KR101665561B1 - An Apparatus for Diagnosing Cancer and an Method for The Same - Google Patents

Abstract

The present invention relates to an oligonucleotide group for cancer detection of a specific sequence hybridized with a nucleic acid of a cancer cell, a cancer diagnostic kit comprising an oligonucleotide group of a specific sequence, and a method for providing information necessary for diagnosis or prognosis of cancer. The method of the present invention makes it possible to early diagnose a specific cancer according to the mutation of a specific gene using RNA, unlike the case of examining a somatic mutation using existing gDNA. According to the method of the present invention, it is possible to detect a mutation that has not been detected by gDNA, and all cancer-related gene mutation tests can be performed with a single RNA extraction, thereby making it possible to perform an accurate and quick diagnosis. In particular, the method of the present invention enables simultaneous detection of all mutations associated with thyroid cancer, thereby enabling the diagnosis of thyroid cancer to be rapidly and accurately performed.

Description

An Apparatus for Diagnosing Cancer and an Method for The Same}

The present invention relates to a novel device and method for diagnosing cancer.

According to a report by the International Cancer Institute (IARC) under the World Health Organization (WHO), the number of new cancer cases in 184 countries around the world is 12.7 million in 2008, and it is expected to increase to 22.2 million in 2030. Currently, cancer diagnosis in clinical practice is carried out through an interview, physical examination, and clinical pathology examination, and once suspected, it is proceeded with radiographic examination and endoscopy, and finally, it is confirmed by a biopsy. However, with the currently used clinical test method, diagnosis is possible only when the number of cancer cells is 1 billion cells and the diameter of the cancer is 1 cm or more. In many cases, research on an accurate and early diagnosis method is being actively conducted.

In particular, the number of thyroid cancers has been increasing at a very rapid rate in the last 10 years. According to the annual report of the National Cancer Registration Project published from 1999 to 2005, the incidence of thyroid cancer among all cancers was 3.3% (7th place) in 1999. In 2005, it increased to 8.9% (5th place), and especially in women, it became the most common cancer among female cancers, surpassing breast cancer from 6.5% (7th) in 1999 to 16.7% (1st) in 2005.

Thyroid cancer has a higher incidence than other cancers, but if the diagnosis is made early and surgery can be performed early, the survival rate is high. Therefore, it is also very important to establish an early and accurate diagnosis method.

In the conventional method for diagnosing thyroid cancer, ultrasound reading or histopathological reading of thyroid cells or thyroid tissue has been used, but there are many problems in cases where reading is difficult or unclear.

In addition, the method of detecting and diagnosing a gene mutation related to cancer is BRAF (mutation appears in thyroid cancer, melanoma, colorectal cancer, ovarian cancer, hairy cell leukemia), KRAS (thyroid cancer, Mutations appear in pancreatic cancer, colon cancer, lung cancer, breast cancer, and prostate cancer), NRAS (mutations appear in thyroid cancer, acute leukemia, chronic leukemia, brain tumors), or HRAS (mutations appear in thyroid cancer, gastric cancer, bladder cancer, and prostate cancer) genes RET/PTC or PAX8/PPARγ gene rearrangement mutations (which are thyroid cancer specific mutations), which cannot be checked for mutations and tested using DNA, require separate RNA to be tested, so it is inconvenient to separate DNA and RNA separately. There is a problem in that it takes a long time to diagnose, and when only a mutation of one of the genes is detected, the accuracy of cancer diagnosis is lowered.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors have made intensive research efforts to develop a method for accurately and conveniently diagnosing cancer. As a result, when using the oligonucleotide group of primers and probes having a specific sequence of the present invention, RNA extracted from cancer cells or tissues or cDNA obtained by reverse transcription thereof is used as a template to detect mutations of BRAF, KRAS, NRAS and HRAS genes as well as RET The present invention was completed by confirming that both the rearrangement detection of /PTC and PAX8/PPARγ genes are possible, so that the diagnosis of cancer can be accurately and conveniently.

Accordingly, an object of the present invention is to provide a group of oligonucleotides for detection of cancer that hybridizes with a nucleic acid of a cancer cell.

Another object of the present invention is to provide a cancer diagnostic kit comprising a specific group of oligonucleotides.

Another object of the present invention is to provide a method of providing information necessary for diagnosis or prognosis of cancer.

Other objects and advantages of the present invention will become more apparent by the following detailed description, claims and drawings.

According to an aspect of the present invention, the present invention provides (i) an oligonucleotide group consisting of primers of the first and second sequences of the sequence listing; (Ii) a group of oligonucleotides consisting of a primer selected from the group consisting of the primers of SEQ ID NOs: 4 to 7 and a primer of SEQ ID NO: 8; (Iii) a group of oligonucleotides consisting of primers selected from the group consisting of primers of SEQ ID NOs: 10 to 12 of Sequence Listing and primers of SEQ ID NO: 13; (Iv) a group of oligonucleotides consisting of primers of SEQ ID NOs: 15, 16, and 17 of the Sequence Listing; (V) an oligonucleotide group consisting of a primer of SEQ ID NO: 18 and a primer of SEQ ID NO: 19 or 20; (Vi) a group of oligonucleotides consisting of primers of SEQ ID NOs: 23 and 25; (Vii) a group of oligonucleotides consisting of primers of SEQ ID NOs: 24 and 25; And (viii) an oligonucleotide group selected from the group consisting of an oligonucleotide group consisting of primers of SEQ ID NOs: 28, 29, and 30 of the Sequence Listing, wherein the oligonucleotide group is used for detection of cancer hybridizing to nucleic acids of cancer cells. Provides a group of oligonucleotides.

The present inventors have made intensive research efforts to develop a method for accurately and conveniently diagnosing cancer. As a result, when using the oligonucleotide group of primers and probes having a specific sequence of the present invention, RNA extracted from cancer cells or tissues or cDNA obtained by reverse transcription thereof is used as a template to detect mutations of BRAF, KRAS, NRAS and HRAS genes as well as RET It was confirmed that both the rearrangement detection of /PTC and PAX8/PPARγ genes are possible, so that the diagnosis of cancer can be accurately and conveniently.

In the specification of the present invention, the term "primer" is complementary to the 5'-end sequence or the 3'-end sequence of the target nucleic acid site to be amplified during the nucleic acid amplification reaction, and in suitable conditions (ie, It refers to a single-stranded oligonucleotide capable of serving as an initiation point of a polymerase reaction of a template-indicating nucleic acid under four different nucleoside triphosphates and polymerase). Suitable lengths of primers are typically 15-30 nucleotides, although varying depending on various factors such as temperature and application of the primer. Short primer molecules generally require lower temperatures to form sufficiently stable hybridization complexes with the template. The primer used in the present invention is hybridized or annealed at one site of the template to form a double-chain structure. Conditions for nucleic acid hybridization suitable for forming such a double-stranded structure are Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization. , A Practical Approach, IRL Press, Washington, DC (1985).

In the present specification, the term “nucleic acid” has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic structural units in nucleic acid molecules, are not only natural nucleotides, but also analogs with modified sugar or base sites. (analogue) (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)).

According to an embodiment of the present invention, the sequence list of the present invention is the first sequence, the fourth to the seventh sequence, the 10th to the 12th sequence, the 15th sequence, the 16th sequence, the 18th sequence, the 23rd sequence, SEQ ID NOs: 24, 28, and 29 are forward primers.

According to an embodiment of the present invention, the second sequence, the 8th sequence, the 13th sequence, the 17th sequence, the 19th sequence, the 20th sequence, the 25th sequence, and the 30th sequence of the present invention are reverse primers (reverse primers). primer).

