KR20060000840A - Kit for quantitative analysis of pml-rara - Google Patents

Abstract

The present invention provides PCR reaction mixtures, probe and primer mixtures capable of detecting PML-RARa fusion genes, probe and primer mixtures capable of detecting ABL genes, PML-RARa standard plasmid DNA, ABL standard plasmid DNA, PML-RARa negative The present invention relates to a method for quantifying PML-RARa fusion gene using a control kit and a PML-RARa quantitative kit including an ABL negative control group and an electric kit. When the PML-RARa quantitative kit of the present invention is used, the degree of recombination of the PML-RARa fusion gene which causes acute promyelocytic leukemia can be more easily and accurately quantified, and thus can be widely used for accurate diagnosis of acute promyelocytic leukemia. Could be.

PML-RARa fusion gene, PML-RARa standard plasmid DNA, ABL standard plasmid DNA

Description

Kit for Quantitative Analysis of PML-RARa}             

1 is a genetic map showing one embodiment of PML-RARa standard plasmid DNA.

Figure 2 is a genetic map showing one embodiment of ABL standard plasmid DNA.

The present invention relates to a PML-RARa quantitative kit. More specifically, the present invention provides PCR reaction mixtures, probe and primer mixtures capable of detecting PML-RARa fusion genes, probe and primer mixtures capable of detecting ABL genes, PML-RARa standard plasmid DNA, ABL standard plasmid DNA The present invention relates to a method for quantifying PML-RARa fusion gene using PML-RARa quantitative kit and electric kit including PML-RARa negative control group and ABL negative control group.

A gene rearrangement in which the RARa gene of human chromosome 17 is translocated and fused to the PML gene region of chromosome 15 is called PML-RARa rearrangement and is called acute promyelocytic leukemia: APL). Acute promyelocytic leukemia corresponds to the M3 subtype of acute myelocytic leukemia (AML), with PML-RARa rearrangement in more than 95% of acute promyelocytic leukemias. In the presence of the electrical gene rearrangement, it is known that the therapeutic effect by the all-trans retinoic acid (ATRA) agent is good. When the leukocytes in which the PML-RARa gene rearrangement occurs are exposed to ATRA, the PML-RARa fusion protein is depleted. Secreted, activating the transcription of the retinoic acid reactive gene, resulting in the differentiation of acute promyelocytic leukemia cells. The PML-RARa rearrangement is difficult to find cytologically, but is important in determining the treatment direction and prognosis of the patient, the molecular biological results have an important meaning.

Accordingly, in order to confirm the PML-RARa rearrangement, a method of quantifying the degree of recombination of the PML-RARa fusion gene is mainly used, and a method of more easily quantifying the degree of recombination of the PML-RARa fusion gene is used. Various studies are underway to develop. For example, diagnostic methods combining cytogenetics and fluorescent in situ hybridization (FISH) (Mancini et al, British Journal of Haematology, 91: 878-884, 1995), and methods using PCR for somatic DNA (see: Tashiro et al, Japanese Journal of Cancer Research, 84: 110-113, 1993) and methods using RT-PCR (Castaigne et al., Blood, 79: 3110-3115, 1991; Fukutani et al, Leukemia, 9 : 588-593, 1995). However, FISH is a method of directly detecting individual cells in which PML-RARa rearrangement has occurred, and it is difficult to detect in the early stage of acute promyelocytic leukemia, and the method by PCR or RT-PCR has excellent sensitivity. Errors may be due to contamination, etc., and there is a disadvantage in that it is not possible to accurately determine the progress of the disease.

As a method for overcoming the above-mentioned disadvantages, recently, PML-RARa diagnostic kits have been developed and marketed using a real-time RT-PCR method capable of quantifying target RNA from a small amount of sample. However, the diagnostic kits may include stability and reproducibility problems by providing RNA as a standard gene, do not include primers and probes for quantifying reference genes used for standardization, or primers and probes for quantifying reference genes. Even if it is included, since it contains primers and probes for quantifying glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene or beta actin gene irrelevant to leukemia quantification, it is difficult to perform accurate PML-RARa quantification.

Therefore, there is a constant need to develop a quantitative kit that can more easily and accurately quantify the degree of recombination of PML-RARa fusion genes.

Accordingly, the present inventors have made intensive studies to develop a quantitative kit that can more easily and accurately quantify the degree of recombination of PML-RARa fusion genes. As a result, the PML-RARa fusion gene-specific primers and probes and the ABL gene specific control group When using the PML-RARa quantitative kit comprising a primer and a probe, it was confirmed that the degree of recombination of the PML-RARa fusion gene can be quantified more simply and accurately, and completed the present invention.

After all, the main object of the present invention is to provide a PML-RARa quantitative kit that is simple and has improved accuracy and reproducibility.

Another object of the present invention is to provide a method for quantifying a PML-RARa fusion gene using an electric kit.

