KR20120021691A - Methods and kits for the pik3ca mutant detection using pna mediated real-time pcr clamping - Google Patents

Abstract

PURPOSE: A method for selectively detecting mutant and a kit used from the same are provided to ensure sensitivity and specificity. CONSTITUTION: A method for detecting PIK3CA gene mutant comprises: a step of performing real-time PCR of PIK3CA genes under the presence of a PIK3CA gene clamping primer set for amplifying PIK3CA exon 9 or exon 20 and a PNA clamping probe which complementarily binds to exon 9 or exon 20 wild type; and a step of analyzing gene amplification to determine presence or concentration of the mutant. The probe has 15-20 mer bases. The method is used for diagnosing colon cancer, lung cancer, stomach cancer, head squamous cell carcinoma, glioblastoma, ovarian cancer, or breast cancer.

Description

Method and kits for the PIK3CA mutant detection using PNA mediated Real-time PCR clamping}

The present invention relates to mutation detection of a PIK3CA (Phosphatidylinositol 3-kinase, catalytic, alpha polypeptide) gene using a peptide nucleic acid (Peptide Nucleic Acid) probe, and more particularly, to exon 9 of a PIK3CA gene. Or a PNA probe that specifically binds to a wild type for the detection of 20 mutations, thereby detecting only a small amount of the mutant with high sensitivity by inhibiting amplification of the wild type and a kit for use in the method.

Since the oncogene and tumor suppressor gene mutations are known to be involved in the development of cancer, various studies have been conducted to detect mutations of various oncogenes and tumor suppressor genes. Accordingly, many mutations have been found that are involved in the development of cancer and prognosis of drugs. Among these, mutations in the PIK3CA gene result in activation of the phosphatidylinositol 3-kinase (PI3K) protein, which activates the signaling system of the tyrosine kinase receptor. Activated tyrosine kinases are involved in the carcinogenic process by activating proteins that inhibit the survival and division of cells and promote the metabolism and inhibit the function of proteins that support cell growth (Serena Di et al., Clin Cancer Res. 2009 August 15; 15 (16), 5017-5019). 75% of the PIK3CA gene mutations are found as point mutations at exon 9, which translates into the helical region of the PI3K protein, and exon 20, which translates into the kinase region; (Ligresti G et al., Cell cycle 2009 May 1; 8: 9, 1352-1358). These PIK3CA gene mutations are found in a wide variety of cancers, especially in colorectal cancer, gastric cancer, lung cancer, pancreatic cancer, head squamous cell carcinoma, glioblastoma, endometrial cancer, ovarian cancer, breast cancer, etc. (Ligresti G et al., Cell cycle 2009 May 1; 8: 9, 1352-1358, Barbi S et al., J Exp Clin Cancer Res. 2010 Apr 16; 29, 32, Kalinsky K et al., Clin Cancer Res. 2009 August 15; 15 (16), 5049-5059, Gallia GL et al., Mol Cancer Res. 2006 Oct; 4 (10): 709-14, Ogino S et al., J Clin Oncol. 2009 Mar 20 27 (9): 1477-84, Oda K et al. Cancer Res. 2005 Dec 1; 65 (23): 10669-73). PIK3CA gene mutations worsen the patient's prognosis by speeding cancer progression and increasing the frequency of metastasis. It is also known to be involved in the resistance of the targeted therapeutics of the HER family to belong to the signaling system downstream of the Human Epidermal Growth Factor (HER family) (Wee S et al., Cancer Res. 2009 May 15; 69 (10): 4286-93, Guo XN et al., Cancer Res. 2007 Jun 15; 67 (12): 5851-8, Ogino S et al., J Clin Oncol. 2009 Mar 20; 27 (9): 1477-84). According to a recently reported paper, the prescription of a drug called Herceptin, a type of tyrosine kinase, improves the prognosis for breast cancer with HER2 gene amplification, but in addition to amplification of the HER2 gene, Herceptin when a mutation of the PIK3CA gene is carried out. It is reported to be resistant to (O'Brien NA et al., Mol Cancer Ther. 2010 Jun; 9 (6): 1489-502). PI3K is an important factor in the development of therapeutics that target it, and its importance is increasing. Therefore, PIK3CA mutation detection is an important predictor of the therapeutic effect in the treatment of human epidermal growth factor as a target and may play a decisive role in a good prognosis.

The detection method of the PIK3CA mutation is a variation detection method through sequencing after polymerase chain reaction (PCR), and the change in electrophoretic movement distance according to the three-dimensional conformation difference between wild type and mutant genes. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) method (EI-Habr EA et al., Clin Neuropathol. 2010 Jul-Aug; 29 (4) : 239-45) and the like have been used. However, the above method is a method of detecting mutations through a process of cleavage with restriction enzymes after PCR, electrophoresis, sequencing, and thus, a reaction time is long, cumbersome and expensive. In addition, since clinical samples often contain very few mutations compared to wild-type, although the detection of a small amount of mutations is very important, the above method has low detection sensitivity, making it difficult to detect very small amounts of mutations.