According to one embodiment of the present invention, the cancer of the present invention is thyroid cancer, melanoma, colon cancer, ovarian cancer, hairy cell leukemia, pancreatic cancer, lung cancer, breast cancer, prostate cancer, acute leukemia, Chronic leukemia, brain tumor, gastric or bladder cancer. According to a specific embodiment of the present invention, the cancer of the present invention is thyroid cancer.

According to one embodiment of the present invention, the oligonucleotide group (i) of the present invention is hybridized with nucleic acids of thyroid cancer, melanoma, colon cancer, ovarian cancer or hairy cell leukemia cells.

According to one embodiment of the present invention, the oligonucleotide group (ii) of the present invention is hybridized with nucleic acids of thyroid cancer, pancreatic cancer, colon cancer, lung cancer, breast cancer or prostate cancer cells.

According to one embodiment of the present invention, the oligonucleotide group (iii) of the present invention is hybridized with nucleic acids of thyroid cancer, acute leukemia, chronic leukemia or brain tumor cells.

According to one embodiment of the present invention, the oligonucleotide group (iv) or (v) of the present invention is hybridized with a nucleic acid of thyroid cancer, gastric cancer, bladder cancer or prostate cancer.

According to an embodiment of the present invention, the oligonucleotide group of the present invention further comprises a probe having the oligonucleotide group (i) of the present invention having the third sequence of the sequence listing; The oligonucleotide group (ii) of the present invention further comprises a probe having SEQ ID NO: 9; Or the oligonucleotide group (iii) of the present invention further comprises a probe having the 14th sequence of the sequence listing; Or the oligonucleotide group (iv) of the present invention further comprises a probe having the 21st sequence of sequence listing; The oligonucleotide group (v) of the present invention further comprises a probe having SEQ ID NO: 22; The oligonucleotide group (vi) of the present invention further comprises a probe having SEQ ID NO: 26; The oligonucleotide group (vii) of the present invention further comprises a probe having SEQ ID NO: 27; Alternatively, the oligonucleotide group (viii) of the present invention further includes a probe having the 31st sequence of SEQ ID NO.

According to another embodiment of the present invention, the group of oligonucleotides of the present invention further comprises a probe having the third sequence of the oligonucleotide group (i) of the present invention; The oligonucleotide group (ii) of the present invention further comprises a probe having SEQ ID NO: 9; The oligonucleotide group (iii) of the present invention further comprises a probe having the 14th sequence of the sequence listing; The oligonucleotide group (iv) of the present invention further comprises a probe having the 21st sequence of Sequence Listing; The oligonucleotide group (v) of the present invention further comprises a probe having SEQ ID NO: 22; The group of oligonucleotides of the present invention (vi) further comprises a probe having the 26th sequence of Sequence Listing; The oligonucleotide group (vii) of the present invention further comprises a probe having SEQ ID NO: 27; And the oligonucleotide group (viii) of the present invention additionally includes a probe having the 31st sequence in the sequence listing.

In the present specification, the term "probe" is a single-stranded nucleic acid molecule, and includes a sequence complementary to a target nucleotide sequence. According to an embodiment of the present invention, the probe of the present invention can be modified within a range in which the advantages of the probe of the present invention, that is, hybridization specificity, are not impaired. For example, a reporter fluorescent substance or a fluorescent inhibitor (quencher) may be tagged at the end of the probe oligonucleotide to be used as a probe label. In the probe sequence of the present invention, the label of the probe may be changed to a variety of labels used in the art. Suitable labels are fluorophores (e.g., fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia)), chromophores, chemiluminescent groups, magnetic particles, radioactive isotopes. Elements (P ³² and S ³⁵ ), mass labels, electron congesting particles, enzymes (alkaline phosphatase or horseradish peroxidase), cofactors, substrates for enzymes, heavy metals (e.g. gold) and antibodies, streptavidin , Biotin, digoxigenin, and hapten having a specific binding partner such as a chelating group, but are not limited thereto. Labeling can be carried out by various methods commonly practiced in the art, such as nick translation method, random priming method (Multiprime DNA labeling systems booklet, "Amersham" (1989)) and kineation method (Maxam & Gilbert, Methods. in Enzymology , 65:499 (1986)). The label provides a signal that can be detected by fluorescence, radioactivity, colorimetric measurement, gravimetric measurement, X-ray diffraction or absorption, magnetic, enzymatic activity, mass analysis, binding affinity, high frequency hybridization, and nanocrystals.

According to another aspect of the present invention, the present invention provides (i) an oligonucleotide group consisting of primers of the first and second sequences of the sequence listing; (Ii) a group of oligonucleotides consisting of a primer selected from the group consisting of the primers of SEQ ID NOs: 4 to 7 and a primer of SEQ ID NO: 8; (Iii) a group of oligonucleotides consisting of primers selected from the group consisting of primers of SEQ ID NOs: 10 to 12 of Sequence Listing and primers of SEQ ID NO: 13; (Iv) a group of oligonucleotides consisting of primers of SEQ ID NOs: 15, 16, and 17 of the Sequence Listing; (V) an oligonucleotide group consisting of a primer of SEQ ID NO: 18 and a primer of SEQ ID NO: 19 or 20; (Vi) a group of oligonucleotides consisting of primers of SEQ ID NOs: 23 and 25; (Vii) a group of oligonucleotides consisting of primers of SEQ ID NOs: 24 and 25; And (viii) provides a cancer diagnostic kit comprising an oligonucleotide group selected from the group consisting of the oligonucleotide group consisting of the primers of SEQ ID NOs: 28, 29, and 30 of the Sequence Listing.

According to one embodiment of the present invention, the cancer of the present invention is thyroid cancer, melanoma, colon cancer, ovarian cancer, hairy cell leukemia, pancreatic cancer, lung cancer, breast cancer, prostate cancer, acute leukemia, Chronic leukemia, brain tumor, gastric or bladder cancer. According to a specific embodiment of the present invention, the cancer of the present invention is thyroid cancer.

According to another embodiment of the present invention, the diagnostic kit for thyroid cancer, melanoma, colon cancer, ovarian cancer or hairy cell leukemia of the present invention comprises an oligonucleotide group (i).

According to another embodiment of the present invention, the diagnostic kit for thyroid cancer, pancreatic cancer, colon cancer, lung cancer, breast cancer or prostate cancer of the present invention includes an oligonucleotide group (ii).

According to one embodiment of the present invention, the diagnostic kit for thyroid cancer, acute leukemia, chronic leukemia or brain tumor of the present invention includes an oligonucleotide group (iii).

According to one embodiment of the present invention, the diagnostic kit for thyroid cancer, gastric cancer, bladder cancer or prostate cancer of the present invention includes an oligonucleotide group (iv) and/or (v).

According to one embodiment of the present invention, the diagnostic kit of the present invention further comprises a probe having the oligonucleotide group (i) of the present invention having the third sequence of the sequence listing; The oligonucleotide group (ii) of the present invention further comprises a probe having SEQ ID NO: 9; Or the oligonucleotide group (iii) of the present invention further comprises a probe having the 14th sequence of the sequence listing; Or the oligonucleotide group (iv) of the present invention further comprises a probe having the 21st sequence of sequence listing; The oligonucleotide group (v) of the present invention further comprises a probe having SEQ ID NO: 22; The oligonucleotide group (vi) of the present invention further comprises a probe having SEQ ID NO: 26; The oligonucleotide group (vii) of the present invention further comprises a probe having SEQ ID NO: 27; Alternatively, the oligonucleotide group (viii) of the present invention further includes a probe having the 31st sequence of SEQ ID NO.