PML-RARa quantitative kit of the present invention (i) PCR reaction mixture; (Ii) a PML-RARa probe and primer mixture consisting of two sense primers (SEQ ID NOs: 1 and 2) and an antisense primer (SEQ ID NO: 3) and a probe (SEQ ID NO: 4) specific for the PML-RARa fusion gene; (Iii) an ABL probe and primer mixture consisting of the ABL gene specific, senseprimer (SEQ ID NO: 5) and antisense primer (SEQ ID NO: 6) and probe (SEQ ID NO: 7); (Iii) PML-RARa standard plasmid DNA comprising the PML-RARa fusion gene; (Iii) ABL standard plasmid DNA comprising the ABL gene; (Iii) a PML-RARa negative control group comprising an electro PCR reaction mixture and a PML-RARa probe and primer mixture; And, (iii) an ABL negative control comprising an electro PCR reaction mixture and an ABL probe and primer mixture: wherein the PCR reaction mixture may further include sterile water, and the PCR reaction mixture is not particularly limited thereto. HCl (pH 8.3) buffer, DNA polymerase, KCl, MgCl ₂ , dATP, dCTP, dGTP and dUTP, and (NH ₄ ) ₂ SO ₄ , calf serum albumin, EDTA, ROX (6) -carboxy-X-rhodamine) or mixtures thereof. In addition, the probe is not particularly limited thereto, but 6-carboxyfluorescein, hexachloro-6-carboxyfluorescein, or tetrachloro-6-carboxyfluorine at the 5 'end thereof. Labeled with tetrachloro-6-carboxyfluorescein, thymine in the 10-20th base from 5 ', labeled with 6-carboxytetramethyl-rhodamine, and phosphorylated at the 3' end. It is preferable to use. In addition, the PML-RARa standard plasmid DNA is not particularly limited thereto, but it is preferable to include the PML-RARa fusion gene of SEQ ID NO: 10 and to have a genetic map of FIG. 1, and the ABL standard plasmid DNA is not particularly limited thereto. However, it is preferable to use an ABL gene of SEQ ID NO: 13 and having a genetic map of FIG. 2.

The inventors of the present invention have sought to improve the conventional kits while variously studying methods for more effectively quantifying the degree of recombination of the PML-RARa fusion gene.

The kit for quantifying the degree of recombination of the PML-RARa fusion gene used up to now is mainly using a real-time polymerase chain reaction device, the technical principle of quantification is as follows: First, the PML-RARa fusion gene present in the sample As a template, when performing a polymerase chain reaction using an appropriate primer, a probe capable of binding to the PML-RARa fusion gene is added. In this case, the probe used is manufactured to label two different types of fluorescent materials at the 3 'end and the 5' end, respectively, and when the two used fluorescent materials are present within a predetermined interval, the fluorescence generated therein may cause mutual interference. In this case, a fluorescence does not develop. As a result, when the polymerase chain reaction is carried out, the polymerase chain reaction is sequentially performed by a DNA polymerase which binds a primer to one end of the template and recognizes it, and in the middle of the template, two kinds of fluorescent substances Labeled probes are bound. Subsequently, when the polymerase reaches the site to which the electric probe is bound, the polymerization reaction is continuously performed while the probe is sequentially decomposed from the 5 'end labeled with the fluorescent material, so that the fluorescent material labeled at the 5' end is freed. Results in dissociation. Accordingly, the interference phenomenon disappears, and the electrodissociated fluorescent substance performs a color reaction, indicating that the BCR-ABL fusion gene is amplified.

First of all, the inventors have noticed the shortcomings of the probe among the above-described technical principles. That is, as the length of the probe increases, the specificity of the PML-RARa fusion gene is improved, and thus the PML-RARa fusion gene can be more accurately quantified. However, when the fluorescent materials present at each end of the electric probe are spaced apart from each other by a certain distance or more, they are colored and cannot be used for quantification, and since the length of the probe is substantially limited, the accuracy of PML-RARa fusion gene quantification is improved. It is limited.

Accordingly, the inventors of the present invention have found a method of increasing the length of the probe while substantially improving the accuracy of PML-RARa fusion gene quantification while maintaining the interval at which the color development of the fluorescent material may be interfered with. When the material is labeled at the 5 'end and the remaining fluorescent material is labeled at the base of 10 to 20bp from the 5' end, it was found that the interference of the fluorescent material can be effectively used while increasing the length of the probe. At this time, when the fluorescent material is labeled at a base of less than 10 bp from the 5 'end, the color development time of the fluorescence is too short, resulting in low reliability, and the fluorescent material is added at a base of 20 bp or more from the 5' end. In the case of labeling, it was found that interference could not occur and quantification could not be performed.

In addition, the present inventors were able to improve the accuracy when correcting the quantitative value of the measured PML-RARa fusion gene using a comparison group. In other words, when amplifying the PML-RARa fusion gene, even when the sample amount and the experimental conditions were minutely changed, different result values were shown. Therefore, a method for correcting the amount of change due to the sample amount and the experimental conditions was searched. . As a result, the ABL gene showing the most stable expression level was amplified under the same conditions as the PML-RARa fusion gene, and the value calculated by dividing the color development of the PML-RARa fusion gene by the color development of the ABL gene was changed even if the amount of the sample changed. It was confirmed that the same level, it was possible to increase the reliability of the measured results.

On the other hand, using the PML-RARa quantitative kit, it is possible to quantify the degree of recombination of the PML-RARa fusion gene present in the sample, the method of quantifying the degree of recombination of the PML-RARa fusion gene present in the sample of the present invention Silver (i) PML-RARa standard plasmid DNA and samples were mixed with PML-RARa probe and primer mixture and PCR reaction mixture, respectively, and then PCR was performed with PML-RARa negative control to obtain PML-RARa standard PCR product and PML. Obtaining each of the -RARa sample PCR products; (Ii) mixing the ABL standard plasmid DNA and the sample with the ABL probe and primer mixture and the PCR reaction mixture, respectively, and then performing PCR with the ABL negative control to obtain the ABL standard PCR product and the ABL sample PCR product, respectively; (Iii) obtaining the PML-RARa value by measuring the amount of the PML-RARa sample PCR product, which was used based on the amount of the PML-RARa standard PCR product obtained previously; (Iii) obtaining the ABL value by measuring the amount of the ABL sample PCR product, which was used based on the amount of the ABL standard PCR product obtained previously; And (iii) dividing the PML-RARa value obtained earlier by the ABL value to obtain a measured value.