High-resolution melting to selectively detect mutations only by using melting temperature differences between wild-type and mutations to increase sensitivity curve analysis, HRMA, scorpion real-time allele specific PCR (DxS'scorpions? and ARMS?) methods to selectively detect mutations using scorpion probes (Simi L et al., Am J Clin Pathol. 2008 Aug; 130 (2): 247-53, Board RE et al., Clin Chem. 2008 Apr; 54 (4): 757-60). This technique can be easily and quickly applied to various diagnostics, and is a good technique for diagnosing and analyzing mutations of cancer-related genes (Bernard et al., Clin Chem. 2002 Aug; 48 (8): 1178-85). However, the above-described method is cumbersome in that several reactions are required to detect a mutation because each probe or primer must be used at each site where the mutation occurs to detect a mutation (Simi L et al., Am J). Clin Pathol. 2008 Aug; 130 (2): 247-53, Board RE et al., Clin Chem. 2008 Apr; 54 (4): 757-60).

Recently, a technique for selectively detecting a mutant type, unlike the method described above, uses a PNA probe that specifically binds to a wild type to inhibit amplification of a large amount of wild type. Technology has been developed. PNA was first reported in 1991 as analogous DNA with nucleic acid bases linked by peptide bonds rather than phosphate bonds (Nielsen PE et al., Science 1991 Dec 6; 254 (5037): 1497-500). PNA is synthesized chemically and is not found in nature. PNAs hybridize with native nucleic acids of complementary base sequences to form double strands. PNA / DNA strands are more stable than DNA / DNA strands and PNA / RNA strands are more stable than DNA / RNA strands when the number of nucleic acid bases is the same. As the basic skeleton of PNA, N- (2-aminoethyl) glycine is most commonly used by amide bonds. In this case, the backbone of peptide nucleic acid is different from that of negatively charged natural nucleic acid. It is electrically neutral. The four nucleic acid bases present in the PNA occupy a similar space as the nucleic acid bases of DNA and the distances between the nucleic acid bases are almost the same as those of natural nucleic acids. PNA is not only chemically more stable than natural nucleic acid but also biologically stable because it is not degraded by nuclease or protease. Since PNA is also electrically neutral, the stability of PNA / DNA, PNA / RNA double strands is not affected by salt concentration. Because of this property, PNAs can recognize complementary nucleic acid sequences better than natural nucleic acids and are therefore used for diagnostic or other biological and medical purposes. The PNA clamping technique uses the advantages of the above-described PNA, so that when the PNA probe is completely bound, the amplification reaction does not occur because the enzyme is not recognized, and in the case of a point mutation, the amplification reaction cannot be perfectly bound. As a method using the principle that occurs, it is widely used because it can quickly and accurately detect a very small amount of mutations compared to wild type.

US Patent Publication No. 2008/0176226 discloses PCR in the presence of a primer set that amplifies a selected site of a target nucleic acid and a PNA probe that perfectly binds to the selected site of a wild type, and performs a melt curve analysis on the obtained PCR product. Methods and kits for detecting mutations in target nucleic acids are disclosed. However, the method distinguishes the mutant types by the difference in the melting curve rather than the number of cycles of the amplification reaction. In addition to the PNA clamping probe, a donor fluorophore and an acceptor can detect fluorescence for measuring the melting curve. Requires a probe to which is attached. As described above, a large amount of fluorescently labeled probes are detected to increase the cost for analysis. In addition, the U.S. Patent Publication discloses a 17mer PNA (sensor probe) labeled with fluorescein at the N-terminus and a 44mer DNA (anchor probe) labeled with LC-Red 640 at the 3'-end to detect K-ras mutations. Only specific examples are disclosed, and no teaching or suggestion is made regarding the detection of mutations in the PIK3CA gene.

The inventors of the PIK3A gene use a relatively long (eg, 15mer or larger) PNA clamping probe to detect mutants using only amplification cycle differences with the wild type. In addition, we have completed the PIK3CA mutation detection technology using PNA-based real-time PCR clamping, which can quickly and accurately detect very small amounts of mixed mutations by completely inhibiting the amplification of a large amount of wild type to improve the detection sensitivity of the variant. .

An object of the present invention is to provide a method for detecting PIK3CA mutation using PNA-based real-time PCR clamping.

Another object of the present invention is to provide a PIK3CA mutation detection kit using PNA-based real-time PCR clamping.

One aspect of the invention

For the PIK3CA gene, in the presence of a set of PIK3CA gene clamping primers that amplify the exon 9 or exon 20 sites of the PIK3CA gene and a Peptide Nucleic Acid (PNA) clamping probe that fully binds to the wild type of exon 9 or exon 20 of the PIK3CA gene Performing real-time polymerase chain reaction (PCR);

Gene amplification by the real-time PCR is analyzed to determine the presence or absence of mutations in the PIK3CA gene:

It relates to a method for detecting mutation of the PIK3CA gene, comprising the step.

In a preferred embodiment of the present invention, the _Ct (cycle threshold) value of real-time PCR is measured to determine the presence or absence of mutation of the PIK3CA gene.

The PNA clamping probe used in the present invention may be preferably composed of a nucleotide sequence of 15 to 30mer, more preferably 17 to 24mer in length, for example, one consisting of the nucleotide sequence of SEQ ID NOs: 1 to 31 Can be.

The PIK3CA gene clamping primer set used in the present invention is a forward primer specifically binding to the upstream portion of PIK3CA gene exon 9 wild type codon 542, 545 or 546, or exon 20 wild type codon 1047, and specifically downstream thereof It may be to include a reverse primer, for example, the forward primer is made of any one of SEQ ID NO: 36, 37, 41 and 42, the reverse primer is made of any one of SEQ ID NO: 33, 35, and 38 to 40 Can be.