According to another embodiment of the present invention, the diagnostic kit of the present invention further comprises a probe in which the oligonucleotide group (i) of the present invention has the third sequence of Sequence Listing; The oligonucleotide group (ii) of the present invention further comprises a probe having SEQ ID NO: 9; The oligonucleotide group (iii) of the present invention further comprises a probe having the 14th sequence of the sequence listing; The oligonucleotide group (iv) of the present invention further comprises a probe having the 21st sequence of Sequence Listing; The oligonucleotide group (v) of the present invention further comprises a probe having SEQ ID NO: 22; The oligonucleotide group (vi) of the present invention further comprises a probe having the 26th sequence of SEQ ID NO; The oligonucleotide group (vii) of the present invention further comprises a probe having SEQ ID NO: 27; And the oligonucleotide group (viii) of the present invention additionally includes a probe having the 31st sequence in the sequence listing.

According to one embodiment of the present invention, the diagnostic kit of the present invention additionally includes a reverse transcriptase.

According to another aspect of the present invention, the present invention comprises the steps of: (a) obtaining a nucleic acid from a biological sample isolated from a human; And (b) the nucleic acid of step (a) (i) detection of a mutation of the BRAF gene, (ii) detection of a mutation of the KRAS gene, (iii) detection of a mutation of the NRAS gene, (iv) detection of a mutation of the HRAS gene, (v) It provides a method of providing information necessary for diagnosis or prognosis of cancer comprising the step of performing a detection selected from the group consisting of) rearrangement detection of RET/PTC gene and (vi) rearrangement detection of PAX8/PPARγ gene. .

According to one embodiment of the present invention, the cancer of the present invention is thyroid cancer, melanoma, colon cancer, ovarian cancer, hairy cell leukemia, pancreatic cancer, lung cancer, breast cancer, prostate cancer, acute leukemia, Chronic leukemia, brain tumor, gastric or bladder cancer. According to a specific embodiment of the present invention, the cancer of the present invention is thyroid cancer.

The method of the present invention will be described in detail for each step as follows:

Step (a): Acquisition of nucleic acids from biological samples isolated from humans

According to the present invention, a nucleic acid is first obtained from a biological sample isolated from humans.

In the present specification, the term “biological sample” refers to a biological sample of an animal separated from the body, and refers to a sample of blood, plasma, serum, urine, bone marrow, cells, hair, or tissue, and according to one embodiment of the present invention, the present invention The biological sample of is a specific cell or tissue sample. According to another embodiment of the present invention, the biological sample of the present invention is a thyroid cell or tissue sample.

According to one embodiment of the present invention, the nucleic acid of the present invention is RNA.

Step (b): detection of specific gene mutation and/or gene rearrangement

After performing step (a), (i) mutation of BRAF gene, (ii) mutation of KRAS gene, (iii) mutation of NRAS gene, (iv) mutation of HRAS gene of nucleic acid obtained in step (a), ( V) Rearrangement of the RET/PTC gene and/or (vi) rearrangement of the PAX8/PPARγ gene is detected.

According to one embodiment of the present invention, the detection of the present invention is carried out using an amplification reaction.

In the present specification, the term “amplification reaction” refers to a reaction to amplify a gene or nucleic acid molecule. Various amplification reactions have been reported in the art, which are polymerase chain reaction (hereinafter referred to as PCR) (U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reaction (hereinafter referred to as RT-PCR). (Sambrook et al., Molecular Cloning.A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001)), Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822), ligase chain reaction ( ligase chain reaction; LCR) (17, 18), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) (19) ( WO 88/10315), self sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (US Patent No. 6,410,276) , Consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Patent No. 5,413,909) And 5,861,245), nucleic acid sequence based amplification (NASBA) (U.S. Patent Nos. 5,130,238, 5,409,818, 5,554,517, and 6,063,603), strand displacem ent amplification (21, 22) and loop-mediated isothermal amplification; LAMP) 23, but is not limited thereto. Other amplification methods that can be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09/854,317.

Polymerase chain reaction (PCR) is the most well-known gene or nucleic acid amplification method, and its many modifications and applications have been developed. In the diagnostic method of the present invention, not only the traditional PCR method, but also the touchdown PCR 24 and the hot start PCR 25 and 26, which have modified the traditional PCR procedure to improve the specificity or sensitivity of PCR. , Nested PCR (2) and booster PCR (27) can be used. In addition, real-time PCR, differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction: IPCR), vectorette PCR, TAIL-PCR (thermal asymmetric interlaced PCR), and multiplex PCR may also be used. For more information on PCR, see McPherson, MJ, and Moller, SG PCR . BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

According to a specific embodiment of the present invention, the detection of the present invention is carried out using real-time PCR. Real-time PCR is a technique that monitors and analyzes the increase of PCR amplification products in real-time (Levak KJ, et al., PCR Methods Appl. , 4(6): 357-62(1995)). The PCR reaction can be monitored by recording the amount of fluorescence emission in each cycle during the exponential phase where the increase in PCR product is proportional to the initial amount of the target template. The higher the starting copy number of the nucleic acid target, the faster the increase in fluorescence is observed and has a lower Ct value (threshold cycle). A distinct increase in fluorescence above the baseline measured between 3-15 cycles indicates the detection of accumulated PCR products. Compared to conventional PCR methods, real-time PCR has the following advantages: (a) Conventional PCR is measured in a plateau, whereas real-time PCR is performed during the exponential growth phase. Data can be obtained; (b) the increase in reporter fluorescence signal is directly proportional to the number of amplicons generated; (c) the disassembled probe provides permanent record amplification of the amplicon; (d) an increase in detection range; (e) requires at least 1,000 times less nucleic acid than conventional PCR methods; (f) detection of amplified DNA is possible without separation through electrophoresis; (g) it is possible to obtain increased amplification efficiency by using a small amplicon size; And (h) the risk of contamination is low.

When the amount of PCR amplification product reaches the amount detectable by fluorescence, an amplification curve starts to occur, the signal rises exponentially, and then reaches a stagnation state. As the initial amount of DNA increases, the number of cycles to reach the detectable amount of amplification product decreases, so the amplification curve appears faster. Therefore, when a real-time PCR reaction is performed using a stepwise diluted standard sample, amplification curves arranged at equal intervals in the order of a large amount of initial DNA are obtained. Here, if a threshold is set at an appropriate point, the point Ct value where the threshold and the amplification curve intersect is calculated. In real-time PCR, PCR amplification products are detected through fluorescence. Detection methods are largely an interchelating method (SYBR Green I method), a method using a fluorescently labeled probe (TaqMan probe method), and the like. Since the interchelating method detects all double-stranded DNA, there is no need to prepare a probe for each gene, so it is possible to construct a reaction system at low cost. While the method using a fluorescently labeled probe is expensive, it has high detection specificity, so that even similar sequences can be distinguished and detected. According to certain embodiments of the present invention, the methods and kits of the present invention utilize the TaqMan probe method.

First, the interchelating method is a method using a double-stranded DNA binding die, and non-specific amplification using a non-sequence-specific fluorescent intercalating reagent (SYBR Green I or ethidium bromide) and amplicon production including a primer-dimer complex Is to quantify. This reagent does not bind to ssDNA. SYBR Green I is a fluorescent die that binds to the minor groove of double-stranded DNA. It hardly shows fluorescence in solution, but is an interchelator that shows strong fluorescence when bound to double-stranded DNA (Morrison TB, Biotechniques., 24(6): 954-8, 960, 962(1998)). Therefore, since fluorescence is emitted through the binding between SYBR Green I and double-stranded DNA, the amount of amplification product produced can be measured. SYBR Green real-time PCR is accompanied by an optimization process such as melting point or dissociation curve analysis for amplicon identification. Normally, SYBR green is used for a singleplex reaction, but can be used for a multiplex reaction when accompanied by a melting curve analysis (Siraj AK, et al., Clin Cancer Res. , 8( 12): 3832-40 (2002); And Vrettou C., et al., Hum Mutat. , Vol 23 (5): 513-521 (2004)).