In addition, this measurement method can be performed using a commercial real-time polymerase chain reaction device, the real-time polymerase chain reaction device is not particularly limited to this, LightCycler ^TM (Roche, Germany), ABI PRISM ^TM 7000/7700 (Applied Biosystems, USA), iCycler ^™ (Bio-Rad, USA) and the like are preferably used.

When the PML-RARa quantitative kit of the present invention is used, the degree of recombination of the PML-RARa fusion gene which causes acute myeloid leukemia can be more easily and accurately quantified, and thus can be widely used for accurate diagnosis of acute promyeloid leukemia. There will be.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . In particular, in the following examples, as a PML-RARa standard plasmid DNA, a plasmid DNA containing the PML-RARa fusion gene of SEQ ID NO: 10 and having a genetic map of FIG. 1 was constructed and used as the ABL standard plasmid DNA. Plasmid DNA containing the ABL gene of No. 13 and having a genetic map of FIG. 2 was constructed and used, but this is only an embodiment for explaining the present invention, and the scope of the present invention is not limited thereto. something to do.

Example 1 Preparation of PML-RARa Quantitative Kit Applicable to Real-Time Polymerase Chain Reactor and Measurement of Recombination Degree of PML-RARa Fusion Gene Using the Same (I)

Example 1-1 Preparation of PML-RARa Quantification Kit Applicable to LightCycler ^™

1 ml PCR reaction mixture, 200 μl PML-RARa probe and primer mixture, 200 μl ABL probe and primer mixture, 96 μl PML-RARa standard plasmid DNA, 96 μl ABL standard plasmid DNA, 160 μl PML-RARa negative control, ABL negative PML-RARa quantitative kit consisting of 160ul control and 1ml sterile water was prepared.

Example 1-1-1 : Preparation of PCR reaction mixture, PML-RARa probe and primer mixture and ABL probe and primer mixture

First, the PCR reaction mixture was 100 mM Tris / HCl (pH 8.3), 20 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , ₇ mM MgCl ₂ , 300 ng calf serum albumin (BSA), 2.5 U DNA polymerase, 1.6 mM dUTP, 0.8 Prepared by mixing mM dATP, 0.8 mM dCTP and 0.8 mM dGTP.

Next, the PML-RARa probe and primer mixtures are PML-RARa fusion gene specific, two sense primers (SEQ ID NOs: 1 and 2), antisense primers (SEQ ID NO: 3) and probes (SEQ ID NO: 3) 4) was prepared by mixing.

bcr1 RQ-S: 5'-cccgtcataggaagtgaggt-3 '(SEQ ID NO: 1)

bcr3 RQ-S: 5'-acctcagctcttgcatcacc-3 '(SEQ ID NO: 2)

bcr1 RQ-AS: 5'-ttgtagatgcggggtagagg-3 '(SEQ ID NO: 3)

Probe: 5'-tatctcttcagaactgctgctctgggtctc-3 '(SEQ ID NO: 4)

Finally, the ABL probe and primer mixture was prepared by mixing the ABL gene specific, sense primer (SEQ ID NO: 5), antisense primer (SEQ ID NO: 6), and probe (SEQ ID NO: 7) having the following nucleotide sequence.

ABL RQ-S: 5'-gcctcagggtctgagtgaag-3 '(SEQ ID NO: 5)

ABL RQ-AS: 5'-acaccattccccattgtgat-3 '(SEQ ID NO: 6)

Probe: 5'-agagtgttatctccactggccacaaaatca-3 '(SEQ ID NO: 7)

At this time, the experimental group A in which the 5 'end of the probe (SEQ ID NOs: 4 and 7) was labeled with 6-carboxyfluorescein as a fluorescent substance, and the 3' end was labeled with 6-carboxytetramethyl-rhodamine as a fluorescent substance; 5 'end was labeled with 6-carboxyfluorescein as a fluorescent substance, 5th base was labeled with 6-carboxytetramethyl- rhodamine as a fluorescent substance at the 5' end, and phosphorylated at the 3 'end; 5 'end was labeled with 6-carboxyfluorescein as a fluorescent substance, 10th base from the 5' end was labeled with 6-carboxytetramethyl- rhodamine as a fluorescent substance, and 3 'terminal was phosphorylated; 5 'end was labeled with 6-carboxyfluorescein as a fluorescent substance, 15th base from the 5' end was labeled with 6-carboxytetramethyl- rhodamine as a fluorescent substance, and 3 'end was phosphorylated; 5 'end was labeled with 6-carboxyfluorescein as a fluorescent substance, 20th base from the 5' end was labeled with 6-carboxytetramethyl- rhodamine as a fluorescent substance, and 3 'terminal was phosphorylated; And the 5 'end was labeled with 6-carboxyfluorescein as a fluorescent substance, and the 25th base from the 5' end was labeled with 6-carboxytetramethyl-rhodamine as a fluorescent substance and the 3 'end was phosphorylated. F was prepared respectively.

