Promer for Real-Time Detection of Nucleic Acid or Protein and Method of detecting Nucleic Acid or Protein Using the Same

Abstract

The present invention relates to a promer that has a structure of X-Y-Z and that has a detectable marker attached to both ends or the inside thereof and also that is used as a primer and a probe during real-time detection of nucleic acid or protein, and to a method for real-time detection of nucleic acid, having amplifying a nucleic acid to be detected using the promer, and then measuring the amount of fragments of the promer cleaved, or to a method for real-time detection of protein. The method for detection of nucleic acid or protein according to the present invention uses a small amount of oligos compared to a conventional detection method, does not require a separate probe for real-time detection, and thus can achieve real-time detection of the nucleic acid or protein to be detected in a cost-effective and simple manner. Furthermore, mutations in the Y region can be detected through amplification after cleavage of the Y region of the promer, and multiplex detection of nucleic acids or proteins larger than the number of fluorescent labels attached to the promer is possible. Thus, the present invention can be effectively used for diagnosis of various diseases and for prognostic diagnosis.

Claims

1. A promer which is used as a primer and a probe for real-time detection of nucleic acid or protein, has a structure of X-Y-Z, comprises a detectable marker attached to both ends or an inside thereof, and forms a complex by binding to a target nucleic acid or a specific region of a target protein to be detected in real time, wherein when Y region of the promer is cleaved by a specific enzyme, X region act as primer in maintaining a complex with the target nucleic acid or the target protein and Y and Z regions are separated from the specific region of the target nucleic acid or protein, wherein when any one of X, Y and Z is DNA, one or more of the other two are RNA, wherein X, Y and Z are a DNA or RNA consisting of nucleotides, and wherein when any one of X, Y and Z is DNA, one or more of the other two are RNA.

2. The promer of claim 1, wherein X is a DNA or RNA comprising 1 to 60 nucleotides, Y is a DNA or RNA comprising 1 to 10 nucleotides, Z is a DNA or RNA comprising 0 to 10 nucleotides, and when Z is null, Y is a DNA or RNA comprising 3 to 10 nucleotides, and when Z is 1, Y is a DNA or RNA comprising 2 to 10 nucleotides.

3. The promer of claim 1, wherein X, Y and Z are DNA, RNA and DNA, respectively, or are RNA, DNA and RNA, respectively.

4. The promer of claim 1, wherein the detectable marker is either a fluorescent label that binds to the promer by covalent binding or non-covalent binding, or a fluorescent pair of the fluorescent label and a quencher.

5. The promer of claim 1, wherein X, Y and Z of the promer are synthesized so as to be wholly or partially methylated to prevent nonspecific cleavage.

6. The promer of claim 1, wherein X of the promer is a DNA or RNA comprising 10 to 30 nucleotides, Y is a DNA or RNA comprising consisting of 1 to 10 nucleotides, and Z is a DNA or RNA comprising 2 to 10 nucleotides.

7. The promer of claim 1, wherein when the Y region of the promer is DNA, the enzyme is DNA nuclease (DNase), specifically DNase I, DNase II, S1 nuclease, nuclease P1, AP endonuclease, or UvrABSC nuclease, and when the Y region is RNA, the enzyme is ribonuclease (RNase), specifically RNase II, RNase III, RNase IV, RNase H, or RNase T.sub.2.

8. The promer of claim 1, wherein the promer is used as: i) an RT primer for synthesizing cDNA from RNA among nucleic acids; or ii) a forward primer for amplifying cDNA synthesized from nucleic acid (DNA or RNA); or iii) a reverse primer for amplifying cDNA synthesized from nucleic acid (DNA or RNA); or iv) forward and reverse primers for cDNA synthesized from nucleic acid (DNA or RNA); or v) a probe for real-time detection of a nucleic acid (DNA or RNA) or protein to be detected.

9. A kit for detection of nucleic acid, comprising: the promer of claim 1; and an enzyme capable of cleaving the Y region of the promer.

10. The kit of claim 9, wherein when the Y region of the promer is DNA, the enzyme is DN A nuclease (DNase), specifically DNase I, DNase II, S1 nuclease, nuclease P1, AP endonuclease, or UvrABSC nuclease, and when the Y region is RNA, the enzyme is ribonuclease (RNase), specifically RNase II, RNase III, RNase IV, RNase H, or RNase T.sub.2.