The Ct (cycle threshold) value refers to the number of cycles in which the fluorescence generated in the reaction exceeds the threshold, which is inversely proportional to the logarithm of the initial copy number. Therefore, the Ct value assigned to a particular well reflects the number of cycles in which a sufficient number of amplicons have accumulated in the reaction. The Ct value is the cycle in which an increase in ΔRn is first detected. Rn refers to the size of the fluorescence signal generated during PCR at each time point, and ΔRn refers to the fluorescence emission intensity (normalized reporter signal) of the reporter die divided by the fluorescence emission intensity of the reference die. The Ct value is also called Cp (crossing point) in LightCycler. The Ct value represents the point at which the system begins to detect an increase in the fluorescence signal associated with the exponential growth of the PCR product in the log-linear phase. This period provides the most useful information about the reaction. The slope of the log-linear step represents the amplification efficiency (Eff) (http://www.appliedbiosystems.co.kr/).

On the other hand, the TaqMan probe is typically a primer (e.g., 20-30 nucleotides) comprising a fluorophore at the 5'-end and a quencher at the 3'-end (e.g., TAMRA or non-fluorescent quencher (NFQ)). ) Is a longer oligonucleotide. Excited fluorescent material transfers energy to a nearby quencher rather than emitting fluorescence (FRET = Frster or fluorescence resonance energy transfer; Chen, X., et al., Proc. Natl Acad Sci USA , 94(20): 10756-61(1997)). Therefore, when the probe is normal, no fluorescence is generated. TaqMan probes are designed to anneal to the internal sites of PCR products. According to a specific embodiment of the present invention, the TaqMan probe of the present invention is SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 14, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 26 , Sequence listing may be designed as a sequence comprising the 27th sequence or the sequence listing 31st sequence.

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

The reporter molecule and quencher molecule bound to the TaqMan probe include a fluorescent substance and a non-fluorescent substance. Fluorescent reporter molecules and quencher molecules that can be used in the present invention may be any known in the art, and examples thereof are as follows (the number in parentheses is the maximum emission wavelength expressed in nanometers): Cy2 ^TM (506), YOPRO ^TM -1 (509), YOYO ^TM -1 (509), Calcein (517), FITC (518), FluorX ^TM (519), Alexa ^TM (520), Rhodamine 110 (520), 5 -FAM (522), Oregon Green ^TM 500 (522), Oregon Green ^TM 488 (524), RiboGreen ^TM (525), RhodamineGreen ^TM (527), Rhodamine 123 (529), Magnesium Green ^TM (531), Calcium Green ^TM (533), TO-PRO ^TM -1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY ^TM R (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3 ^TM (570), Alexa ^TM 546 (570), TRITC (572), Magnesium Orange ^TM (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange ^TM (576) , Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red ^TM (590), Cy3.5 ^TM (596), ROX (608), Calcium Crimson ^TM (615), Alexa ^TM 594 (615) , Texas Red (615), Nile Red (628), YO-PRO ^TM -3 (631), YOYO ^TM -3 (631), R-phycocyanin (642), CPhycocyanin (648), TO-PRO ^TM -3 ( 660), TOTO3 (660), D iD DilC(5) (665), Cy5 ^TM (670), Thiadicarbocyanine(671), Cy5.5 (694), HEX (556), TET (536), VIC(546), BHQ-1(534), BHQ -2 (579), BHQ-3 (672), BiosearchBlue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL FluorRed 610 (610), CAL Fluor Red 635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705) and Quasar 705 (610). According to certain embodiments of the present invention, the reporter molecule and the quencher molecule comprise VIC and FAM labels.

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

In addition, the non-fluorescent material used for the reporter molecule and the quencher molecule bound to the TaqMan probe may include a minor groove binding (MGB) moiety. The term "MGB-conjugate probe" as used herein refers to a probe conjugated with MGB at the 3'-end of the probe. MGB is a substance that binds to the minor grooves of DNA with high affinity.Netropsin, distamicin, lexitropsin, mithramycin, chromomycin A3, olivo Mycin, anthramycin, sibiromycin, pentamidine, stilbamidine, berenil, CC-1065, Hoechst 33258, DAPI (4-6- diamidino-2-phenylindole), dimers, trimers, tetramers and pentamers of CDPI, N-methylpyrrole-4-carbox-2-amide (MPC) and dimers, trimers, tetramers and pentamers thereof. It does not become.

Conjugation of the probe and MGB significantly increases the stability of the hybrid formed between the probe and its target. More specifically, the increased stability (ie, increased degree of hybridization) results in an increased melting temperature (Tm) of the hybrid duplex formed by the MGB-conjugate probe compared to the normal probe. Therefore, MGB stabilizes the Van der Waals force to increase the melting temperature (Tm) of the MGB-conjugate probe without increasing the probe length, thereby allowing shorter probes (e.g., 21 or less) in Taqman real-time PCR under more stringent conditions. Nucleotides).

In addition, the MGB-conjugate probe removes the background fluorescence more efficiently.

According to certain embodiments of the present invention, the 5'-end of the probe of the present invention is labeled with a fluorophore of VIC or FAM, and the 3'-end is one selected from the group consisting of MGBNFQ and IntZEN-3IABIK. It can be transformed into a quencher.

More specifically, the 3rd, 14th, 27th, 31st and 37th sequences of the sequence listing of the present invention use VIC as a 5'-end fluorescent material and MGBNFQ as a quencher at the 3'-end And, SEQ ID NO: 9 of the present invention uses FAM as a fluorescent substance at the 5'-end and MGBNFQ as a quencher at the 3'-end, and SEQ ID NO: 21, 22, 26 and Sequence 34 uses FAM as a fluorescent material at the 5'-end and IntZEN-3IABIK as a quencher at the 3'-end.

According to another embodiment of the present invention, when the nucleic acid of the present invention is RNA, step (b) of the present invention further includes a reverse transcription step (pre-b) of transcribing the RNA to cDNA. According to certain embodiments of the present invention, step (pre-b) of the present invention is carried out at 45-55° C. for 20-40 minutes. According to certain embodiments of the present invention, when the present invention additionally includes a step (pre-b), the detection of the present invention is carried out using a polymerase chain reaction. The detection of the present invention can also be carried out by reverse transcription polymerase chain reaction, and the reverse transcription polymerase chain reaction is the same as the reaction in which the step (pre-b) and the polymerase chain reaction are combined.

According to a specific embodiment of the present invention, the polymerase chain reaction of the present invention comprises the steps of (a') sustaining at 90-100° C. for 5 minutes to 15 minutes; (b') sustaining for 10-20 seconds at 90-100° C. and 40-50 seconds at 55-65° C.; And (c') repeating the step (b') 40-50 times.

In addition to the above-described gene amplification method, the detection of the present invention may be performed using hybridization, immunoassay, or microarray.

According to an embodiment of the present invention, (i) detection of mutations in the BRAF gene of the present invention is performed using a group of oligonucleotides consisting of primers of the first and second sequences of the sequence list; (Ii) Mutation detection of the KRAS gene of the present invention is carried out using a group of oligonucleotides consisting of a primer selected from the group consisting of the primers of SEQ ID NOs: 4 to 7 and the primers of SEQ ID NO: 8; (Iii) Mutation detection of the NRAS gene of the present invention is carried out by using a group of oligonucleotides consisting of a primer selected from the group consisting of the primers of SEQ ID NOs: 10 to 12 and the primers of SEQ ID NO: 13; (Iv) The detection of mutations in the HRAS gene of the present invention includes an oligonucleotide group consisting of primers of SEQ ID NOs: 15, 16 and 17; Or using a group of oligonucleotides comprising a primer of SEQ ID NO: 18 and a primer of SEQ ID NO: 19 or 20; (V) The detection of rearrangement of the RET/PTC gene of the present invention includes an oligonucleotide group consisting of primers of SEQ ID NOs: 23 and 25; Or by using a group of oligonucleotides consisting of primers of SEQ ID NOs: 24 and 25; Or (vi) the rearrangement detection of the PAX8/PPARγ gene of the present invention is carried out using a group of oligonucleotides consisting of primers of SEQ ID NOs: 28, 29, and 30 of the Sequence Listing.