Example 1-1-2 Preparation of PML-RARa Standard Plasmid DNA

PML-RARa standard plasmid DNA was prepared as follows: First, RNA was extracted from an NB4 cell line using RNAqueous (Ambion, USA), and the cDNA synthesis kit using p (dN) ₆ as a primer was extracted NB4 RNA. First strand cDNA synthesis kit, Roche, Germany) and synthesized cDNA, using the electrosynthesized cDNA as a template, using a sense primer (SEQ ID NO: 8) and an antisense primer (SEQ ID NO: 9) having the following sequence PCR was performed to obtain a 406 bp PML-RARa fusion gene (SEQ ID NO: 10).

sense: 5'-aaaggcccttcctatggaga-3 '(SEQ ID NO: 8)

antisense: 5'-ccccatagtggtagcctgag-3 '(SEQ ID NO: 9)

The 406 bp PML-RARa fusion gene obtained above was introduced into the pCR 2.1-TOPO vector (Invitrogen Corporation Carlsbad, California USA) to prepare PML-RARa standard plasmid DNA (see FIG. 1). 1 is a genetic map of PML-RARa standard plasmid DNA. To facilitate the use of the previously prepared PML-RARa standard plasmid DNA, it was serially diluted to have copy numbers of 2x10 ⁶ , 2x10 ⁴ , 2x10 ³ and 2x10 ² , and diluted PML-RARa standard plasmid DNA 24 μl each was aliquoted separately.

As described above, the PML-RARa standard plasmid DNA prepared in the present invention was easily deposited by a conventional method in the art by inserting a known gene into a commercially available vector, and was not separately deposited.

Example 1-1-3 Preparation of ABL Standard Plasmid DNA

Next, ABL standard plasmid DNA was prepared as follows: First, K562 cDNA was used as a template, and PCR was performed using a sense primer (SEQ ID NO: 11) and an antisense primer (SEQ ID NO: 12) having the following nucleotide sequence: A 500 bp ABL gene (SEQ ID NO: 13) was obtained.

sense: 5'-cgcctcagggtctgagtgaag-3 '(SEQ ID NO: 11)

antisense: 5'-ccgttgaatgatgatgaacc-3 '(SEQ ID NO: 12)

An ABL standard plasmid DNA was prepared by introducing the 500 bp ABL gene obtained previously into the pCR 2.1-TOPO vector (see FIG. 2). 2 is a genetic map of ABL standard plasmid DNA. To facilitate the use of the previously prepared ABL standard plasmid DNA, it was serially diluted to have 2x10 ⁶ , 2x10 ⁴ , 2x10 ³ and 2x10 ² replicates, and the diluted ABL standard plasmid DNA was individually dispensed in 24 μl each. It was.

As described above, the ABL standard plasmid DNA prepared in the present invention was easily deposited by a conventional method in the art by inserting a known gene into a commercially available vector, and was not separately deposited.

Example 1-1-4 : Preparation of PML-RARa negative control group and ABL negative control group

The PCR reaction mixture prepared in Example 1-1-1 was added to each PML-RARa probe and primer mixture to prepare a PML-RARa negative control, and the electrical PCR reaction mixture was added to each ABL probe and primer mixture to ABL. A negative control was prepared.

Example 1-2 Measurement of Recombination Degree of PML-RARa Fusion Genes Using PML-RARa Quantitative Kit Applicable to LightCycler ^™

Example 1-2-1 : Preparation of Sample to be Measured

First, cDNA (experimental group 1-1) obtained by extracting RNA from the bone marrow of treated acute myeloid leukemia patient and applying the extracted RNA to the cDNA synthesis kit using p (dN) ₆ as a primer CDNA obtained by applying 10 ng to a cDNA synthesis kit using p (dN) ₆ as a primer and 1 ng of extracted RNA to a cDNA synthesis kit using p (dN) ₆ as a primer CDNA obtained by extracting RNA from the bone marrow of the patient (Experimental Group 1-3) and the bone marrow of a patient diagnosed with acute myeloid leukemia and not yet treated, and applying 1 μg of the extracted RNA to a cDNA synthesis kit using p (dN) ₆ as a primer. (experiment 2-1), the extracted RNA 10ng p (dN) ₆ to the RNA 1ng obtained cDNA (experiment 2-2) and extracted by applying a cDNA synthesis kit was used as the primer p (dN) ₆ primers CDNA (Experimental Group 2-3) obtained by applying to the cDNA synthesis kit used was prepared respectively.

Example 1-2-2 : Measurement of PML-RARa Value of Sample

10 μl of the PCR reaction mixture prepared in Example 1-1, 4 μl of PML-RARa probe and primer mixture (Experimental Groups A to F), and 4 μl of sterile water were dispensed into a tube for PCR, and then 2 × 10 ⁶ , 2 × 10 ⁴ , Each sample is prepared by dispensing 2 μl of PML-RARa standard plasmid DNA having 2x10 ³ and 2x10 ² copy number, and cDNA of Experimental Groups 1-1 to 2-3, and preparing PML-RARa negative control group in a separate tube. 20 μl was added to prepare each PML-RARa PCR sample. Subsequently, each prepared sample was applied to LightCycler ^™ (Roche, Germany) which is a real-time polymerase chain reaction device, and PCR was performed to obtain PML-RARa standard PCR product and PML-RARa sample PCR product, respectively. In this case, PCR was denatured for 10 minutes at 95 ° C., amplified by 45 cycles of a series of reactions for 10 seconds at 95 ° C., 10 seconds at 60 ° C., and 30 seconds at 72 ° C. It was carried out by cooling for 30 seconds at 40 ℃.

Subsequently, the amount of PML-RARa standard PCR product and the amount of PML-RARa sample PCR product thus obtained were respectively measured, and the relative amount of PML-RARa sample PCR product was calculated based on the amount of PML-RARa standard PCR product. By this, PML-RARa value of the sample with respect to experiment groups A-F was obtained.

