KR100652247B1 - - Kit for Quantitative Analysis of BCR-ABL - Google Patents

Abstract

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

BCR-ABL fusion gene, BCR-ABL standard plasmid DNA, ABL standard plasmid DNA

Description

Kit for Quantitative Analysis of BCR-ABL             

1 is a genetic map showing an example of the BCR-ABL standard plasmid DNA.

Fig. 2 is a genetic map showing an example of ABL standard plasmid DNA.

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

Chronic myelogenous leukemia (CML) has been known to cause a variety of pathogens, but the most common cause is caused by translocation of the chromosome. It is known to be the main cause of illness. In fact, in 95% of patients with chronic myeloid leukemia, translocations between chromosome 9 and chromosome 22 are found. It is formed by translocation to the breakpoint cluster region (bcr) gene present at the 2nd position, and this rearrangement is called BCR-ABL rearrangement. Chronic myeloid leukemia caused by the previous BCR-ABL rearrangement is very effective in treating imatinib mesylate sold under the trademark Glivec ^TM as a special drug. This is very important for determining the treatment direction and prognostic judgment.

In order to identify the BCR-ABL rearrangement, a method of quantifying the degree of recombination of the BCR-ABL fusion gene is mainly used, and to develop a method of more easily quantifying the degree of recombination of the BCR-ABL fusion gene. Various studies are in progress. For example, diagnostic methods using fluorescent in situ hybridization (FISH) (Tkachuk et al., Science, 250 (4980): 559-562, 1990), methods using PCR for somatic cell DNA (Dobrovic et al. al., Blood, 72 (6): 2063-2065, 1998), methods using RT-PCR (Lee et al., Blood, 73 (8): 2165-2170, 1989) and nested Methods using PCR have been developed (Hessner et al., Genet. Anal. Tech. Appl., 11 (4): 90-94, 1994). However, FISH is a method of directly detecting individual cells in which BCR-ABL rearrangement has occurred, and it is difficult to detect in the early stage of chronic myeloid leukemia, and the method by PCR or RT-PCR has excellent sensitivity, but contaminated the sample. This may result in a false-positive response, and has the disadvantage of not being able to accurately determine the progress of the disease.

As a method for overcoming the above-mentioned disadvantages, recently, BCR-ABL diagnostic kits using a real-time RT-PCR method capable of quantifying target RNA from a small amount of sample have been developed and marketed. However, the diagnostic kits include a stability and reproducibility problem by providing RNA as a standard gene, do not include the reference gene quantification primers and probes used for standardization, or the reference gene quantification primers and probes 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 BCR-ABL quantification.

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

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 the BCR-ABL fusion gene. As a result, the BCR-ABL fusion gene specific primers and probes and the control group are specific for the ABL gene. When using the BCR-ABL quantitative kit including a primer and a probe, it was confirmed that the degree of recombination of the BCR-ABL 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 BCR-ABL quantitative kit which is simple and has improved accuracy and reproducibility.

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

BCR-ABL quantitative kit of the present invention (i) PCR reaction mixture; (Ii) a BCR-ABL probe and primer mixture consisting of two sense primers (SEQ ID NOs: 1 and 2), an antisense primer (SEQ ID NO: 3) and a probe (SEQ ID NO: 4) specific for the BCR-ABL fusion gene; (Iii) an ABL probe and primer mixture consisting of an ABL gene specific, sense primer (SEQ ID NO: 5), antisense primer (SEQ ID NO: 6) and probe (SEQ ID NO: 7); (Iii) BCR-ABL standard plasmid DNA comprising cDNA of the BCR-ABL fusion gene; (Iii) ABL standard plasmid DNA comprising the cDNA of the ABL gene; (Iii) a BCR-ABL negative control group comprising an electrical PCR reaction mixture and a BCR-ABL 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 comprise sterile water, but the PCR reaction mixture is not particularly limited thereto. -HCl (pH 8.3) buffer, DNA polymerase, KCl, MgCl ₂ , dATP, dCTP, dGTP and dUTP, (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. Preferably, the BCR-ABL standard plasmid DNA is not particularly limited thereto, but preferably includes the cDNA of BCR-ABL of SEQ ID NO: 10 and has a genetic map of FIG. 1, and the ABL standard plasmid DNA is not particularly limited thereto. However, it is preferred to include the ABL cDNA of SEQ ID NO: 13 and to have a genetic map of FIG.

The inventors of the present invention have sought to improve the conventional kits while researching various methods for more effectively quantifying the degree of recombination of the BCR-ABL fusion gene.

