAGTCATTTTTGTCTT GAATGAACATGGTAATTGGCCGA EML4 TGTCTTGTGTTTCAACAGAAGGAGAATAT GCTGTCATGGTAGAGAAGGATACG EML4 CCTGAGAAGCTCAAACTGGAGT cctggccTGATGTTTCCTTTTTAATTTTT EML4 gcacaccagcgttatgacaaag TGTGTGGATAGAAACTAGATCTCTGGTT EML4 GGTGGTTTGTTCTGGATGCAGA CAATTGCAGTGAAGTGCTGTGAAT EML4 AGTGGATAGGATTCATTCATTAATTGCCA TACAGCCAGGAAGGTACCATCT EML4 ATTTCTCTAGTCAACACTGACCTATTTTATTCT TCCCAATAATTTTACAACTTGTTCTACTTCACT EML4 GTAGCAGTAATTGAATTGATACTTGAAGGAGA CGCTCCAGGTCCAGAAGAAAATATG EML4 GCAAATACCATAATTACATGCGGTAAATCT GCCATGACAACTTGATGCTTATTAAACAAT EML4 TTTTTAAATGGCATTAGTTCTGTGTGCT GCTCAAAAGTGCCAAGTCCAATA EML4 TCTGTTACTCTATCCACACTGCAGAT ACTTCATGGCCACATAAACACAAAAC EML4 AACAGTATTGGCTAGCTGTTGAACT ATACTTACAGTACAATATTTCATAGTCTCCCGA EML4 CCCAGACAACAAGTATATAATGTCTAACTC CCCTGACAGACACATCTTAGCatatatata EML4 CAAGCACTATGATTATACTTCCTGTTTCT ACAAACCACTTCTTTACATCAGGTGT EML4 TTAATAAGCATCAAGTTGTCATGGCAAAAA GGCTCTACAGTAGTTTTGCTCCATA EML4 AGACTCAGGTGGAGTCATGCTTA CCTGGTCTAAGAGATGGGACTGA EML4 ATTTCTGAAACAGGCATGTCAAGAATG CCAGTTGATATCAGGTGACTGTCATTG EML4 AGCCATGTCACCAATGTCAGTTTTA GGCTTTGGTTAGAGTAGTATCCGCTA EML4 AGTTATCTTTGCCTCAGAATGAGACTG GTGGGAGAACTGCTTATTCTACTTTCC EML4 GCCCTTAAATGAGACAGCTGAAGA GCTTATCTCGTTGCATGGCTCTT EML4 GAGACCTTGGTGAGCCTCTTTATG ACATGCAGCTGAAGGAAAAGAGTT EML4 CAGCTATAAATGCAGGCTTCGAGTA GTTCCTCGTAAAATAAAGTTTCGTGATGT EML4 GGAAAGGCAGATCAATTTTTAGTAGGC ACCCTGAAAATGAAAGACACTCATTGTTAT EML4 GCTAATTTTTCTGCATCCCTGTGTT GGGATACTGAAACAGATGGACTTTACAAA EML4 GTGATAGCTGTTGCCGATGACT GAGTATCATGGAGAGGAATCAGTAACCTAT EML4 CTTTTGATGACATTGCATACATTCGAAAGA CAGTTATCTTTTCAGTTCAATGCATGCT PIK3CA ATCCGCAAATGACTTGCTATTATTGATG CCCAGGATTCTTACAGAAAACAAGTG NRAS GGGTGAGGCAGTCTTTACTCAC GCCGTTGTACACTCATCTTCCTAG ALK CCTCACCTCTATGGTGGGATCA GCTCGCCAATTAACCCTGATTACT NRAS CAGGTCTCTCCGGAGCAAA GCCAAGTCCCTGTGTACGA HER2 AGAACCTCTCAACATTGTCAGTTTTCT GCTCTGAGTAGAACCATTGCTCA MET TTGGCACAACAACTGCAGCAAA CCAGAACATTGTTCGCTGCATTG ALK GTCTCTCGGAGGAAGGACTTGA CAGACTCAGCTCAGTTAATTTTGGTTAC ALK CAGCTGGTGACACAGCTTATG CTCCGGAGAGACCTGCAAAGAG HER2 TATGCAGATTGCCAAGGTATGCA AATGGGAAGCACCCATGTAGAC HER2 GGGTGTCTCTCTGTGGCTTTAC ACTCTGTAGGCTGCAGTTCTCA ALK