Example 1-2-3 : Measurement of ABL Values of Samples

10 μl of the PCR reaction mixture prepared in Example 1-1, 4 μl of the ABL probe and primer mixture (Experimental Groups A to F) and 4 μl of sterile water were dispensed into a PCR tube, followed by 2 × 10 ⁶ , 2 × 10 ⁴ , and 2 × 10 ^3. And 2 μl of ABL standard plasmid DNA having ² × 10 ² copy number and cDNA of Experimental Groups 1-1 to 2-3, respectively, to prepare a sample, and 20 μl of ABL negative control group was put into a separate tube. ABL PCR samples were prepared. Subsequently, each prepared sample was applied to LightCycler ^™ , a real-time polymerase chain reaction device, and PCR was performed to obtain an ABL standard PCR product and an ABL sample PCR product, respectively. At this time, PCR was denatured at 95 ° C. for 10 minutes, amplified by repeating a series of 45 times of reaction for 10 seconds at 95 ° C., 10 seconds at 60 ° C., and 30 seconds at 72 ° C., and then 30 seconds at 40 ° C. Cooling was carried out.

Then, the amount of the ABL standard PCR product and the amount of the ABL sample PCR product were measured, respectively, and the relative amounts of the ABL sample PCR product were calculated on the basis of the amount of the ABL standard PCR product. The ABL value of the sample was obtained.

Example 1-2-4 : Calculation and Analysis of Measured Values

The PML-RARa values of the samples obtained in Examples 1-2-2 and Example 1-2-3, respectively, were divided by the ABL values to calculate the measured values (see Table 1).

Comparison of the measured values calculated by PML-RARa value and ABL value of each experimental group sample Probe PML-RARa Value (Replication) ABL value (replicate) Measures Experimental Group 1-1 Experiment group A Experiment group B Experiment group C Experiment group D Experiment group E Experiment group F Not measurable Not measurable 10900 10600 10200 Not measurable Not measurable Not measurable 37600 37100 36900 Not measurable Uncalculated Uncalculated 0.29 0.29 0.28 Uncalculated Experimental group 1-2 Experiment group A Experiment group B Experiment group C Experiment group D Experiment group E Experiment group F Not measured Not measured 115 130 124 Not measured Not measurable Not measurable 451 428 403 Not measurable Uncalculated Uncalculated 0.25 0.31 0.31 Uncalculated Experimental Group 1-3 Experiment group A Experiment group B Experiment group C Experiment group D Experiment group E Experiment group F Not measurable Not measurable 1.1 1.6 1.4 Not measurable Not measurable Not measurable 4.9 5.3 5.7 Not measurable Uncalculated Uncalculated 0.22 0.30 0.25 Uncalculated Experimental Group 2-1 Experiment group A Experiment group B Experiment group C Experiment group D Experiment group E Experiment group F Not measurable Not measurable 1064580 1062880 1058420 Not measurable Not measurable Not measurable 71300 72800 72040 Not measurable Uncalculated Uncalculated 14.93 14.60 14.69 Uncalculated Experimental Group 2-2 Experiment group A Experiment group B Experiment group C Experiment group D Experiment group E Experiment group F Not Measurable 10510 10476 10492 Not Measurable Not measurable Not measurable 694 726 734 Not measurable Uncalculated Uncalculated 15.14 14.43 14.29 Uncalculated Experimental Group 2-3 Experiment group A Experiment group B Experiment group C Experiment group D Experiment group E Experiment group F Not measurable Not measurable 141 135 138 Not measurable Not measurable Not measurable 9.5 9.2 8.8 Not measurable Uncalculated Uncalculated 14.84 14.71 15.68 Uncalculated

As shown in Table 1, the measured values of the treated patients (experimental groups 1-1 to 1-3) showed low values, while the measured values of untreated patients (experimental groups 2-1 to 2-3) were significantly higher. Numerical values were shown. The reason for the high measured values of the diagnosed patients was that the PML-RARa fusion gene was expressed at a higher level than the normally expressed ABL gene. In addition, even when the amount of the RNA sample used was changed, the same measured value was shown, and it was confirmed that reliable results could be obtained even when the amount of the sample changed or a very small amount of the sample was used.

In addition, a primer labeled with 6-carboxytetramethyl-rhodamine as a fluorescent substance at the 3 'end (Experimental Group A), the 5th base (Experimental Group B) from the 5' end and the 25th base (Experimental Group F) from the 5 'end was In the case of use, appropriate measurement values could not be obtained, but the primers labeled with the fluorescent substance 6-carboxytetramethyl-rhodamine at the 5 'end to the 10th, 15th and 20th bases (Experimental Groups C, D and E) were used. In that case, an appropriate measured value could be obtained.

These results were found to be caused by the characteristics of the fluorescent material labeled on the primer. That is, 6-carboxyfluorescein and 6-carboxytetramethyl-rhodamine, which are labeled on the electric primers, exhibit fluorescence when present alone, but when present within a certain distance from each other, the fluorescence generated by each other interferes with each other. It causes a phenomenon and prevents color development.

According to the results of Table 1, it can be seen that when 6-carboxyfluorescein and 6-carboxytetramethyl-rhodamine exist at intervals of 10 to 20 bp, they cause interference with each other and maintain the narrower intervals. In this case, the color development time is too short, it can be seen that when maintaining a wider interval does not cause interference with each other.