11. The kit of claim 9, further comprising reagents required for amplification of DNA.

12. A method for real-time detection of RNA, comprising the steps of: (a) extracting RNA from a sample; (b) adding an RT primer or the promer of claim 1 to the RNA extracted in step (a), thereby synthesizing cDNA; (c) adding, to the cDNA synthesized in step (b), (i) the kit, wherein the promer contained in the kit has a nucleotide sequence capable of complementarily binding to a portion of a nucleotide sequence of the cDNA synthesized in step (b) and serves as a forward primer and a probe, and a reverse primer having a nucleotide sequence capable of complementarily binding to a portion of a nucleotide sequence of the cDNA synthesized in step (b); or (ii) the kit wherein the promer contained in the kit has a nucleotide sequence capable of complementarily binding to a portion of a nucleotide sequence of the cDNA synthesized in step (b) and serves as a reverse primer and a probe, and a forward primer having a nucleotide sequence capable of complementarily binding to a portion of a nucleotide sequence of the cDNA synthesized in step (b); or (iii) the kit, wherein the promer contained in the kit has a nucleotide sequence capable of complementarily binding to a portion of a nucleotide sequence of the cDNA synthesized in step (b) and serves as a forward primer, a reverse primer and a probe, and amplifying the cDNA by extension; and (d) measuring an amount of fragments of the promer cleaved through step (c).

13. The method of claim 12, wherein step (c) is a step in which the Y region of the promer bound to the cDNA synthesized in step (b) is cleaved by the enzyme contained in the kit so that the Y and Z regions are separated from the cDNA and the X region serves as the primer for amplification.

14. A method for real-time detection of DNA, comprising the steps of: (a) extracting DNA from a sample; (b) adding, to the DNA extracted in step (a), (i) the kit of claim 10, wherein the promer contained in the kit has a nucleotide sequence capable of complementarily binding to a portion of a nucleotide sequence of the DNA extracted in step (a) and serves as a forward primer and a probe, and a reverse primer having a nucleotide sequence capable of complementarily binding to a portion of a nucleotide sequence of the DNA synthesized in step (a); or (ii) the kit, wherein the promer contained in the kit has a nucleotide sequence capable of complementarily binding to a portion of a nucleotide sequence of the DNA extracted in step (a) and serves as a reverse primer and a probe, and a forward primer having a nucleotide sequence capable of complementarily binding to a port ion of a nucleotide sequence of the DNA extracted in step (a); or (iii) the kit, wherein the promer contained in the kit has a nucleotide sequence capable of complementarily binding to a portion of a nucleotide sequence of the DNA extracted in step (a) and serves as a forward primer, a reverse primer and a probe, and amplifying the DNA by extension; and (c) measuring an amount of fragments of the promer cleaved through step (b).

15. The method of claim 14, wherein step (b) is a step in which the Y region of the promer bound to the DNA extracted in step (a) is cleaved by the enzyme contained in the kit so that the Y and Z regions are separated from the cDNA and the X region serves as the primer for amplification.

16. A method for real-time detection of protein in a sample, the method comprising the steps of: (a) preparing an antibody having attached thereto a nucleic acid having a nucleotide sequence complementary to the promer of claim 1; (b) binding the antibody, prepared in step (a), to a sample containing a protein to be detected, thereby forming a protein-antibody complex; (c) hybridizing the kit to the protein-antibody complex of step (b), thereby forming a protein-antibody-promer complex; and (d) measuring an amount of fragments of the promer cleaved through step (c).

17. The method of claim 16, wherein step (d) is a step in which the Y region of the promer bound to the antibody prepared in step (a) is cleaved by the enzyme contained in the kit so that the Y and Z regions are separated from the cDNA and the X region serves as the primer for amplification.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0145] FIG. 1 illustrates a process of detecting a target nucleic acid using a promer according to the present invention.

[0146] FIG. 2 shows the results of a polymerase chain reaction performed using the primers of SEQ ID NOs: 2 and 7, prepared in an example of the present invention, to measure expression of CK-18 gene in a SW620 human cell line and PBMC (a peripheral blood mononuclear cell).