In the present invention, gene amplification is analyzed using DNA intercalating fluorescent material, for example SYBR Green I, Evergreen, Ethidium Bromide (EtBr), BEBO, YO-PRO-1, TO-PRO -3, consisting of LC green, SYTO-9, SYTO-13, SYTO-16, SYTO-60, SYTO-62, SYTO-64, SYTO-82, POPO-3, TOTO-3, BOBO-3 and SYTOX orange One or more selected from the group can be used. Any fluorescent material capable of DNA intercalation can be used, and there is no particular limitation.

The method according to the invention can be used to determine or diagnose the treatment of colorectal cancer, gastric cancer, lung cancer, pancreatic cancer, head squamous cell carcinoma, glioblastoma, endometrial cancer, ovarian cancer or breast cancer.

Another aspect of the invention

PNA clamping probe of any one of SEQ ID NOs: 1 to 31:

It relates to a kit for use in the mutation detection method of the PIK3CA gene according to the present invention.

According to the present invention, mutations in the PIK3CA gene, which are expected to be involved in cancer development, prognosis, and drug resistance, can be detected within a short time even a mutation contained in a very small amount with excellent sensitivity and specificity. In addition, the PNA itself, which is used as a probe, is very stable to biological enzymes and physical elements, and the detection method is very simple and it is expected to be very easy for mass analysis and clinical use because the detection is performed in a short time.

1 is a schematic diagram showing the PNA-based PCR clamping principle;
Figure 2 is a graph comparing the detection sensitivity (ΔC _t ) according to the probe to select a probe for detecting the PIK3CA exon 9 codon 542 mutation using the PNA probe of SEQ ID NO: 7 to 11 designed in the present invention;
Figure 3 compares the detection sensitivity (ΔC _t ) according to the probe to select a probe for detecting the PIK3CA exon 9 codon 545 mutation using the PNA probe of SEQ ID NO: 12, 13, 14, 16 and 17 designed in the present invention It is a graph;
Figure 4 is a graph comparing the detection sensitivity (ΔC _t ) according to the probe to select a probe for detecting the PIK3CA exon 20 codon 1047 mutation using the PNA probe of SEQ ID NO: 1 to 6 designed in the present invention;
5 is a graph comparing the detection sensitivity (ΔC _t ) according to the sample application amount using a PNA probe of SEQ ID NOs: 12 and 13 designed in the present invention in a cell line having a PIK3CA exon 9 codon 545 mutation;
Figure 6 is a graph comparing the detection sensitivity (ΔC _t ) according to the mutation containing concentration using a PNA probe of SEQ ID NO: 4 designed in the present invention in a cell line having a PIK3CA exon 20 codon 1047 mutation.

The present invention provides a method for detecting a small amount of a mutant with high sensitivity by inhibiting amplification of a wild type by a PNA probe that specifically binds to a wild type for detecting a mutation of exon 9 or 20 of the PIK3CA gene and a kit for use in the method. It is about. 1 schematically shows the principle of the method according to the invention. Specifically, the present invention is for the detection of mutations in exon 9 or 20 of the PIK3CA genes listed in Table 1 below.

Exon protein amino acid Sequence Exon9 Helical Gly542lys 1624 Exon9 Helical Gly545lys 1633 Exon9 Helical Gly545gly 1634 Exon9 Helical Gly545asp 1635 Exon9 Helical Gln546Gly, Gln546Lys 1636 Exon9 Helical Gln546Pro, Gln546Arg 1637 Exon20 Kinase His1047Tyr 3139 Exon20 Kinase His1047Leu, His1047Arg 3140

One. PNA Clamping Of the probe  Design and build

The PNA probes of the present invention are perfectly matched to the wild-type gene sequence in which the mutations (including substitution) of PIK3CA exons 9 and 20 occur, and are preferably 15 or more, preferably 15 to 30, More preferably, it is characterized by consisting of 17 to 27, most preferably 17 to 24 base sequences. The PNA probe of the present invention is preferably designed such that the wild type gene region of the region where mutations (including substitution) of PIK3CA exons 9 and 20 occur is located in the center of the probe. For example, the PNA probe of the present invention may be composed of any one of SEQ ID NOs: 1 to 31 shown in Table 2 below. PNA probe sequences within the range that can be easily modified by those skilled in the art from the nucleotide sequence using conventional knowledge should be considered to be within the scope of the present invention, using the PNA-based real-time PCR clamping according to the present invention As long as the amplification cycle difference alone can effectively detect mutations of the PIK3CA gene exons 9 and 20, it is included within the scope of the present invention.

Specifically, SEQ ID NOs: 7 to 11 are probes for completely binding with the wild type of codon 542 of PIK3CA exon 9 to inhibit wild type amplification and detect mutations. Substitution at codon 542 results in mutations in the helical structure portion of the PI3K protein by replacing guanine at nucleotide 1624 with adenine and wild type glutamic acid at codon 542 with lysine. SEQ ID NOs: 7 to 11 are designed to hybridize specifically to the 1624 th base comprising codon 542 of PIK3CA exon 9. SEQ ID NOs: 9-31 are probes for completely binding with the wild type of the codon 545 portion of PIK3CA exon 9 to inhibit amplification of the wild type and to detect mutations. Substitution at codon 545 includes the substitution of adenine for guanine at nucleotide 1633 with the substitution of lysine for wild type gutamic acid at codon 545, for the replacement of wild type glutamic acid at codon 545 with glycine for adenine at nucleotide 1634. Guanine of nucleotide 1635 is substituted with thymine such that the wild type glutamic acid of codon 545 is replaced with aspartic acid. SEQ ID NOs: 1 to 6 were designed to hybridize specifically to the 3140th base comprising codon 1047 of exon 20. SEQ ID NOs: 1 to 6 are probes for completely binding with wild type of codon 1047 of PIK3CA exon 20 to inhibit amplification of wild type and to detect mutations. Substitution at codon 1047 results in mutations in the kinase portion of the PI3K protein by the substitution of adenine at nucleotide 3140 with guanine or thymine and substitution of arginine or leucine with wild-type histidine of codon 1047.