Example 2 Preparation of PML-RARa Quantification Kit Applicable to Real-Time Polymerase Chain Reactor and Measurement of Recombination Degree of PML-RARa Fusion Gene Using the Same (II)

Example 2-1 ABI PRISM ^? Preparation of PML-RARa Quantification Kit Applicable to 7000/7700

PCR reaction mixture 1.225 ml, 200 μl PML-RARa probe and primer mixture, 200 μl ABL probe and primer mixture, 160 μl PML-RARa standard plasmid DNA, 160 μl ABL standard plasmid DNA, 200 μl PML-RARa negative control, ABL negative A PML-RARa quantitative kit consisting of 200 μl of control and 1 ml of sterile water was prepared: wherein the PCR reaction mixture was 100 mM Tris / HCl (pH 8.3), 20 mM KCl, 20 mM EDTA, 120 nM ROX (6-carboxy-X-rhodamine ), 7mM MgCl ₂ , 2.5U DNA polymerase, 1.6mM dUTP, 0.8mM dATP, 0.8mM dCTP, 0.8mM dGTP and 0.5U UNG (uracil-N-glycosylase) was prepared by mixing, Example 1 as a primer PML-RARa probe and primer mixture, ABL probe and primer mixture, PML-RARa standard plasmid DNA, ABL standard plasmid DNA, PML-RARa except for using experimental group D of each primer prepared in -1-1 The negative control group and the ABL negative control group were prepared in Examples 1-1-1 to 1-1-4. The same one was used.

Example 2-2 ABI PRISM ^? Recombination level measurement of PML-RARa fusion gene using PML-RARa quantitative kit applicable to 7000/7700

Using the experimental group 1-1 and experimental group 1-2 prepared in Example 1-2-1 as a sample to be measured, the degree of recombination of the PML-RARa fusion gene was calculated and analyzed.

Example 2-2-1 : Measurement of PML-RARa Value of Sample

12.5 μl of the PCR reaction mixture prepared in Example 2-1, 4 μl of the PML-RARa probe and primer mixture, and 4 μl of sterile water were dispensed into the PCR tube, followed by 2 × 10 ⁶ , 2 × 10 ⁴ , 2 × 10 ^3, and 2 × 10 ² . Samples were prepared by dispensing 4 μl of PML-RARa standard plasmid DNA having a copy number, cDNA of Experimental Group 1-1 and cDNA of Experimental Group 1-2, and putting 25µl of PML-RARa negative control group in a separate tube. Each PML-RARa PCR sample was prepared. Subsequently, each prepared sample was subjected to ABI PRISM ^? 7000 (Applied Biosystems, USA) and PCR were performed to obtain PML-RARa standard PCR product and PML-RARa sample PCR product, respectively. At this time, PCR was performed for 2 minutes at 50 ° C, denatured at 95 ° C for 10 minutes, and then amplified by repeating a series of 45 times of reaction at 95 ° C for 15 seconds and at 60 ° C for 1 minute.

Subsequently, the amount of PML-RARa standard PCR product and the amount of PML-RARa sample PCR product thus obtained were respectively measured, and the relative amount of PML-RARa sample PCR product was calculated based on the amount of PML-RARa standard PCR product. This obtained the PML-RARa value of a sample.

Example 2-2-2 : Measurement of ABL Values of Samples

Dispense 12.5 μl of the PCR reaction mixture prepared in Example 2-1, 4 μl of the ABL probe and primer mixture, and 4 μl of sterile water into a tube for PCR, and then replicate the numbers of ² × 10 ⁶ , 2 × 10 ⁴ , 2 × 10 ^3, and 2 × 10 ² . Samples were prepared by dispensing 4 μl of ABL standard plasmid DNA, cDNA of Experimental group 1-1 and cDNA of Experimental group 1-2, respectively, and putting 25µl of ABL negative control group into separate tubes. Was prepared. Subsequently, each prepared sample was subjected to ABI PRISM ^? It was applied to 7000 and PCR was performed to obtain an ABL standard PCR product and an ABL sample PCR product, respectively. At this time, PCR was performed for 2 minutes at 50 ° C, denatured at 95 ° C for 10 minutes, and then amplified by repeating a series of 45 times of reaction at 95 ° C for 15 seconds and at 60 ° C for 1 minute.

Subsequently, the amount of ABL standard PCR product and the amount of ABL sample PCR product obtained above were measured, respectively, and the relative amount of ABL sample PCR product was calculated based on the amount of ABL standard PCR product, thereby obtaining the ABL value of the sample. .

Example 2-2-3 : Calculation and Analysis of Measured Values

The PML-RARa value of the sample obtained in Example 2-2-1 was divided by the ABL value of the sample obtained in Example 2-2-2 to calculate the measured value (see Table 2).

Comparison of the measured values calculated by PML-RARa value and ABL value of each experimental group Experimental group Amount of RNA                                              PML-RARa Value (Replication) ABL value (replicate) Measures Experimental Group 1-1 Experimental Group 1-2 1µg 10ng 11682.9 182.9 102787 1360.5 0.114 0.134

As shown in Table 2, when the amount of RNA obtained from the treated patient was measured differently, as in the result of Table 1, when the PML-RARa value was measured only, a significant difference appeared, but as the ABL value. The measured values measured by calibration showed the same level of measured values regardless of the amount of RNA used.

Therefore, when using the PML-RARa quantitative kit of the present invention, it was confirmed that the amount of PML-RARa fusion gene can be measured more accurately.

Example 3 Preparation of PML-RARa Quantitative Kit Applicable to Real-Time Polymerase Chain Reactor and Measurement of Recombination Degree of PML-RARa Fusion Gene Using the Same (III)

Example 3-1 Preparation of PML-RARa Quantification Kit Applicable to iCycler ^™

PCR reaction mixture 1.225 ml, 200 μl PML-RARa probe and primer mixture, 200 μl ABL probe and primer mixture, 160 μl PML-RARa standard plasmid DNA, 160 μl ABL standard plasmid DNA, 200 μl PML-RARa negative control, ABL negative A PML-RARa quantitative kit consisting of 200 μl of control and 1 ml of sterile water was prepared: wherein the PCR reaction mixture was 100 mM Tris / HCl (pH 8.3), 20 mM KCl, 20 mM EDTA, 7 mM MgCl ₂ , _2.5 U DNA polymerase, Prepared by mixing 1.6 mM dUTP, 0.8 mM dATP, 0.8 mM dCTP, 0.8 mM dGTP and 0.5 U UNG, except using Experimental Group D of each primer prepared in Example 1-1-1 above as a primer. PML-RARa probe and primer mixture, ABL probe and primer mixture, PML-RARa standard plasmid DNA, ABL standard plasmid DNA, PML-RARa negative control group and ABL negative control group are described in the above Examples 1-1-1 to 1-1. The same one as prepared in -4 was used.