The kit for quantifying the degree of recombination of the BCR-ABL fusion gene used so far mainly uses a real-time polymerase chain reaction device. The technical principle of quantification is as follows: First, the BCR-ABL fusion gene present in the sample is templated. When performing the polymerase chain reaction using an appropriate primer, a probe capable of binding to the BCR-ABL 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 types of fluorescent materials are present within a predetermined interval, the fluorescence that is generated is mutually interfering. 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 for the BCR-ABL fusion gene is improved, so that the BCR-ABL 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 predetermined 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 quantifying the BCR-ABL fusion gene is improved. It is limited.

Accordingly, the inventors of the present invention have found ways to improve the accuracy of quantifying BCR-ABL fusion genes by increasing the length of the probe while maintaining the interval at which the color development of the fluorescent material may be interfered. When the 5 'end is labeled and the remaining fluorescent material is labeled with the intermediate base of the probe, it can be seen that the interference of the fluorescent material can be used while increasing the length of the probe.

Accordingly, the present inventors quantified the BCR-ABL fusion gene while varying the distance between the fluorescent materials, in order to find an appropriate interval at which the interference of the fluorescent material can be performed, and as a result, the fluorescent material may be labeled at intervals of 10 to 20 bp. In this case, it can be seen that the most effective interference response appears. At this time, when the fluorescent material is labeled at intervals of 10 bp or less, the color development time of the fluorescence is too short, resulting in low reliability. When the fluorescent substance is labeled at an interval of 20 bp or more, interference does not occur. It was found that the quantification could not be performed.

In addition, the present inventors were able to improve the accuracy when the quantitative value of the measured BCR-ABL fusion gene is corrected using the comparison group. In other words, when amplifying the BCR-ABL 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 according 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 BCR-ABL fusion gene, and the value calculated by dividing the color development of the BCR-ABL fusion gene by the color development of the ABL gene was changed. Even if the same level was confirmed that it was possible to increase the reliability of the measured results.

Using the BCR-ABL quantitative kit, it is possible to quantify the degree of recombination of the BCR-ABL fusion gene present in the sample: That is, the method of quantifying the degree of recombination of the BCR-ABL fusion gene present in the sample is (i) BCR-ABL standard plasmid DNA, BCR-ABL negative control and sample were mixed with BCR-ABL probe and primer mixture and PCR reaction mixture, respectively, and then PCR was performed to perform BCR-ABL standard PCR product and BCR-ABL sample PCR product. Obtaining each of them; (Ii) mixing ABL standard plasmid DNA, ABL negative control and sample with ABL probe and primer mixture and PCR reaction mixture, respectively, followed by PCR to obtain ABL standard PCR product and ABL sample PCR product, respectively; (Iii) obtaining the BCR-ABL value by measuring the amount of the BCR-ABL sample PCR product, which was used based on the amount of the BCR-ABL 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 obtained BCR-ABL value 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 BCR-ABL quantitative kit of the present invention is used, the degree of recombination of the BCR-ABL fusion gene which causes chronic myelogenous leukemia can be more easily and accurately quantified. It may be widely used.

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, in particular, in the following examples, a plasmid DNA comprising the BCR-ABL fusion gene of SEQ ID NO: 10 and having the genetic map of FIG. 1 was constructed and used as the BCR-ABL standard plasmid DNA. As ABL standard plasmid DNA, a plasmid DNA comprising the ABL gene of SEQ ID NO: 13 and having a genetic map of FIG. 2 was constructed and used, but this is only one embodiment for explaining the present invention, and the scope of the present invention is It will be apparent that this is not limitative.

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

Example 1-1 Preparation of BCR-ABL Quantification Kit Applicable to LightCycler ^™

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

Example 1-1-1 : Preparation of PCR Reaction Mixture, BCR-ABL 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 BCR-ABL probe and primer mixture is a BCR-ABL fusion gene specific two sense primers (SEQ ID NOs: 1 and 2), an antisense primer (SEQ ID NO: 3) and a probe (SEQ ID NO: 4) was prepared by mixing.