SEQ ID NO: name Sequence (5 '→ 3') Length One H1047-1 AATTATGCACATCATGGTGGC 21 2 H1047-2 AATTATGCACATCATGGTGGCT 22 3 H1047-3 GATTCACATCATGGTGGC 18 4 H1047-4 ATTATGCACATCATGGTGGC 20 5 H1047-5 TTATGCACATCATGGTG 17 6 H1047-6 ATTCACATCATGGTGGC 17 7 E542-1 ATCCTCTCTCTGAAATCACTGA 22 8 E542-2 TCTCTGAAATCACTGAGCAG 20 9 E542_E545-1 CCTCTCTCTGAAATCACT 18 10 E542_E545-2 GATCCTCTCTCTGAAATCACT 21 11 E542_E545-3 ATCCTCTCTCTGAAATCACTG 21 12 E545? 546-1-as TCTTTCTCCTGCTCAGTSATTT 22 13 E545? 546-2-as TCTTTCTCCTGCTCAGTSATTTAG 24 14 E545? 546-3-as TTTCTCCTGCTCAGTSATTT 20 15 E545? 546-4-s CTTAAATCACTGAGCAGGA 19 16 E545? 546-5-s AATCACTGAGCASSAGA 17 17 E545? 546-6-s TCTTAAATCACTGAGCAGG 19 18 E545? 546-7-s AAATCACTGAGCAGGAGAAA 20 19 E545? 546-8-s AAATCATTGAGCAGGAGAAA 20 20 E545? 546-9-s AAATCACTGASCAGGAGAAA 20 21 E545? 546-10-s AAATCACTGASCAGSAGAAA 20 22 E545? 546-11-as TTCTCCTGCTCAGTGATTT 19 23 E545? 546-12-as TTTCTCCTGCTCAGTGATTTTAG 23 24 E545? 546-13-as CTTTCTCCTGCTCAGTGATTT 21 25 E545? 546-14-as TCTTTCTCCTGCTCATTGATTT 22 26 E545? 546-15-as AATCTTTCTCCTGCTCATTGATTT 24 27 E545? 546-16-as TCTTTCTCCTGCTCAGTGATTT 22 28 E545? 546-17-s AAATCACTGAGCAGGAGAAAGA 22 29 E545-1-as TTCTCCTGCGCAGTGATTT 19 30 E545-2-as TTTCTCCTGCGCAGTGATTTTAG 23 31 E545-3-as CTTTCTCCTGCGCAGTGATTT 21

The PNA probe of the present invention may include a hydrophilic functional group at the N-terminal or C-terminal in order to increase reaction efficiency and solubility, for example, N-terminal or C-terminal It may contain one to several hydrophilic linkers, amino acids, or amine groups in Shakeel et al., J. Chem. Technol. Biotechnol., 2006, 81, 892-899; Gildea et al., Tetrahedron Lett., 1998, 39, 7255-7258; Demidov et al., PNAS, 2002, 99, 5953-5958; Wang et al., Anal. Chem., 1997, 69, 5200-5202. Specifically, in the present invention, a probe in which one lysine is attached to the N-terminus or one lysine is attached to the N-terminus and the C-terminus is used.

The PNA oligomer used in the present invention is a PNA monomer protected with Bts (Benzothiazolesulfonyl) group, or a PNA monomer protected with known Fmoc (9-flourenylmethloxycarbonyl) or t-Boc (t-butoxycarbonyl) according to the method of Korean Patent No. 464,261. (Dueholm et al., J Org chem. 59 (19): 5767-5773, 1994; Christensen J peptide Sci 1 (3): 175-183, 1995; Thomson et al., Tetrahedron 51 (22): 6179-6194, 1995).

2. PIK3CA  gene Clamping Of primer  Design and build

In the present invention, "PIK3CA gene clamping primer" refers to amplification of a mutant gene that inhibits amplification of a wild-type gene that is perfectly bound to a PNA probe and that is not completely bound to the PNA probe (ie, a mismatched sequence exists). Point to PCR primers. The clamping primer of the present invention is not particularly limited, but in order to detect mutations with higher sensitivity and specificity, a portion of the clamping primer overlaps with the PNA probe in one direction and the other direction is to be detected based on the PNA clamping probe. Including the site, it is preferable to devise in consideration of the size of the PCR amplification product. In addition, considering the T _m with the PNA probe, the length is between 17mer and 30mer, it is preferable to design lower than the T _m of the PNA probe. In order to maximize the diagnostic sensitivity and specificity, it is desirable to design to include the part immediately before the base of the mutation in the PNA clamping probe sequence that binds complementarily with the wild type. According to a specific example, the clamping primers were designed to overlap the PNA probes of SEQ ID NOs: 1 to 31 and 5 to 12 base sequences. The forward primer of SEQ ID NO: 36 exemplified herein is designed to specifically recognize the upstream partial base of codon 542 of the PIK3CA gene exon 9 of SEQ ID NOs: 7-11. The reverse primer of SEQ ID NO: 33 in combination with the forward primer of SEQ ID NO: 36 exemplified herein was designed to specifically recognize the 61-80th base of the 9 region of the PIK3CA gene intron. The reverse primers of SEQ ID NOs: 39 and 40 were designed to specifically recognize the downstream bases of codons 545 and 546 of the PIK3CA gene exon 9 of SEQ ID NOs: 9-31. The forward primer of SEQ ID NO: 37 in combination with the reverse primers of SEQ ID NOs: 39 and 40 exemplified herein is designed to specifically recognize the 32-59th base of the PIK3CA gene exon 9. The forward primers of SEQ ID NOs: 41 and 42 were designed to specifically recognize the upstream partial base of codon 1047 of the PIK3CA gene exon 20 of SEQ ID NOs: 1-6. The reverse primer of SEQ ID NO: 35 in combination with the forward primers of SEQ ID NOs: 41 and 42 exemplified in the present invention was designed to specifically recognize the 9th to 29th bases of the 21 region of the PIK3CA gene intron. The length of the primer was designed to be between 20mer and 28mer so that the size of the amplification product of the primer combination was 50bp to 500bp, respectively.