According to another embodiment of the present invention, (i) detection of mutations in the BRAF gene of the present invention is carried out using an oligonucleotide group consisting of primers of the first and second sequences of the sequence list; (Ii) Mutation detection of the KRAS gene of the present invention is carried out using a group of oligonucleotides consisting of a primer selected from the group consisting of the primers of SEQ ID NOs: 4 to 7 and the primers of SEQ ID NO: 8; (Iii) Mutation detection of the NRAS gene of the present invention is carried out by using a group of oligonucleotides consisting of a primer selected from the group consisting of the primers of SEQ ID NOs: 10 to 12 and the primers of SEQ ID NO: 13; (Iv) The detection of mutations in the HRAS gene of the present invention includes an oligonucleotide group consisting of primers of SEQ ID NOs: 15, 16 and 17; Or using a group of oligonucleotides comprising a primer of SEQ ID NO: 18 and a primer of SEQ ID NO: 19 or 20; (V) The detection of rearrangement of the RET/PTC gene of the present invention includes an oligonucleotide group consisting of primers of SEQ ID NOs: 23 and 25; Or by using a group of oligonucleotides consisting of primers of SEQ ID NOs: 24 and 25; And (vi) rearrangement detection of the PAX8/PPARγ gene of the present invention is carried out using a group of oligonucleotides consisting of primers of SEQ ID NOs: 28, 29, and 30 of the Sequence Listing.

According to another embodiment of the present invention, (i) the detection of mutations in the BRAF gene of the present invention additionally uses a probe having the third sequence of the sequence listing; (Ii) The detection of mutations in the KRAS gene of the present invention additionally uses a probe having SEQ ID NO: 9; (Iii) The detection of mutations in the NRAS gene of the present invention additionally uses a probe having SEQ ID NO: 14; (Iv) The detection of mutations in the HRAS gene of the present invention additionally uses a probe having a sequence listing of SEQ ID NO: 21 or 22; (V) The detection of rearrangement of the RET/PTC gene of the present invention additionally uses a probe having the 26th sequence or the 27th sequence of the sequence listing; Or (vi) the detection of rearrangement of the PAX8/PPARγ gene of the present invention additionally uses a probe having the 31st sequence of the sequence listing.

According to certain embodiments of the present invention, (i) the detection of mutations in the BRAF gene of the present invention additionally uses a probe having the third sequence of sequence listing; (Ii) The detection of mutations in the KRAS gene of the present invention additionally uses a probe having SEQ ID NO: 9; (Iii) The detection of mutations in the NRAS gene of the present invention additionally uses a probe having SEQ ID NO: 14; (Iv) The detection of mutations in the HRAS gene of the present invention additionally uses a probe having a sequence listing of 21 or 22; (V) The detection of rearrangement of the RET/PTC gene of the present invention additionally uses a probe having the 26th sequence or the 27th sequence of the sequence listing; And (vi) the rearrangement detection of the PAX8/PPARγ gene of the present invention additionally uses a probe having the 31st sequence in the sequence list.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a cancer diagnostic kit comprising a group of oligonucleotides for detection of cancer of a specific sequence hybridized with a nucleic acid of a cancer cell, a group of oligonucleotides of a specific sequence, and a method of providing information necessary for diagnosis or prognosis of cancer do.

(b) The method of the present invention enables early diagnosis of a specific cancer according to the mutation of a specific gene using RNA, unlike the conventional gDNA test for somatic mutation.

(c) According to the method of the present invention, since it is possible to detect mutations that have not been detected with gDNA, it is possible to test all cancer-related gene mutations with a single RNA extraction, and thus, there is an advantage of enabling accurate and rapid diagnosis.

(d) In particular, since the method of the present invention can simultaneously test all mutations related to thyroid cancer, it is effective to quickly and accurately diagnose thyroid cancer.

1 is an overview of a novel method of providing information necessary for diagnosis or prognosis of cancer (eg, thyroid cancer).
2 shows a method of designing primers and probes for detecting BRAF V600E mutations.
3 shows a method of designing primers and probes for KRAS mutation detection.
4 shows a method of designing primers and probes for detecting NRAS mutations.
5 shows a method of designing primers and probes for detecting HRAS mutations.
6 shows a method of designing primers and probes for RET/PTC1 mutation detection.
7 shows a method of designing primers and probes for RET/PTC3 mutation detection.
8 shows a method of designing primers and probes for detecting PAX8/PPARγ mutations.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1: RNA sample preparation

Using HighPure RNA Isolation Kit (Roche, Switzerland) from each positive cell line containing KRAS G12S/G12C/G12D/G12A/G12V/G13D, NRAS Q61R/Q61K/Q61L, HRAS G12V/Q61R, and BRAF V600E mutations. RNA was used after isolation. Sources of the positive cell line are listed in Table 1 below.

In the case of RET/PTC1, RET/PTC3, PAX8/PPARg, KRAS G12R, HRAS G13R/Q61K mutations that did not obtain a positive cell line, genes were artificially synthesized through Integrated DNA Technologies to include some of the mutations (gBlock Gene Fragment ), and then the plasmid DNA introduced into pGEM®easy Vector (Promega, Madison, USA) was used. The synthesized gene sequence is SEQ ID NO: 38 (RET/PTC1 mutation), SEQ ID NO: 39 (RET/PTC3 mutation), SEQ ID NO: 40 (PAX8/PPARg exon 7 mutation), SEQ ID NO: 41 ( PAX8/PPARg exon 9 mutation), SEQ ID NO: 42 (KRAS G12R mutation) and SEQ ID NO: 43 (HRAS G13R/Q61K mutation).

gene Mutation Cell line/Gene fragment source RET/PTC1 CCDC6/RET gBlock Gene fragment IntegratedDNATechnologies RET/PTC3 NCOA4/RET gBlock Gene fragment IntegratedDNATechnologies PAX8/PPARg PAX8 exon7/PPARg gBlock Gene fragment Integrated DNA Technologies PAX8 exon9/PPARg gBlock Gene fragment Integrated DNA Technologies KRAS G12S A549 KCLB G12C MIA-PACA KCLB G12D SNU-C2B KCLB G12A SW-1116 KCLB G12V SW-620 KCLB G13D LOVO KCLB G12R gBlock Gene fragment Integrated DNA Technologies NRAS Q61R SK-MEL-2 KCLB Q61K H9 KCLB Q61L HL-60 KCLB HRAS G12V T24 KCLB G13R gBlock Gene fragment Integrated DNA Technologies Q61R MC/CAR ATCC Q61K gBlock Gene fragment Integrated DNA Technologies BRAF V600E SK-MEL-28 ATCC

* KCLB (Korean Cell Line Bank, Korea Cell Line Bank, Korea)

* ATCC (American Type Culture Collection, USA)

Example 2: cDNA sample preparation

RNA of the cell line isolated in Example 1 was synthesized as cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland).

Example 3: for mutation detection primer And Probe device

BRAF Gene mutation detection

In order to identify the V600E mutation in the BRAF gene exon 15, a forward primer (BRAF-PF3), a reverse primer (BRAF-PR3-1), and a fluorescent probe (BRAF-PP1) are present in exon 15, and RNA is templated. The test was made possible. Primer and probe sequences are shown in Table 2.