[0147] FIG. 3 shows the results of a polymerase chain reaction performed using the primers of SEQ ID NOs: 3 and 8, prepared in an example of the present invention, to measure expression of CK-19 gene in a SW620 human cell line and PBMC.

[0148] FIG. 4 shows the results of a polymerase chain reaction performed using the primers of SEQ ID NOs: 6 and 9, prepared in an example of the present invention, to measure expression of GAPDH gene in a SW620 human cell line and PBMC.

[0149] FIG. 5 shows the results of a polymerase chain reaction performed using the promer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 2, prepared in an example of the present invention, to measure expression of CK-18 gene in a SW620 human cell line and PBMC.

[0150] FIG. 6 shows the results of a polymerase chain reaction performed using the forward primer of SEQ ID NO: 3 and the promer of SEQ ID NO: 4, prepared in an example of the present invention, to measure expression of CK-19 gene in a SW620 human cell line and PBMC.

[0151] FIG. 7 shows the results of a polymerase chain reaction performed using the promer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6, prepared in an example of the present invention, to measure expression of GAPDH gene in a SW620 human cell line and PBMC.

[0152] FIG. 8 shows the results of a polymerase chain reaction performed using the promer of SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 12, prepared in an example of the present invention, to measure G12D mutant in KRAS gene.

[0153] FIG. 9 shows the results of a polymerase chain reaction performed using the promer of SEQ ID NO: 10 and the reverse primer of SEQ ID NO: 12, prepared in an example of the present invention, to measure G12V mutant in KRAS gene.

[0154] FIG. 10 shows the results of a polymerase chain reaction performed using the promer of SEQ ID NO: 10 and the reverse primer of SEQ ID NO: 12, prepared in an example of the present invention, to measure G12D mutant in KRAS gene.

[0155] FIG. 11 shows the results of detecting T529C mutant in OPN1MW gene after performing a polymer chain reaction using the promer of SEQ ID NO: 14 and the reverse primer of SEQ ID NO: 19, prepared in an example of the present invention.

[0156] FIG. 12 shows the results of detecting T529C mutant in OPN1MW gene after performing a polymer chain reaction using the promer of SEQ ID NOs: 14 and 15 and the reverse primer of SEQ ID NO: 19, prepared in an example of the present invention.

[0157] FIG. 13 shows the results of detecting T529C mutant in OPN1MW gene after performing a polymer chain reaction using the promers of SEQ ID NOs: 14 and 16 and the reverse primer of SEQ ID NO: 19, prepared in an example of the present invention.

[0158] FIG. 14 shows the results of detecting T529C mutant in OPN1MW gene after performing a polymer chain reaction using the promers of SEQ ID NOs: 14 and 17 and the reverse primer of SEQ ID NO: 19, prepared in an example of the present invention.

[0159] FIG. 15 shows the results of detecting T529C mutant in OPN1MW gene after performing a polymer chain reaction using the promers of SEQ ID NOs: 14 and 18 and the reverse primer of SEQ ID NO: 19, prepared in an example of the present invention.

[0160] FIG. 16 shows the results of detecting G12D mutant in KRAS gene after performing a polymer chain reaction using the promers of SEQ ID NOs: 20 and 21 and the reverse primer of SEQ ID NO: 12, prepared in an example of the present invention.

[0161] FIG. 17 shows the results of detecting G12V mutant in KRAS gene after performing a polymer chain reaction using the promers of SEQ ID NOs: 22 and 23 and the reverse primer of SEQ ID NO: 12, prepared in an example of the present invention.

[0162] FIG. 18 show the results of confirming whether or not the Salmonella Set 33 was amplified using Catacleave probe according to the prior art.