Example 3-2 Measurement of Recombination Degree of PML-RARa Fusion Gene Using PML-RARa Quantitative Kit Applicable to iCycler ^™

Using the experimental group 1-1 and experimental group 1-2 prepared in Example 1-2-1 as a sample to be measured, the degree of recombination of the PML-RARa fusion gene was calculated and analyzed.

Example 3-2-1 : Measurement of PML-RARa Value of Sample

10 μl of the PCR reaction mixture prepared in Example 3-1, 4 μl of the PML-RARa probe and primer mixture, and 4 μl of sterile water were dispensed into the tubes for PCR, followed by 2 × 10 ⁶ , 2 × 10 ⁴ , 2 × 10 ^3, and 2 × 10 ² . Each sample was prepared by dispensing 2 μl of PML-RARa standard plasmid DNA having a copy number, cDNA of Experimental Group 1-1 and cDNA of Experimental Group 1-2, and adding 20µl of PML-RARa negative control group to a separate tube. Each PML-RARa PCR sample was prepared. Subsequently, each prepared sample was applied to iCycler ^TM (Bio-Rad, USA), which is a real-time polymerase chain reaction device, and PCR was performed to obtain PML-RARa standard PCR product and PML-RARa sample PCR product, respectively. At this time, PCR was performed for 2 minutes at 50 ° C, denatured at 95 ° C for 10 minutes, and then amplified by repeating a series of 45 times of reaction at 95 ° C for 15 seconds and at 60 ° C for 1 minute.

Subsequently, the amount of PML-RARa standard PCR product and the amount of PML-RARa sample PCR product thus obtained were respectively measured, and the relative amount of PML-RARa sample PCR product was calculated based on the amount of PML-RARa standard PCR product. This obtained the PML-RARa value of a sample.

Example 3-2-2 : Measurement of ABL Values of Samples

10 μl of the PCR reaction mixture prepared in Example 3-1, 4 μl of the ABL probe and primer mixture and 4 μl of sterile water were dispensed into the PCR tube, followed by copying ² × 10 ⁶ , 2 × 10 ⁴ , 2 × 10 ^3, and 2 × 10 ² . Samples were prepared by dispensing 2 μl of ABL standard plasmid DNA, cDNA of Experimental Group 1-1 and cDNA of Experimental Group 1-2, and putting 20µl of ABL negative control group into separate tubes. Was prepared. Subsequently, each prepared sample was applied to iCycler ^™ , which is a real-time polymerase chain reaction device, and PCR was performed to obtain PML-RARa standard PCR product and PML-RARa sample PCR product, respectively. At this time, PCR was performed for 2 minutes at 50 ° C, denatured at 95 ° C for 10 minutes, and then amplified by repeating a series of 45 times of reaction at 95 ° C for 15 seconds and at 60 ° C for 1 minute.

Subsequently, the amount of ABL standard PCR product and the amount of ABL sample PCR product obtained above were measured, respectively, and the relative amount of ABL sample PCR product was calculated based on the amount of ABL standard PCR product, thereby obtaining the ABL value of the sample. .

Example 3-2-3 : Calculation and Analysis of Measured Values

The PML-RARa value of the sample obtained in Example 3-2-1 was divided by the ABL value of the sample obtained in Example 3-2-2 to calculate the measured value (see Table 3).

Comparison of the measured values calculated by PML-RARa value and ABL value of each experimental group Experimental group Amount of RNA                                              PML-RARa Value (Replication) ABL value (replicate) Measures Experimental Group 1-1 Experimental Group 1-2 1µg 10ng 5700 46.55 7550 60 0.75 0.66

As shown in Table 3, as in the results of Table 1 and Table 2, when the amount of RNA obtained from the treated patients were measured differently, a significant difference appeared when only the PML-RARa value was measured. The measured value measured by correcting the ABL value showed the same level of measured value regardless of the amount of RNA used.

Therefore, when using the PML-RARa quantitative kit of the present invention, it was confirmed that the amount of PML-RARa fusion gene can be measured more accurately.

As described and demonstrated in detail above, the present invention provides a PCR reaction mixture, a probe and primer mixture capable of detecting PML-RARa fusion gene, a probe and primer mixture capable of detecting ABL gene, PML-RARa standard plasmid DNA, ABL Provided are methods for quantifying PML-RARa fusion genes using PML-RARa quantitative kits and electrical kits comprising standard plasmid DNA, PML-RARa negative controls and ABL negative controls. When the PML-RARa quantitative kit of the present invention is used, the degree of recombination of the PML-RARa fusion gene which causes acute myeloid leukemia can be more easily and accurately quantified, and thus can be widely used for accurate diagnosis of acute promyeloid leukemia. There will be.