Sequence name order BRAF-PF3 5'-ATATATTTCTTCATGAAGACCTCACAGTAAAA (SEQ ID NO: 1)-3' BRAF-PR3-1 5'-GGACCCACTCCATCGAGATACCTC (SEQ ID NO: 2)-3' BRAF-PP1 5'-VIC-TAGGTGATTTTGGTCTAGCTACA (SEQ ID NO: 3)-MGBNFQ-3'

KRAS Gene mutation detection

Forward primers (KRAS-c34-F10, KRAS-C34T-F1, KRAS-c35-F11 or KRAS-c38-F2), reverse primers (KRAS-1R) and fluorescent probes (MGB3 (FAM)) in the KRAS gene exon 2 It was designed to exist so that RNA could be tested as a template. Primer and probe sequences are shown in Table 3.

Sequence name order KRAS-c34-F10 5'-CTTGTGGTAGTTGGAGGTH (SEQ ID NO: 4)-3' KRAS-C34T-F1 5'-CTTGTGGTAGTTGGAGGTT (SEQ ID NO: 5)-3' KRAS-c35-F11 5'-TTGTGGTAGTTGGAGCTCH (SEQ ID NO: 6)-3' KRAS-c38-F2 5'-GTGGTAGTTGGAGCTGGTCA (SEQ ID NO: 7)-3' KRAS-1R 5'-TTGTTGGATCATATTCGTCCAC (SEQ ID NO: 8)-3' MGB3(FAM) 5'-FAM-ACGATACAGCTAATTCA (SEQ ID NO: 9)-MGBNFQ-3'

NRAS Gene mutation detection

RNA was tested as a template by designing the presence of forward primers (NRAS Q61L-F6, NRAS Q61K-F6 or NRAS Q61R-F8), reverse primers (NRAS R1) and fluorescent probes (NRAS Probe (VIC)) in the NRAS gene exon 3 Was made possible. Primer and probe sequences are shown in Table 4.

Sequence name order NRAS Q61L-F6 5'-ACTGGATACAGCTGGAGT (SEQ ID NO: 10)-3' NRAS Q61K-F6 5'-CATACTGGATACAGCTGTAA (SEQ ID NO: 11)-3' NRAS Q61R-F8 5'-CTGGATACAGCTGGTCG (SEQ ID NO: 12)-3' NRAS R1 5'-CCTTCGCCTGTCCTCATGTATT (SEQ ID NO: 13)-3' NRAS Probe (VIC) 5'-VIC-CAGTGCCATGAGAGAC (SEQ ID NO: 14)-MGBNFQ-3'

HRAS Gene mutation detection

In the HRAS gene exon 2, a forward primer (HRAS G12V-F5 or HRAS G13R-F4), a reverse primer (HRAS 12,13-R1) and a fluorescent probe (HRAS 12,13_ZEN probe (FAM)) were designed to exist, and exon 3 A forward primer (HRAS 61-F1), a reverse primer (HRAS Q61K-R3 or HRAS Q61R-R10) and a fluorescent probe (HRAS 61_ZEN probe (FAM)) were designed to exist in the inside, so that RNA could be tested as a template. Primer and probe sequences are shown in Table 5.

Sequence name order HRAS G12V-F5 5'-GGTGGTGGGCGCCTT (SEQ ID NO: 15)-3' HRAS G13R-F4 5'-TAGTGGGCGCCGTCC (SEQ ID NO: 16)-3' HRAS 12,13-R1 5'-GGGTCGTATTCGTCCACAAAA (SEQ ID NO: 17)-3' HRAS 61-F1 5'-CGGAAGCAGGTGGTCATTG (SEQ ID NO: 18)-3' HRAS Q61K-R3 5'-GGCGCTGTACTCCTCATT (SEQ ID NO: 19)-3' HRAS Q61R-R10 5'-GCGCTGTACTCCTCGC (SEQ ID NO: 20)-3' HRAS 12,13_ZEN probe(FAM) 5'-FAM-TGCGCTGACCATCCAGCTGATCC (SEQ ID NO: 21)-IntZEN-3IABIK-3' HRAS 61_ZEN probe(FAM) 5'-FAM-TGTCCAACAGGCACGTCTCCCCA (SEQ ID NO: 22)-IntZEN-3IABIK-3'

RET/PTC rearrangement mutation gene mutation detection

RET/PTC rearrangement mutations can be classified into RET/PTC1 type and RET/PTC3 type.RET/PTC1 rearrangement mutations are forward primers (R/P1-F1) and fluorescent probes (ZEN( P1)) was located, and reverse primers (R/P1,P3-R2) were designed to be located at exon 12 of the RET gene to enable the CCDC6/RET rearrangement mutation test.

The RET/PTC3 rearrangement mutation causes the forward primer (R/R3-F1) and the fluorescent probe (MGB-P3) to be located in exon 11 of the NCOA4 gene, and the reverse primers (R/P1, P3-R2) are It was designed to be located in exon 12 to enable NCOA4/RET rearrangement mutation testing.

Primer and probe sequences are shown in Table 6.

Sequence name order R/P1-F1 5'-GGAGACCTACAAACTGAAGTGCAA (SEQ ID NO: 23)-3' R/R3-F1 5'-CCAGTGGTTATCAAGCTCCTTACA (SEQ ID NO: 24)-3' R/P1,P3-R2 5'-AACCAAGTTCTTCCGAGGGAA (SEQ ID NO: 25)-3' ZEN(P1) 5'-FAM-TGGCTTTGCGCAGGTCGCG (SEQ ID NO: 26)-IntZEN-3IABIK-3' MGB-P3 5'-VIC-TTGGATAAGCCAGTCCT (SEQ ID NO: 27)-MGBNFQ-3'

PAX8 / PPARγ Rearrangement mutant gene mutation detection

In the case of RET/PTC rearrangement mutations, exon 9 of the PAX8 gene and exon 1 of PPARγ can be rearranged, and exon 7 of the PAX8 gene and exon 1 of PPARγ are rearranged.The forward primer is exon 7 of the PAX8 gene. (PASX8-7-F2) and exon 9 (PAX8-9-F3-3 (KIS)) were placed at two positions, and a fluorescent probe (PAX8 MGB (VIC)) and a reverse primer (PPARg-1R) were used for the PPARγ gene. By designing to be located in exon 1, it was possible to detect two PAX8/PPARγ rearrangement mutations at once. Primer and probe sequences are shown in Table 7.

Sequence name order PASX8-7-F2 5'-AACCTCTCGACTCACCAGACCTA (SEQ ID NO: 28)-3' PAX8-9-F3-3(KIS) 5'-CACCAGCGGACAGGGC (SEQ ID NO: 29)-3' PPARg-1R 5'-CAAAGTTGGTGGGCCAGAA (SEQ ID NO: 30)-3' PAX8 MGB (VIC) 5'-VIC-TTGACACAGAGATGCC (SEQ ID NO: 31)-MGBNFQ-3'

Example 4: real time Reverse transcription Polymerase chain reaction (Real time reverse transcription polymerase chain reaction) or real time Polymerase chain reaction (Real time polymerase chain reaction)

In order to identify the major mutations in the gene of Example 3, real-time reverse transcription polymerase chain reaction (one-step real-time reverse transcription PCR) using RNA isolated from the target group or after synthesizing the RNA into cDNA in real time Polymerase chain reaction (real-time PCR) was performed. The RNA was synthesized into cDNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Switzerland).

The primer and probe sequences used in the real-time reverse transcription polymerase chain reaction or the real-time polymerase chain reaction are the same as in Example 3.

Real-time reverse transcription polymerase chain reaction of KRT7 or GAPDH gene was performed as a control in order to confirm whether RNA was well extracted from the cells and contained in an appropriate amount for the test and there was no problem in the polymerase chain reaction. The primers and probe sequences for the KRT7 or GAPDH gene are shown in Table 8.