DETAILED DESCRIPTION OF THE INVENTION

[0163] Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to those skilled in the art that these examples are illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: Real-Time Analysis of Nucleic Acid Using Promer of the Present Invention

[0164] In order to measure the expression of CK-18 (cytokeratin-18), CK-19 (cytokeratin-19) and GAPDH (glyceraldehyde 3-phophate dehydrogenase) genes, the promers according to the present invention and primers were constructed by IDT (Integrated DNA Technologies, USA) as shown in Table 1 below (see Table 1). Herein, for the promers, FAM (fluorescein succinimidyl ester) was attached to the 5 end, and 3IABkFG was attached to the 3 end. For ribonucleic acid (RNA), the letter r was added to the front of the sequence for discrimination from deoxyribonucleic acid (DNA). As controls, the primers to be used in the SYBR method were prepared by IDT as shown in Table 2 below.

TABLE-US-00001 TABLE1 HumanCK18 5-ATCTTGGTGATG SEQID F-Promer CCTTrGrGAC-3 NO:1 HumanCK18 5-CCTGCTTCTGCT SEQID Rprimer GGCTTAAT-3 NO:2 HumanCK19 5-GTCACAGCTGAG SEQID FPrimer CATGAAAG-3 NO:3 HumanCK19 5-TCACTATCAGCTC SEQID R-Promer GCArCrATC-3 NO:4 HumanGAPDH 5-AAGGTGAAGGTCG SEQID F-Promer GAGrUrCAA-3 NO:5 HumanGAPDH 5-AATGAAGGGGTCA SEQID Rprimer TTGATGG-3 NO:6

TABLE-US-00002 TABLE2 HumanCK18 5-ATCTTGGTGA SEQID Fprimer TGCCTTGGAC-3 NO:7 HumanCK18 5-CCTGCTTCTG SEQID Rprimer CTGGCTTAAT-3 NO:2 HumanCK19 5-GTCACAGCTG SEQID Fprimer AGCATGAAAG-3 NO:3 HumanCK19 5-TCACTATCAG SEQID Rprimer CTCGCACATC-3 NO:8 HumanGAPDH 5-AAGGTGAAGG SEQID Fprimer TCGGAGTCAA-3 NO:9 HumanGAPDH 5-AATGAAGGGG SEQID Rprimer TCATTGATGG-3 NO:6

[0165] RNA from each of the SW620 and PMBC obtained from human blood was subjected to reverse transcription polymerase chain reaction (RT-PCR) to synthesize cDNAs. 10 ng of each cDNA, 10 m units of heat-resistant RNase H and 4 l of AptaTaq DNA master (Roche) were added to a tube, and the total volume was adjusted to 20 l using triple distilled water. Next, the cDNA was subjected to a polymerase chain reaction (PCR) was performed in the presence of 1 l of 10 M concentration of each promer and each primer, shown in Tables 1 and 2. Herein, the PCR reaction was performed for 40 cycles (SYBR method) and 45 cycles (the promer of the present invention), each cycle consisting of 5 min at 95 C., 60 secs at 62 to 63 C. and 10 sec at 95 C. The results of the PCR are shown in FIGS. 2 to 7.

[0166] As a result, it could be seen that, when expression of CK-18, CK-19 and GAPDH was measured using the SYBR method, a nonspecific amplification curve for CK-18 and CK-19 excluding GAPDH appeared in the negative control, and nonspecific amplification also occurred in the positive control (see FIGS. 2 to 4). On the contrary, when the promer according to the present invention was used, problems such as nonspecific amplification did not occur in the case of not only GAPDH but also CK-18 and CK-19. Thus, it can be seen that the promer according to the present invention can be used in gene expression and analysis without causing any nonspecific reaction, unlike the conventional SYBR method.

Example 2: Measurement of Mutant Using the Promer of the Present Invention

[0167] Using the promer of the present invention, G12D and G12V mutants in KRAS gene were measured. Specifically, the promers according to the present invention and a reverse primer were constructed by IDI as shown in Table 3 below. For the promers, FAM (fluorescein succinimidyl ester) was attached to the 5 end, and 3IABkFG was attached to the 3 end. For ribonucleic acid (RNA), the letter r was added to the front of the sequence for discrimination from deoxyribonucleic acid (DNA).