On the other hand, the forward primer of SEQ ID NO: 32 provided in the present invention for gene identification through sequencing of exon 9 of the PIK3CA gene was designed to specifically recognize the -63 ~-44th base of the PIK3CA gene intron 8 site , The reverse primer in combination with the primer was designed to specifically recognize the 61-80th base of the PIK3CA gene intron 9 site by SEQ ID NO: 33. These primers were designed to be combined so that the size of the amplification product was 269 bp. In order to identify genes by sequencing exon 20 of the PIK3CA gene, the forward primer of SEQ ID NO: 34 provided in the present invention is designed to specifically recognize the 69th to 88th bases of the PIK3CA gene exon 20 region, and the primers The reverse primer in combination with was designed to specifically recognize the 9th to 29th bases of the 21 region of the PIK3CA gene intron with SEQ ID NO. These primers were designed to be combined so that the size of the amplification product was 202 bp. The properties of each primer are summarized in Table 3 below.

order
number part location name direction Sequence (5 '→ 3') Length 32 Intron 8 -63 to -44 E542? 545-F Forward CTGTGAATCCAGAGGGGAAA 20 33 Intron 9 61 to 80 E542? 545-R Reverse ACATGCTGAGATCAGCCAAA 20 34 Exon 20 69-88 H1047-F Forward CTCAATGATGCTTGGCTCTG 20 35 Intron 21 9 to 29 H1047-R Reverse TCAGTTCAATGCATGCTGTTT 21 36 Exon 9 59-73 E542K clamp-F Forward CAATTTCTACACGAGATCCTCTCTC 25 37 Exon 9 32 to 59 E545F2 Forward GGGAAAATGACAAAGAACAGCTCAAAGC 28 38 Exon 9 86-109 E542 clamp-R2 Reverse AATCTTTCTCCTGCTCAGTGATTT 24 39 Exon 9 97-120 E545 clamp-R3 Reverse ACTCCATAGAAAATCTTTCTCCTG 24 40 Exon 9 99-122 E546 clamp-R4 Reverse TGACTCCATAGAAAATCTTTCTCC 24 41 Exon 20 240 to 262 H1047RL clamp-F Forward CATGAAACAAATGAATGATGCAC 23 42 Exon 20 240 to 261 H1047YRL clamp-F Forward CATGAAACAAATGAATGATGCA 22

3. PNA  Based real-time PCR Clamping  Used PIK3CA  Mutation detection

PIK3CA gene mutation detection method according to the invention

(a) performing real-time PCR on the PIK3CA gene in the presence of a set of PIK3CA gene clamping primers and a PNA clamping probe; And

(b) analyzing the gene amplification by real-time PCR to determine the presence or absence of mutation of the PIK3CA gene:

It includes.

The PIK3CA gene used in step (a) is prepared by extracting from the subject sample. In the present invention, there is no particular limitation on nucleic acid extraction, and any nucleic acid extraction method generally used may be used, and DNA may be prepared by extracting DNA from blood or tumor samples of a patient using commercially available nucleic acid extraction kits.

In step (a), mutations in the PIK3CA gene are detected using a real-time PCR method. Real-time PCR method provides more accurate quantitative analysis because the amount of initial sample with exponential amplification is expressed as the number of cycles (Cycle threshold, hereinafter referred to as 'C _t ') where the exponential increase in fluorescence is detected. The reaction can be analyzed in real time. This method eliminates the step of measuring the intensity with an electrophoresis image analyzer and can quickly and easily diagnose by automating and quantifying the amplification degree of the amplification product.

In the present invention, it is preferable that the PNA clamping probe in the reaction product of real-time PCR clamping has a final concentration of 1 to 1000 nM.

In the present invention, fluorescence is detected by using an intercalating method. In this method, a fluorescent label is intercalated on the amplified double-stranded DNA to emit fluorescence. The amount of product produced is measured. This can be applied to any PCR device and can detect mutations in the PIK3CA gene with high sensitivity and specificity even without the preparation of a primer.