Sequence name order KRT7-F1 5'-CCACCCACAATCACAAGAAGATT (SEQ ID NO: 32)-3' KRT7-R1 5'-TCACTTTCCAGACTGTCTCACTGTCT (SEQ ID NO: 33)-3' KRT7-ZEN probe(FAM) 5'-FAM-CCACCCCTGCCTCCCATGCC (SEQ ID NO: 34)-IntZEN-3IABIK-3' GAPDH-F1(020713) 5'-CCACCCATGGCAAATTCC (SEQ ID NO: 35)-3' GAPDH-R1(020713) 5'-TGACAAGCTTCCCGTTCTCA (SEQ ID NO: 36)-3' GAPDH-Probe(020713) 5'-VIC-TGGCACCGTCAAGG (SEQ ID NO: 37)-MGBNFQ-3'

A fluorescent material such as Fluorescein (FAM) or VIC (Applied Biosystems) was attached to the probe so as to confirm the size of the amplified product.

1 μL of the forward primer, 1 μL of the reverse primer, 5 μL of RNA sample, 3.5 μL of distilled water, 12.5 μL of 2X RT-PCR premix (Nanohelix, Korea), 1 μL of RT-PCR enzyme mixture (Nanohelix, Korea) are mixed, ① 50° C. (30 minutes, once); ② 95°C (10 minutes, 1 time); ③ Real-time reverse transcription polymerase chain reaction was performed under the conditions of 95°C (15 seconds) -60°C (45 seconds) (45 times). When performing real-time polymerase chain reaction using a cDNA sample instead of an RNA sample, only steps ② and ③ were performed without performing the above step ①.

Example 5: mutation identification

From the results of using the positive cell line, it was confirmed that both KRT7 and GAPDH were amplified within the range of 11.3-27.4 Ct (threshold cycle, meaning the number of cycles of the fluorescent signal indicating the contact point), and there was no problem in RNA extraction and cDNA synthesis. If it is less than Ct 38, it can be determined as a positive mutation. In the case of the KRAS mutant cell line, KRAS Ct was amplified within the range of 22.7-26.2, confirming the positive KRAS mutation. In the case of the NRAS mutant cell line, NRAS Ct was amplified within the range of 25.8-30.5, confirming the positive NRAS mutation. In the case of the HRAS mutant cell line, HRAS Ct was amplified within the range of 23.3-31.5, confirming the positive HRAS mutation. When the positive plasmid DNA was used, KRT7 and GAPDH Ct could not be identified, and Ct 20.7-22.7 for each mutation was confirmed to be positive for each mutation.

As a result, it is possible to confirm all the positive mutations of the gene using the primers and real-time polymerase chain reaction conditions of the present invention. Diagnosis of cancer having a mutation for the gene is possible using each primer or a combination thereof. I could confirm that it is possible

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is clear that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION BIOSEWOOM, INC. <120> An Apparatus for Diagnosing Cancer and an Apparatus for The Same <130> PN140031 <160> 43 <170> KopatentIn 2.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> BRAF-PF3 <400> 1 atatatttct tcatgaagac ctcacagtaa aa 32 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> BRAF-PR3-1 <400> 2 ggacccactc catcgagata cctc 24 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> BRAF-PP1 <400> 3 taggtgattt tggtctagct aca 23 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> KRAS-c34-F10 <400> 4 cttgtggtag ttggaggth 19 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> KRAS-C34T-F1 <400> 5 cttgtggtag ttggaggtt 19 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> KRAS-c35-F11 <400> 6 ttgtggtagt tggagctch 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KRAS-c38-F2 <400> 7 gtggtagttg gagctggtca 20 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> KRAS-1R <400> 8 ttgttggatc atattcgtcc ac 22 <210> 9 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> MGB3 (FAM) <400> 9 acgatacagc taattca 17 <210> 10 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> NRAS Q61L-F6 <400> 10 actggataca gctggagt 18 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NRAS Q61K-F6 <400> 11 catactggat acagctgtaa 20 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> NRAS Q61R-F8 <400> 12 ctggatacag ctggtcg 17 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NRAS R1 <400> 13 ccttcgcctg tcctcatgta tt 22 <210> 14 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> NRAS Probe (VIC) <400> 14 cagtgccatg agagac 16 <210> 15 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HRAS G12V-F5 <400> 15 ggtggtgggc gcctt 15 <210> 16 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> HRAS G13R-F4 <400> 16 tagtgggcgc cgtcc 15 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> HRAS 12,13-R1 <400> 17 gggtcgtatt cgtccacaaa a 21 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> HRAS 61-F1 <400> 18 cggaagcagg tggtcattg 19 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> HRAS Q61K-R3 <400> 19 ggcgctgtac tcctcatt 18 <210> 20 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> HRAS Q61R-R10 <400> 20 gcgctgtact cctcgc 16 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> HRAS 12,13_ZEN probe (FAM) <400> 21 tgcgctgacc atccagctga tcc 23 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> HRAS 61_ZEN probe (FAM) <400> 22 tgtccaacag gcacgtctcc cca 23 <210> 23 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> R/P1-F1 <400> 23 ggagacctac aaactgaagt gcaa 24 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> R/R3-F1 <400> 24 ccagtggtta tcaagctcct taca 24 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> R/P1,P3-R2 <400> 25 aaccaagttc ttccgaggga a 21 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ZEN(P1) <400> 26 tggctttgcg caggtcgcg 19 <210> 27 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> MGB-P3 <400> 27 ttggataagc cagtcct 17 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> PASX8-7-F2 <400> 28 aacctctcga ctcaccagac cta 23 <210> 29 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PAX8-9-F3-3 (KIS) <400> 29 caccagcgga cagggc 16 <210> 30 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PPARg-1R <400> 30 caaagttggt gggccagaa 19 <210> 31 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> PAX8 MGB (VIC) <400> 31 ttgacacaga gatgcc 16 <210> 32 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> KRT7-F1 <400> 32 ccacccacaa tcacaagaag att 23 <210> 33 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> KRT7-R1 <400> 33 tcactttcca gactgtctca ctgtct 26 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KRT7-ZEN probe (FAM) <400> 34 ccacccctgc ctcccatgcc 20 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-F1(020713) <400> 35 ccacccatgg caaattcc 18 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-R1(020713) <400> 36 tgacaagctt cccgttctca 20 <210> 37 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-Probe (020713) <400> 37 tggcaccgtc aagg 14 <210> 38 <211> 400 <212> DNA <213> Artificial Sequence <220> <223> RET/PTC1 <400> 38 catctcgccg ttccgcctgg aggagctcac caaccgcctg gcctcgctgc agcaagagaa 60 caaggtgctg aagatagagc tggagaccta caaactgaag tgcaaggcac tgcaggagga 120 gaaccgcgac ctgcgcaaag ccagcgttac catcgaggat ccaaagtggg aattccctcg 180 gaagaacttg gttcttggaa aaactctagg agaaggggaa tttggaaaag tggtcaaggc 240 aacggccttc catctgaaag gcagaggagg gtacaccacg gtggccgtga agatgctgaa 300 agagaacgcc tccccgagtg agcttcgaga cctgctgtca gagttcaacg tcctgaagca 360 ggtcaaccac cctcatgtca tcaaattgta tggggcctgc 400 <210> 39 <211> 480 <212> DNA <213> Artificial Sequence <220> <223> RET/PTC3 <400> 39 accattcaaa ttcctgagca cttgatggct catgctagtt cagcaaatat tgggcccttc 60 ctggagaaga gaggctgtat ctccatgcca gagcagaagt cagcatccgg tattgtagct 120 gtccctttca gcgaatggct ccttggaagc aaacctgcca gtggttatca agctccttac 180 atacccagca ccgaccccca ggactggctt atccaaaagc agaccttgga gaacagtcag 240 gaggatccaa agtgggaatt ccctcggaag aacttggttc ttggaaaaac tctaggagaa 300 ggggaatttg gaaaagtggt caaggcaacg gccttccatc tgaaaggcag aggagggtac 360 accacggtgg ccgtgaagat gctgaaagag aacgcctccc cgagtgagct tcgagacctg 420 ctgtcagagt tcaacgtcct gaagcaggtc aaccaccctc atgtcatcaa attgtatggg 480 480 <210> 40 <211> 494 <212> DNA <213> Artificial Sequence <220> <223> PAX8/PPARg exon7 <400> 40 gactcacaga gcagcagcag cggaccccga aagcaccttc gcacggatgc cttcagccag 60 caccacctcg agccgctcga gtgcccattt gagcggcagc actacccaga ggcctatgcc 120 tcccccagcc acaccaaagg cgagcagggc ctctacccgc tgcccttgct caacagcacc 180 ctggacgacg ggaaggccac cctgacccct tccaacacgc cactggggcg caacctctcg 240 actcaccaga cctaccccgt ggtggcagat gaccatggtt gacacagaga tgccattctg 300 gcccaccaac tttgggatca gctccgtgga tctctccgta atggaagacc actcccactc 360 ctttgatatc aagcccttca ctactgttga cttctccagc atttctactc cacattacga 420 agacattcca ttcacaagaa cagatccagt ggttgcagat tacaagtatg acctgaaact 480 tcaagagtac caaa 494 <210> 41 <211> 495 <212> DNA <213> Artificial Sequence <220> <223> PAX8/PPARg exon9 <400> 41 aaagcaggaa acccccgagg tgtccagttc tagctccacc ccttgctctt tatctagctc 60 cgcccttttg gatctgcagc aagtcggctc cggggtcccg cccttcaatg cctttcccca 120 tgctgcctcc gtgtacgggc agttcacggg ccaggccctc ctctcagggc gagagatggt 180 ggggcccacg ctgcccggat acccacccca catccccacc agcggacagg gcagctatgc 240 ctcctctgcc atcgcaggca tggtggcaga tgaccatggt tgacacagag atgccattct 300 ggcccaccaa ctttgggatc agctccgtgg atctctccgt aatggaagac cactcccact 360 cctttgatat caagcccttc actactgttg acttctccag catttctact ccacattacg 420 aagacattcc attcacaaga acagatccag tggttgcaga ttacaagtat gacctgaaac 480 ttcaagagta ccaaa 495 <210> 42 <211> 200 <212> DNA <213> Artificial Sequence <220> <223> KRAS G12R <400> 42 ataaggcctg ctgaaaatga ctgaatataa acttgtggta gttggagctc gtggcgtagg 60 caagagtgcc ttgacgatac agctaattca gaatcatttt gtggacgaat atgatccaac 120 aatagaggta aatcttgttt taatatgcat attactggtg caggaccatt ctttgataca 180 gataaaggtt tctctgacca 200 <210> 43 <211> 480 <212> DNA <213> Artificial Sequence <220> <223> HRAS G13R, Q61K <400> 43 tcgcgcctgt gaacggtggg gcaggagacc ctgtaggagg accccgggcc gcaggcccct 60 gaggagcgat gacggaatat aagctggtgg tggtgggcgc cggccgtgtg ggcaagagtg 120 cgctgaccat ccagctgatc cagaaccatt ttgtggacga atacgacccc actatagagg 180 attcctaccg gaagcaggtg gtcattgatg gggagacgtg cctgttggac atcctggata 240 ccgccggcaa ggaggagtac agcgccatgc gggaccagta catgcgcacc ggggagggct 300 tcctgtgtgt gtttgccatc aacaacacca agtcttttga ggacatccac cagtacaggg 360 agcagatcaa acgggtgaag gactcggatg acgtgcccat ggtgctggtg gggaacaagt 420 gtgacctggc tgcacgcact gtggaatctc ggcaggctca ggacctcgcc cgaagctacg 480 480