TABLE-US-00003 TABLE3 G12DForward- 5-CTTGTGGTAGTT SEQID Promer GGAGCTGrATG-3 NO:10 G12VForward- 5-ACTTGTGGTAGT SEQID Promer TGGAGCTGrATG-3 NO:11 Uni-reverse 5-CATATTCGTCCAC SEQID primer AAAATGATTCTG-3 NO:12

[0168] In addition, the KRAS gene to be measured was obtained as total DNA from each of SW620 cell line and LS174T cell line. For wild-type gene, total DNA was obtained from the blood of normal persons in the same manner. Herein, SW620 is known as G12V mutant cell-line, and LS174T is known as G12D mutant cell-line.

[0169] Next, 10 m units of heat-resistant RNase H, 4 l of AptaTaq DNA Master (Roche), 40 ng of total DNA extracted from SW620, and each of 4 pg, 40 pg, 400 pg, and 4 ng of total DNA extracted from LS174T were placed in a tube, and then the total volume was adjusted to 20 l using triple distilled water. Thereafter, 1 l of 10 M concentration of the promer of SEQ ID NO: 11 and the primer of SEQ ID No: 12, constructed as described above, were prepared, and then polymerase chain reaction was performed to measure G12D mutant known as LS174T mutant. Herein, the PCR reaction was performed under the conditions of 5 min at 95 C., 60 secs at 62 to 63 C. and 10 secs at 95 C. The results of the measurement are shown in FIG. 8.

[0170] In addition, G12V mutant known as SW620 mutant was measured in the presence of the promer of SEQ ID NO: 10 and the primer of SEQ ID NO: 12 under the same conditions as described above, except that each of 4 pg, 40 pg, 400 pg, and 4 ng of total DNA extracted from SW620 was added to 40 ng of total DNA extracted from LS174T, instead of adding total DNA extracted from SW620 to total DNA extracted from LS174T. The results of the measurement are shown in FIG. 9.

[0171] In addition, G12D mutant known as LS174T mutant was measured in the presence of the promer of SEQ ID NO: 10 and the primer of SEQ ID NO: 12 under the same conditions as described above, except that 40 pg of total DNA extracted from LS174T cell line was added to 40 ng of total RNA from normal persons, instead of adding total DNA extracted from SW620 to total DNA extracted from LS174T. The results of the measurement are shown in FIG. 10.

[0172] From the above results, it can be seen that, when the promer according to the present invention is used, a mutant can be measured with excellent sensitivity and specificity.

[0173] Namely, in the endpoint genotyping, which is widely used as a method to measure point mutations, only the presence or absence of mutation can be identified through real-time PCR reaction. Respectively. However, in the case of using the promer according to the present invention, it was found that the type and the ratio of the mutation can be analyzed through the real-time PCR reaction as described above. Furthermore, in the case of the conventional method, a maximum of 10.1% mutation can be confirmed, but in the case of the present invention, 0.01% mutation analysis is possible.

Example 3: Determination of Optimal Size of the Promer According to the Present Invention

[0174] The OPN1MW gene (medium-wave-sensitive opsin-1 gene) located in the long arm (Xq28) of chromosome X is a gene associated with color weakness and color blindness, and it is known that mutations occur at four nucleotide locations of the gene to cause red-green color blindness. The positions of the mutations are C282A, T529C, T607C and G989A.

[0175] Accordingly, in order to examine the mutant detection ability of the promer of the present invention according to the length thereof, a plasmid containing a gene (see the DNA sequence of SEQ ID NO: 13) obtained by mutating the T529C position of the OPN1MW gene was constructed, and then diluted to concentrations of 10 fM, 1 fM, 100 aM and 10 aM. 1 l of each of the dilutions was taken, and then five promers and a reverse primer were constructed from each dilution by IDT as shown in Table 4 below. The constructed promers and primer were used for detection. Herein, FAM (fluorescein succinimidyl ester) was attached to the 5 end of the five constructed promers, and 3IABkFG was attached to the 3 end. For ribonucleic acid (RNA), the letter r was added to the front of the sequence for discrimination from deoxyribonucleic acid (DNA). The results obtained using the promer of SEQ ID NO: 14 (see FIG. 11) and the results obtained using the promer of SEQ ID NO: 13 and each of the promers of SEQ ID NOs: 15, 16, 17, and 18 (see FIGS. 12 to 15) are shown in FIGS. 11 to 15.