In the present invention, a DNA-binding fluorophore used in a real-time gene detection method is used as a fluorescent material for identifying gene amplification products, and there is no particular limitation on the type thereof. For example, in addition to SYBR Green I, Evergreen, Ethidium Bromide (EtBr), BEBO, YO-PRO-1, TO-PRO-3, LC Green, SYTO-9, SYTO-13, SYTO-16, SYTO-60, SYTO-62, SYTO-64, SYTO-82, POPO-3, TOTO-3, BOBO-3, SYTOX orange and the like can be used (Gudnason et al., Nucleic Acids Res. 2007; 35 (19): e127, Bengtsson et al., Nucleic Acids Res. 2003; 31 (8): e45; Wittwer et al., Clinical Chemistry 49 (6): 853-860).

In step (b), the amplification of the PIK3CA gene is determined by analyzing the gene amplification by real-time PCR in step (a). The amplification of the PIK3CA gene may be confirmed by comparing the amplified C _t values. When a PNA probe designed to hybridize with a wild-type gene hybridizes to the PIK3CA mutant codon gene region and inhibits amplification, the amplification is inhibited, resulting in high C _t value. On the other hand, if a mutation occurs in the PIK3CA mutant codon region, the hybridization cannot be hybridized with the PNA probe, resulting in a low C _t value. By checking the value of ΔC _t obtained by subtracting the C _t value obtained from the unknown sample from the C _t value obtained from the positive control sample, the presence or absence of mutation of each codon is confirmed (see Equation 1 below).

[Equation 1]

△ C _t = C _t obtained from a positive control sample value - C _t value obtained from the sample of unknown

[Positive control sample: wild type gene sample]

Since the larger the mutant gene is included, the lower the C _t value is. Therefore, the greater the ΔC _t difference, the larger the mutation.

PIK3CA mutation detection method using PNA-based real-time PCR clamping of the present invention can be used to examine tumors such as colon cancer, gastric cancer, lung cancer, pancreatic cancer, head squamous cell carcinoma, glioblastoma, endometrial cancer, ovarian cancer, breast cancer, etc. In addition to tumor research, it can be very useful for studying the mechanisms involved in the PIK3CA signaling system. It can also be effectively applied to studies that require large sample analysis, such as population-based studies.

Hereinafter, the present invention will be described in more detail with reference to Examples, which are intended to aid the understanding of the present invention but are not intended to limit the scope of the present invention in any way.

Example 1 Synthesis of PNA Probes to Inhibit Amplification of Codons 542, 545, and 1047 Wild Type Present in PIK3CA Exons 9 and 20

Twenty-eight PNA probes that fully bind to the wild type of exon 9 codons 542, 545 and exon 20 codon 1047 of the PIK3CA gene were constructed as shown in Table 2 above. Probes that perfectly bind to the wild type of each codon are designed so that the mutant sequence is located in the middle of the probe for effective isolation from the mutation. PNA probes were synthesized according to the method described in Korean Patent No. 464,261 (Lee H et al., Org Lett. 2007 Aug 16; 9 (17): 3291-3.).

Example 2: Primer Synthesis for Target Nucleic Acid Amplification and Clamping PCR of PIK3CA Exons 9 and 20

Primers were prepared by analyzing exon 9 and 20 sites of PIK3CA gene for amplification and clamping PCR of target nucleic acids of PIK3CA exon 9 and 20. The primers of SEQ ID NO: 36 were synthesized using a primer set consisting of SEQ ID NOs: 32 and 33 and a clamping primer of PIK3CA exon 9 codon 542 to identify wild type and mutant genes of PIK3CA exon 9. As the reverse primer used for clamping exon 9 codon 542, the reverse primer of SEQ ID NO: 33 designed to identify the gene of PIK3CA exon 9 was used identically. SEQ ID NOs: 39 and 40 were synthesized with a clamping primer of PIK3CA exon 9 codon 545, and the primer of SEQ ID NO: 37 was used as a forward primer used for clamping exon 9 codon 545. The primers of SEQ ID NOs: 41 and 42 were synthesized using a primer set consisting of SEQ ID NOs: 34 and 35 to identify wild type and mutant genes of PIK3CA exon 20 and a clamping primer of exon 20 codon 1047. As the reverse primer used for clamping exon 20 codon 1047, the reverse primer of SEQ ID 35 was designed to identify the gene of PIK3CA exon 20. The sequence of the used primer is as shown in Table 3 above. Primer was synthesized by BIONIA (Korea).

Example 3: Mutagenesis and Cloning to Prepare Target Nucleic Acids of PIK3CA Exons 9 and 20

Genes of PIK3CA exons 9 and 20 were amplified using primer sets of SEQ ID NOs: 32 and 33 and primer sets of SEQ ID NOs: 34 and 35 using human whole DNA. The amplified nucleic acid was ligated into pGEM-T easy vector (Promega, USA) and transformed into E. coli JM 109 cells to obtain a large amount of DNA. In order to secure a clone with a mutant gene, a primer for mutation was prepared using a normal clone prepared by the above-described method, and a clone with a mutant gene was obtained using a site-specific mutagenesis kit (Stratagene, USA). The obtained clone was confirmed by its sequencing analysis.

Example 4 Nucleic Acid Extraction from Wild-Type and Mutant Cell Lines of PIK3CA Exons 9 and 20

In order to secure target nucleic acids of wild type and mutant of PIK3CA exons 9 and 20, cell lines were distributed from the Korea Cell Line Bank. HT29 (genomic DNA) human colon cancer cell line [KCLB30038, Korea Cell Line Bank (KCLB), Seoul, Korea] was distributed as a wild type cell line. Mutant cell lines shown in Table 4 below were also sold from Korea Cell Line Bank.