<110> KIM, Dong Wook <120> Kit for Quantitative Analysis of PML-RARa <160> 13 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ccgtcatagg aagtgaggt 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 acctcagctc ttgcatcacc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ttgtagatgc ggggtagagg 20 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 4 tatctcttca gaactgctgc tctgggtctc 30 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcctcagggt ctgagtgaag 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acaccattcc ccattgtgat 20 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 7 agagtgttat ctccactggc cacaaaatca 30 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 aaaggccctt cctatggaga 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ccccatagtg gtagcctgag 20 <210> 10 <211> 406 <212> DNA <213> Artificial Sequence <220> <223> PML-RARa <400> 10 aaaggccctt cctatggaga ggatgtctcc aatacaacga cagcccagaa gaggaagtgc 60 agccagaccc agtgccccag gaaggtcatc aagatggagt ctgaggaggg gaaggaggca 120 aggttggctc ggagctcccc ggagcagccc aggcccagca cctccaaggc agtctcacca 180 ccccacctgg atggaccgcc tagccccagg agccccgtca taggaagtga ggtcttcctg 240 cccaacagca accacgtggc cagtggcgcc ggggaggcag ccattgagac ccagagcagc 300 agttctgaag agatagtgcc cagccctccc tcgccacccc ctctaccccg catctacaag 360 ccttgctttg tctgtcagga caagtcctca ggctaccact atgggg 406 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 cgcctcaggg tctgagtgaa g 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ccgttgaatg atgatgaacc 20 <210> 13 <211> 500 <212> DNA <213> Artificial Sequence <220> <223> ABL <400> 13 gcctcagggt ctgagtgaag ccgctcgttg gaactccaag gaaaaccttc tcgctggacc 60 cagtgaaaat gaccccaacc ttttcgttgc actgtatgat tttgtggcca gtggagataa 120 cactctaagc ataactaaag gtgaaaagct ccgggtctta ggctataatc acaatgggga 180 atggtgtgaa gcccaaacca aaaatggcca aggctgggtc ccaagcaact acatcacgcc 240 agtcaacagt ctggagaaac actcctggta ccatgggcct gtgtcccgca atgccgctga 300 gtatctgctg agcagcggga tcaatggcag cttcttggtg cgtgagagtg agagcagtcc 360 tggccagagg tccatctcgc tgagatacga agggagggtg taccattaca ggatcaacac 420 tgcttctgat ggcaagctct acgtctcctc cgagagccgc ttcaacaccc tggccgagtt 480 ggttcatcat cattcaacgg 500  

Claims (8)

(Iii) PCR reaction mixture (Ii) a PML-RARa probe and primer mixture comprising a PMS-RARa fusion gene specific, sense primer (SEQ ID NO: 1) and antisense primer (SEQ ID NO: 2) and probe (SEQ ID NO: 3); (Iii) ABL probe and primer mixtures comprising ABL gene specific, sense primers (SEQ ID NO: 4) and antisense primers (SEQ ID NO: 5) and probes (SEQ ID NO: 6); (Iii) PML-RARa standard plasmid DNA comprising the PML-RARa fusion gene; (Iii) ABL standard plasmid DNA comprising the ABL gene; (Iii) a PML-RARa negative control group comprising an electro PCR reaction mixture and a PML-RARa probe and primer mixture; And, (Iii) A PML-RARa quantitative kit comprising an ABL negative control comprising an electro PCR reaction mixture and an ABL probe and primer mixture. The method of claim 1, Further comprising a sterile water PML-RARa quantitative kit. The method of claim 1, The PCR reaction mixture is characterized in that it comprises Tris-HCl (pH 8.3) buffer, DNA polymerase, KCl, MgCl ₂ , dATP, dCTP, dGTP and dUTP PML-RARa quantitative kit. The method of claim 3, wherein (NH ₄ ) ₂ SO ₄ , calf serum albumin, EDTA, ROX (6-carboxy-X-rhodamine) or a mixture thereof is further included in the PCR reaction mixture. PML-RARa quantitative kit. The method of claim 1, Electric probes have 6-carboxyfluorescein, hexachloro-6-carboxyfluorescein or tetrachloro-6-carboxyfluorescein at the 5 'end. carboxyfluorescein), thymine from 5 'to 10-20 base is labeled with 6-carboxytetramethyl-rhodamine, and the 3' end is phosphorylated. PML-RARa quantitative kit. The method of claim 1, PML-RARa standard plasmid DNA comprises the PML-RARa fusion gene of SEQ ID NO: 10, characterized in that it has a genetic map of Figure 1 PML-RARa quantitative kit. The method of claim 1, ABL standard plasmid DNA contains the ABL gene of SEQ ID NO: 13 and has a genetic map of FIG. PML-RARa quantitative kit. (Iii) The PML-RARa standard plasmid DNA and the sample described in claim 1 are mixed with the PML-RARa probe and primer mixture and the PCR reaction mixture, respectively, followed by PCR to perform the PML-RARa standard PCR product and the PML-RARa sample. Obtaining each of the PCR products; (Ii) the ABL standard plasmid DNA, ABL and the sample described in claim 1 were mixed with the ABL probe and primer mixture and the PCR reaction mixture, respectively, and then subjected to PCR to obtain the ABL standard PCR product and the ABL sample PCR product, respectively. Doing; (Iii) obtaining the PML-RARa value of the sample by measuring the amount of the PML-RARa sample PCR product based on the amount of the PML-RARa standard PCR product obtained previously; (Iii) obtaining the ABL value of the sample by measuring the amount of the ABL sample PCR product based on the amount of the ABL standard PCR product obtained previously; And, (Iv) present in the sample using the PML-RARa quantitative kit of claim 1, comprising dividing the PML-RARa value of the sample obtained in the preceding steps (v) and (v) by the ABL value to obtain the measured value; Method for quantifying the degree of recombination of the PML-RARa fusion gene.

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