GCCAATGAAGGTGCCATCATTC CTCAGGCATCCCAGGCACAT AKT1 ATTTTACAGAGTAACAGACTAGCTAGAGACA AAAGAAAAAGAAACAGAGAATCTCCATTTTAGC PIK3CA GAAATTTAACAGGGTGTTGTTGTGCA CTGTTCATCTGACAGCTGGGAAT DDR2 GCTGGAGGAGCTAGAGCTTGAT GCTTGTGGGAGACCTTGAACAC MEK1 TGGGTGGTCAGCTGCAAC CATGCTTCAATTAAAGACACACCTTCTTTAA ALK GCTCTGAACCTTTCCATCATACTTAGAAAT ccagactaacaTGACTCTGCCCTATATAAT ALK AAAGAAGGTGTGTCTTTAATTGAAGCAT gggtctaatcccatctccagtct ALK TCAtgttagtctggttcctccaaga gggttatacttgcaacacagtct ALK agggaaggctgggtgaacc actgactttggctccagaacc ALK ggagcctaaggaagtttcagcaag cactgctgtgattgcactgaag ALK ggttctggagccaaagtcagtc aactataggaaacacaactgaccaagatc ALK caatcacagcagtggatttgagg aggcggaattagagcacagatc ALK GAAGAGCCACATCAtgaaaagatctct agttaccatccctgcctacaga ALK ggacctctttggactgcagttt ggtagagctattaggatttttcaaaacca ALK ggttgtcaatgaaatgaattcaccaacata ACAGAATCTACCCACTGAATCACAATTT ALK AAACTCCATGGAAGCCAGAACA ttcattcgatcctcaggtaacccta ALK tggaccgaccgtgatcagat ATCTGCCGGTAGAAGGGAGAT ALK CCTTTGAGGGATGGCACCATAT GAGACATGCCCAGGACAGATG ALK CCTTTCCCTCTGCCCTTTTCAA AGAGAGATAGGAAAATCGGTTTCTGAGTAT ALK GGCTCACAGGCTGAACAGAAAT ACTTCTAGCTCCCACATGCTTC ALK CATTACATAGGGTGGGAGCCAAA TGTGTATCCTCCTGGCTGATCA ALK GCTTTCACCATCGTGATGGACA AAACGGAAGCTCCCAACCTT ALK CTGATCAGCCAGGAGGATACAC CCAAGGTGTCACTTCGTTATGC ALK CCCACCCAATTCCAGGGACTA GGCTTTCTCCGGCATCATGAT ALK TGCTTTTCTAACTCTCTTTGACTGCA GATTGTGGCACAGAGATTCTGATACTT MET CACAGCCTGAGACACTATTCAGTC ACTCTCGCTGATCCTCTCTGT ALK ATGTATTTAACCATGCAGATCCTCAGTTT ATCTTGTTCTGTTTGTGGAAGAACTCT PTEN GGATGAGCTACCTGGAGGATGT CCATCTGCATGGTACTCTGTCT HER2 TCTTTGTCTTCGTTTATAAGCACTGTCA GTGTTCTTGCATAAAAACACTTCAAATG ROS1 TGAAACTTGTTTCTGGTATCCAAAAATCAT CTGGCTTGCAAAAATCCAGTAGTAG ROS1 TTCCTTTAGGAAATGTTAACAGTGCATTTG GATTAAAAATGGTGTAGTATGATTTGTGTACTT ROS1 ACTTACCAAAGGTCAGTGGGATTG CTCTGTGTGCTTAGGTAGAGCTG ROS1 CTGTGACCACACCTGTCATGTA AAGAAGGCAAGACCCTTAAAGGAG RET AGCTCTACCTAAGCACACAGAGTAATA GTGGTTTGTTgctctctgcaaaaa ROS1 GGCCCAATGTGTGGATAGAAC CAGGACAACTTCTCTACATAGCCA RET GTGTGGATAGAACTTTGGTGGGA GAAGTGTCCAGGACAACTTCTCT RET GGGTGGCTATGTAGAGAAGTTGT CTACATGACAGGTGTGGTCACA RET CGAAGTACTGAGTCCAAGCCAT GACAAGTTCCAATGTGCAGAGAAC RET CTTCTGCACTGAAATCTTTCTACAGAATATATT