KCLB No . Mutation Exon Cell line name origin 30038 Wild type HT29 Colorectal cancer 000601 E542K Exon 9 SNU601 Gastric ulcer tumor 30022 E545K Exon 9 MCF7 Breast cancer 10247 H1047R Exon 20 HCT116 Colorectal cancer

Cell lines received were 10% heat-inactivated fetal bovine serum (FBS, Hyclone, Thermo scientific, USA) and 1X penicillin-streptomycin (WelGENE, Korea) in RPMI1640 (Hyclone, Thermo scientific, USA) or MEM (WelGENE, Korea). ) Was incubated in an incubator maintained at 37 ° C., 5% carbon dioxide (CO ₂ ). The cultured cell line was extracted with DNA using a High Pure PCR Template Preparation Kit (Roche, USA) according to the manual provided in the kit to obtain a target nucleic acid. The obtained nucleic acid was quantified using a nanodrop spectrophotometer (ND 2000C, Thermo Scientific, USA) and stored at -20 ° C. Total DNA isolated from human cell lines, respectively, was subjected to amplification of the PIK3CA exon 9 and 20 genes by applying the primer sets of SEQ ID NOs: 32 and 33 and the primer sets of SEQ ID NOs: 34 and 35 described in Table 3 above. The amplified PCR product was purified using Labopass ™ PCR purification kit (Cosmogenetech, Korea), and then genotyped by sequencing. Genotype-identified wild-type and mutant cell lines were used as samples for real-time PCR using the PNA probe of the present invention .

Example  5: PIK3CA  For exon 9 codon 542 PNA Probe  Real time using PCR  Establish clamping method

Using the plasmid DNA extracted from the clone in Example 3 and the DNA extracted from the cell line in Example 4, by performing RT-PCR clamping under the following conditions, PNA probes for detecting mutations in the PIK3CA gene were made and compared. The analysis attempted to find the optimal PNA probe. 1 μl of plasmid DNA solution extracted from clone or 1 μl of template DNA solution (50 ng / μl) extracted from cell line, 1 μl of one clamping sense primer (10 pmole / μl) shown in Table 3, antisense primer (10 pmole / 1 μl), 1 μl of one of the clamping probes (100 nM) of the probes shown in Table 2, 10 μl of 2X IQ Sybr Green Supermix (Bio-Rad, USA), 6 μl of distilled water and a real-time PCR machine, CFX96TM Real-Time PCR System, manufactured by Bio-RAD Co., Ltd., for 3 minutes at 95 ° C., followed by 95 ° C. 30 seconds and 20 seconds at 70 ° C. where PNA can hybridize, and 63 ° C. 30 seconds, 72 ℃ 30 seconds the reaction process was repeated 40 times. Fluorescence was measured at 72 ° C. polymerization step.

Among the probes, the effects of inhibiting amplification and detecting mutations of codon 542 wild type of exon 9 by applying the probes of SEQ ID NOs: 7 to 2 sets of primers (SEQ ID NOs: 33 and 36, and SEQ ID NOs: 37 and 38) The confirmed result is shown in FIG. As shown in FIG. 2, in the forward clamping using SEQ ID NO: 33 and SEQ ID NO: 36, both SEQ ID NO: 7 showed a very good effect, and in the reverse clamping using SEQ ID NO: 37 and 38, the probes of SEQ ID NOs: 8 to 10 were excellent It was confirmed to exhibit an effect.

Among the probes of SEQ ID NOS: 9 to 31, SEQ ID NOs: 12, 13, 14, 16, and 17 were applied to confirm the effects of inhibiting amplification of codon 545 wild type of exon 9 and detecting mutations. As shown in FIG. 3. All the probes applied showed excellent effects, in particular the probes of SEQ ID NOs: 12 and 13 were found to have a better effect in detecting various mutations.

The results of confirming the effect of inhibiting the amplification of codon 1047 wild type of exon 20 and detecting the mutation by applying the probe of SEQ ID NOS: 1 to 6 are shown in FIG. As shown in FIG. 4, the probes of SEQ ID NOs: 1, 2, and 4 exhibited excellent effects, and in particular, the probes of SEQ ID NO: 4 were found to exhibit better effects.

Example 6: Mutation Detection of PIK3CA Gene by Real-Time PCR Clamping Using PNA Probes

By using the method using real-time PCR established in Example 5, the mutant genes were included in the wild-type genes to include 10 ng, 5 ng, 2.5 ng, 1 ng, 0.1 ng, 0.05 ng, and 0.01 ng, respectively. The correlation between the _t values was analyzed to confirm the detection limit of the mutant.

5 shows the results of comparing the detection sensitivity (ΔC _t ) according to the sample application amount using the PNA probes of SEQ ID NOs: 12 and 13 in the cell line having the PIK3CA exon 9 codon 545 mutation. As shown in FIG. 5, as the amount of the mutant gene increases, the C _t value representing the number of reactions in which the fluorescence reaches the threshold value is constantly decreased (ie, the ΔC _t value is increased), thereby increasing the amount of the mutant gene and the C _t value. Correlated with.

Figure 6 shows the results of comparing the detection sensitivity (ΔC _t ) according to the mutation-containing concentration using a PNA probe of SEQ ID NO: 4 in the cell line having a PIK3CA exon 20 codon 1047 mutation. As shown in FIG. 6, as the relative concentration of the mutant gene was increased, the C _t value was constantly decreased (ie, the ΔC _t value was increased), indicating that there was a correlation between the relative concentration of the mutant gene and the C _t value. In addition, it was confirmed that the presence or absence of a mutation present in 0.1% when using the method of the present invention can be detected.