Claims (11)

(i) an oligonucleotide group consisting of a primer selected from the group consisting of primers of Sequence Listing 4 to 7 sequence and a primer of Sequence Listing 8 sequence;
(Ii) a primer selected from the group consisting of primers of SEQ ID NOS: 10 to 12, and an oligonucleotide group consisting of primers of SEQ ID NO: 13;
(Iii) oligonucleotide group consisting of primers of Sequence Listing 15, Sequence 16 and Sequence 17; And
(Iv) a primer of SEQ ID NO: 18, and an oligonucleotide group selected from the group consisting of oligonucleotides consisting of primers of SEQ ID NO: 19 or SEQ ID NO: 20,
Wherein the oligonucleotide group is hybridized with a nucleic acid of a cancer cell.
(i) an oligonucleotide group consisting of a primer selected from the group consisting of primers of Sequence Listing 4 to 7 sequence and a primer of Sequence Listing 8 sequence;
(Ii) a primer selected from the group consisting of primers of SEQ ID NOS: 10 to 12, and an oligonucleotide group consisting of primers of SEQ ID NO: 13;
(Iii) oligonucleotide group consisting of primers of Sequence Listing 15, Sequence 16 and Sequence 17; And
(Iv) an oligonucleotide group selected from the group consisting of a primer of SEQ ID NO: 18, and an oligonucleotide group consisting of primers of SEQ ID NO: 19 or SEQ ID NO: 20.
The diagnostic kit according to claim 2, wherein the thyroid cancer diagnostic kit
Wherein the oligonucleotide group (i) further comprises a probe having the sequence of SEQ ID NO: 9;
Said oligonucleotide group (ii) further comprises a probe having the sequence of SEQ ID NO: 14;
Said oligonucleotide group (iii) additionally comprising a probe having the sequence of SEQ ID NO: 21; or
Wherein the oligonucleotide group (iv) further comprises a probe having the sequence of SEQ ID NO: 22.
[Claim 3] The kit for diagnosing thyroid cancer according to claim 2, wherein the diagnostic kit further comprises a reverse transcriptase.
A method for providing information necessary for diagnosis or prognosis of thyroid cancer including the following steps:
(a) obtaining a nucleic acid from a biological sample separated from a human; And
(b) an oligonucleotide group consisting of a primer selected from the group consisting of primers of the sequence of the forth sequence to the seventh sequence and a primer of the sequence of the sequence listing from the nucleic acid of the step (a) (Ii) a primer selected from the group consisting of primers of the 10th to 12th sequences and an oligonucleotide group consisting of a primer of the 13th sequence of SEQ ID NO: 1, and mutation detection of the NRAS gene and (Iii) oligonucleotide group consisting of primers of Sequence Listing 15, Sequence 16 and Sequence 17; Or detecting the mutation of the HRAS gene using the oligonucleotide group consisting of the primer of SEQ ID NO: 18 and the primer of SEQ ID NO: 19 or SEQ ID NO: 20.
6. The method of claim 5, wherein the nucleic acid is RNA.
6. The method according to claim 5, wherein the step (b) further comprises a reverse transcription step (pre-b) of transcribing RNA as cDNA into the cDNA.
8. The method according to claim 7, wherein said step (pre-b) is carried out at 45-55 DEG C for 20-40 minutes.
6. The method of claim 5, wherein the detection is performed using a polymerase chain reaction.
10. The method of claim 9, wherein the polymerase chain reaction comprises the steps of:
(a ') at 90-100 &lt; 0 &gt; C for 5 minutes to 15 minutes;
(b &apos;) at 90-100 &lt; 0 &gt; C for 10-20 seconds and at 55-65 [deg.] C for 40-50 seconds; And
(c ') repeating the step (b') 40-50 times.
6. The method of claim 5, wherein (i) the mutation detection of the KRAS gene further comprises using a probe having the sequence of SEQ ID NO: 9;
(Ii) detecting the mutation of the NRAS gene further comprises using a probe having the sequence of SEQ ID NO: 14; or
(Iii) the mutation detection of the HRAS gene additionally utilizes a probe having the sequence of SEQ ID NO: 21 or SEQ ID NO: 22.

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