gaatctagataccttgccggtgaag ROS1 cgactagaagcaactccgttca gctcactgctcttccttctctct ROS1 tagattcgctcatcaaaattgactagaagg gcacagttctttggaaaagcaatttc ROS1 gggaaattgcttttccaaagaactg cccagggagttcagtaagcttag ROS1 atcaaaagcaaggtgtttttgcttt ATAATGCCAACTATTTAGTATCCAAAGACTGAG ROS1 AAAAACTAATTAAATCCAGGTAAAAAGCCAT TCGTTTATGGGTGATTTTGACAAATAAGTTTT ROS1 GCCATATATAAGTACACAAATCATACTACACCA GTTTGTTTTGGTTTATTTTGACTCGTTTATGG ROS1 TCACCCATAAACGAGTCAAAATAAACCA CAATGATAAACACTCTTGTACTCTGCaaaa ROS1 CTGCACATTGGAACTTGTCCATG TTCTCCTAGAGTTTTTCCAAGAACCAAG RET GTGGGAATTCCCTCGGAAGAA TGCCTGGCAGGTACCTTTC RET AGTCACTGTCCCTGTGACCAT ACGTAGGGCTATATAATACCAGAAAACTCA RET GCATAGGGACACGTTTCTGTCAT AGGCTGTGCTCATTACACCAG RET CATTTGGAACAGAGGAAAATTTTGACCTC AGaccactattgccctcttacaga RET CCAGTGCCAGCTGGTGTAA GTGGGAAGGCCTGAGAACAC RET GGGCAGTAAATGGCAGTACC TCGGCACCACTGGGTACAG RET TCACATCCAGGTTATATTCCTCAGTAGAAA gtaagcttcgttccaatactitttctacat ROS1 TTTAGGACCAAGAAATCTCAGTCTTTGG GTTTCCTCTACACAACTGAAACTACCT ROS1 CTCAGTCTTTGGATACTAAATAGTTGGCA tcctcatgccatagtttgccag ROS1 GTCCCAACCATGTCAAAATTACAGAC CCATGGGCTAGACACCACT HER2 atttgctcttccatgacaggctta caagtatctcctgatggatgaatgga BIM ccagtgtgtgtatatcacatacttcattta agcaattccacttccaagtatatatccaaaaa BIM AAAGTTAATGTACCGAGGTAAGTTTTCAGT CTGTAGAATGTCCAGAGAAAATTATGGACT BIM GAAAGGACTTAGCCAGATGTGAGTTT CAAAGTCAAGAGAACCACTTATCAACTCA BIM GTCTTAGTTCATGCCTGAAGACCA TGACTTCTTTGTGGAAAATGTATTTTGCAA BIM ATTCTTTACTCAACCCTATCCATGAAGTTC CTGGCTAAGGCGAACCTCTTTAT BIM CATTTCTAAATACCATCCAGCTCTGTCT CATGTGTAGCTGCTGGGATG BIM GCACACCTGTGAGGTGGTG CTGAAATGAGTTCACGAGCAGTAGTA BIM GGGCTGGAAGTTTTATTATTGCTGT CCTGTTAACTCATTTAGTAAGCAAGGATGT BIM GTTAACGTCTTCCTTCTCTCTCTGT TGAGGTTCAGAGCCATGGAC EGFR CATGCGAAGCCACACTGACGT CTTTGTGTTCCCGGACATAGTCCA EGFR TCATCACGCAGCTCATGCCCTT GTGAGGATCCTGGCTCCTTATCTC EGFR gctggtACTTTGAGCCTTCACAG CACCAGCCATCACGTATGCTTC HER2 TCTCCCATACCCTCTCAGCGTA CAGCCATAGGGCATAAGCTGTG HER2

[0232] As is apparent from [Table 3], the primers were 187 forward primers and 187 reverse primers, and the sequence numbers were designated as SEQ ID NO: 9 to SEQ ID NO: 383 in the order described in Table 3 above.