TABLE-US-00004 TABLE14 529CA1 5-TGGGCATTGCCT SEQID Promer TCTCCrCGG-3 NO:14 529CA2 5-GCCTTCT SEQID Promer CCrCGG-3 NO:15 529CA3 5-CAAGCTGGCCATCGTG SEQID Promer GGCATTGCCTTCTCCrCGG-3 NO:16 529CA4 5-TGGGCATTGCCTTCTC SEQID Promer CrCGGATCTGGGC-3 NO:17 529CA5 5-TGGGCATTGCCTTCTCCr SEQID Promer CGGATCTGGGCTGCTGTG-3 NO:18 Reverse 5-GTACCTGCTC SEQID primer CAACCAAAGA-3 NO:19

[0176] As can be seen in FIGS. 11 to 15, when the X region in the X-Y-Z structure of the promer according to the present invention consisted of 9 nucleotides, the X region was also separated from the template after cleavage of the Y region, and thus served only as a probe without serving as a primer (see FIG. 12), and when the X region consisted of 31 nucleotides, a nonspecific amplification reaction was observed (see FIG. 13). Furthermore, when the Z region consisted of 2 or 10 nucleotides, only the Z region was separated from the template after cleavage of the Y region, and the X region was not separated, and thus a normal amplification reaction occurred (see FIGS. 11 and 14), but when the Z region consisted of 17 nucleotides, the Z region was not separated from the template after cleavage of the Y region, and thus a normal amplification reaction did not occur (see FIG. 15). As described above, it could be seen that, when the X and Z regions of the promer according to the present invention consisted of a specific number of nucleotides, a specific amplification reaction could occur.

[0177] In addition, a mutant in KRAS gene was measured in the same manner as the experiment of Example 2, except that, as shown in Table 5 below, the Y region consisted of one RNA and the Z region consisted of one or two DNAs. Specifically, 10 M of the promer of SEQ ID NO: 20 and 1 l of the primer of SEQ ID NO: 10, or 10 M of the promer of SEQ ID NO: 21 and 1 l of the primer of SEQ ID NO: 12, 10 m units of heat-resistant RNase H, 4 l of AptaTaq DNA master (Roche), and each of 120 pg and 1.2 ng of total DNA extracted from LS174T were placed in a test tube, and the total volume was adjusted to 20 l using triple distilled water. Next, a polymerase chain reaction was performed under the conditions of 5 min at 95 C., 60 sec at 62 C. to 63 C. and 10 sec at 95 C., thereby measuring G12D mutant known as LS174T mutant. The results of the measurement are shown in FIG. 16.

[0178] In addition, 10 M of the promer of SEQ ID NO: 22 and 1 l of the primer of SEQ ID NO: 12, or 10 M of the promer of SEQ ID NO: 23 and 1 l of the primer of SEQ ID NO: 12, 10 m units of heat-resistant RNase H, 4 l of AptaTaq DNA master (Roche), and each of 120 pg and 1.2 ng of total DNA extracted from SW620 were placed in a test tube, and the total volume was adjusted to 20 l using triple distilled water. Next, a polymerase chain reaction was performed under the conditions of 5 min at 95 C., 60 sec at 62 C. to 63 C. and 10 sec at 95 C., thereby measuring G12V mutant known as SW620 mutant. The results of the measurement are shown in FIG. 17.

TABLE-US-00005 TABLE5W \DATA\CLDOCS\J-K\59\00 G12DForward- 5-ACTTGTGGTAGT SEQID PromerrYType TGGAGCTGrAT-3 NO:20 G12DForward- 5-CTTGTGGTAGTT SEQID PromerrYYType GGAGCTGrATG-3 NO:21 G12VForward- 5-AACTTGTGGTAG SEQID PromerrYType TTGGAGCTGrUT-3 NO:22 G12VForward- 5-ACTTGTGGTAGT SEQID PromerrYYType TGGAGCTGrUTG-3 NO:23 Uni-reverse 5-CATATTCGTCCA SEQID primer CAAAATGATTCTG-3 NO:12

[0179] As can be seen in FIGS. 16 and 17, when the spacing between the Y region to be cleaved and the Z region was at least 2 bp, the amplification reaction using the promer of the present invention smoothly occurred.