<110> Panagene, Inc. Methods and kits for the PIK3CA mutant detection using          PNA-mediated Real-time PCR clamping <160> 42 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 1 aattatgcac atcatggtgg c 21 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 2 aattatgcac atcatggtgg ct 22 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 3 gattcacatc atggtggc 18 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 4 attatgcaca tcatggtggc 20 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 5 ttatgcacat catggtg 17 <210> 6 <211> 17 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 6 attcacatca tggtggc 17 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 7 atcctctctc tgaaatcact ga 22 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 8 tctctgaaat cactgagcag 20 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 9 cctctctctg aaatcact 18 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 10 gatcctctct ctgaaatcac t 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 11 atcctctctc tgaaatcact g 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 12 tctttctcct gctcagtsat tt 22 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 13 tctttctcct gctcagtsat ttag 24 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 14 tttctcctgc tcagtsattt 20 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 15 cttaaatcac tgagcagga 19 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 16 aatcactgag cassaga 17 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 17 tcttaaatca ctgagcagg 19 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 18 aaatcactga gcaggagaaa 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 19 aaatcattga gcaggagaaa 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 20 aaatcactga scaggagaaa 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 21 aaatcactga scagsagaaa 20 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 22 ttctcctgct cagtgattt 19 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 23 tttctcctgc tcagtgattt tag 23 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 24 ctttctcctg ctcagtgatt t 21 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 25 tctttctcct gctcattgat tt 22 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 26 aatctttctc ctgctcattg attt 24 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 27 tctttctcct gctcagtgat tt 22 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 28 aaatcactga gcaggagaaa ga 22 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 29 ttctcctgcg cagtgattt 19 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 30 tttctcctgc gcagtgattt tag 23 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <220> P223 probe <400> 31 ctttctcctg cgcagtgatt t 21 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 32 ctgtgaatcc agaggggaaa 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 33 acatgctgag atcagccaaa 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 34 ctcaatgatg cttggctctg 20 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 35 tcagttcaat gcatgctgtt t 21 <210> 36 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 36 caatttctac acgagatcct ctctc 25 <210> 37 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 37 gggaaaatga caaagaacag ctcaaagc 28 <210> 38 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 38 aatctttctc ctgctcagtg attt 24 <210> 39 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 39 actccataga aaatctttct cctg 24 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 40 tgactccata gaaaatcttt ctcc 24 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 41 catgaaacaa atgaatgatg cac 23 <210> 42 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 42 catgaaacaa atgaatgatg ca 22

Claims (11)

A set of PIK3CA gene clamping primers that amplify the exon 9 or exon 20 sites of the PIK3CA (Phosphatidylinositol 3-kinase, catalytic, alpha polypeptide) gene, and the PNA (Peptide Nucleic Acid), which completely binds to the wild type of exon 9 or exon 20 of the PIK3CA gene ) Real-time polymerase chain reaction (PCR) on the PIK3CA gene in the presence of the clamping probe;
Gene amplification by the real-time PCR is analyzed to determine the presence or absence of mutations in the PIK3CA gene:
Method of detecting a mutation of the PIK3CA gene, comprising the step. The method of claim 1, wherein the presence or absence of a mutation in the PIK3CA gene is determined by measuring a cycle threshold (C _t ) value of real-time PCR. According to claim 1 or 2, wherein the PNA clamping probe is a mutation detection method of the PIK3CA gene, consisting of 15 to 30mer long nucleotide sequence. The method of claim 3, wherein the PNA clamping probe consists of a nucleotide sequence of 17 to 24mer in length, mutation detection method of the PIK3CA gene. 5. The method of claim 4, wherein the PNA clamping probe consists of any one of the nucleotide sequences of SEQ ID NOs: 1 to 31. 6. 3. The PIK3CA gene clamping primer set of claim 1 or 2, wherein the set of PIK3CA gene clamping primers comprises a forward primer that specifically binds upstream of the PIK3CA gene exon 9 wild type codon 542, 545 or 546, or exon 20 wild type codon 1047, and a downstream portion thereof. It comprises a reverse primer that specifically binds to, the mutation detection method of the PIK3CA gene. The method of claim 6, wherein the forward primer consists of any one of SEQ ID NOs: 36, 37, 41, and 42, and the reverse primer consists of any one of SEQ ID NOs: 33, 35, and 38-40. . The method of claim 1 or 2, wherein the gene amplification is analyzed using DNA intercalating fluorophores. The method of claim 8, wherein the DNA intercalating fluorescent material is SYBR Green I, Evergreen, Ethidium bromide (EtBr), BEBO, YO-PRO-1, TO-PRO-3, LC Green, SYTO-9, PIK3CA, one or more selected from the group consisting of SYTO-13, SYTO-16, SYTO-60, SYTO-62, SYTO-64, SYTO-82, POPO-3, TOTO-3, BOBO-3 and SYTOX Orange Method for detecting mutation of genes. The method of claim 1 or 2, wherein the PIK3CA gene is for use in determining or diagnosing the treatment of colorectal cancer, gastric cancer, lung cancer, pancreatic cancer, head squamous cell carcinoma, glioblastoma, endometrial cancer, ovarian cancer or breast cancer. Mutation Detection Method. PNA clamping probe of any one of SEQ ID NOs: 1 to 31:
A kit for use in a method for detecting mutations in the PIK3CA gene according to claim 5.

Images