BCR RQ-S 1: 5'-cagactgtccacagcattcc-3 '(SEQ ID NO: 1)

BCR RQ-S 2: 5'-cttccatggagacgcagaag-3 '(SEQ ID NO: 2)

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

Probe: 5'-agaccctgaggctcaaagtcagatgctact-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, the 10th base from the 5' end was labeled with 6-carboxytetramethyl- rhodamine as a fluorescent substance, and 3 'terminal was phosphorylated; Experimental group D labeled 5 'end with 6-carboxyfluorescein as a fluorescent substance, 15th base from 5' end with 6-carboxytetramethyl- rhodamine as fluorescent substance, and phosphorylated 3 'end; 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 BCR-ABL Standard Plasmid DNA

The BCR-ABL standard plasmid DNA was prepared as follows: First, BCR-ABL positive K-562 cells (CCL-243, ATCC, USA) using a total RNA isolation kit (RNAqueous Kit, Ambion, USA). ), The whole RNA was extracted, and the extracted RNA was applied to a cDNA synthesis kit (First strand cDNA synthesis kit, Roche, Germany) using p (dN) ₆ as a primer to synthesize cDNA. As a template, PCR was performed using a sense primer (SEQ ID NO: 8) and an antisense primer (SEQ ID NO: 9) having the following nucleotide sequence to obtain a cDNA (SEQ ID NO: 10) of 443 bp BCR-ABL fusion gene.

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

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

The 443 bp BCR-ABL fusion gene obtained above was introduced into the pCR 2.1-TOPO vector (Invitrogen, USA) to prepare a BCR-ABL standard plasmid DNA (see FIG. 1). 1 is a genetic map of BCR-ABL standard plasmid DNA. To facilitate the use of the previously prepared BCR-ABL standard plasmid DNA, it was serially diluted to have copy numbers of 2x10 ⁶ , 2x10 ⁴ , 2x10 ³ and 2x10 ² , and diluted BCR-ABL standard plasmid DNA 24 μl each was aliquoted separately.

As described above, the BCR-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 1-1-3 Preparation of ABL Standard Plasmid DNA

Next, the ABL standard plasmid DNA was prepared as follows: First, a K-562 cDNA synthesized in Example 1-1-2 was used as a template, and a sense primer (SEQ ID NO: 11) and an antisense having the following nucleotide sequence were used. PCR using a primer (SEQ ID NO: 12) was performed to obtain a cDNA (SEQ ID NO: 13) of the ABL gene of 500 bp.

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

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

The ABP gene of 500 bp obtained previously was introduced into the pCR 2.1-TOPO vector to prepare an ABL standard plasmid DNA (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 BCR-ABL negative control group and ABL negative control group

The PCR reaction mixture prepared in Example 1-1-1 was added to the BCR-ABL probe and primer mixture to prepare a BCR-ABL negative control group, and the electrical PCR reaction mixture was added to the ABL probe and primer mixture to prepare an ABL negative control group. It was.

Example 1-2 Determination of Recombination Degree of BCR-ABL Fusion Gene Using BCR-ABL Quantification 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 chronic myelogenous leukemia patient and applying 1 μg of the extracted RNA to a cDNA synthesis kit using p (dN) ₆ as a primer CDNA obtained by applying 100 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 bone marrow of the patient (Experimental Group 1-3) and the patient who has been diagnosed with chronic myelogenous leukemia and not yet treated, and applying 1 ug of extracted RNA to the cDNA synthesis kit using p (dN) ₆ as a primer. (experiment 2-1), the extracted RNA 100ng 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 BCR-ABL Value of Sample

10 μl of the PCR reaction mixture prepared in Example 1-1, 4 μl of the mixture of BCR-ABL probe and primer (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 , Dispense 2 µl each of BCR-ABL standard plasmid DNA having 2x10 ³ and 2x10 ² copy numbers, and cDNAs of Experimental Groups 1-1 to 2-3 to prepare each sample, and prepare the BCR-ABL negative control group in a separate tube. 20 microliters was added, and each BCR-ABL PCR sample was prepared. 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 a BCR-ABL standard PCR product and a BCR-ABL 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 the BCR-ABL standard PCR product and the amount of the BCR-ABL sample PCR product were obtained, respectively, and the relative amounts of the BCR-ABL sample PCR product were calculated based on the amount of the BCR-ABL standard PCR product. By doing so, BCR-ABL values of the samples for the experimental groups A to F were 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 primers (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 , and 2 × 10 ^3. And 2 μl of ABL standard plasmid DNA having 2x10 ² copy number, cDNA of Experimental Groups 1-1 to 2-3 and cDNA of Experimental Group 2, respectively, to prepare samples, and 20µl of ABL negative control group in separate tubes. Was dispensed to prepare each ABL PCR sample. Subsequently, each prepared sample was applied to LightCycler ^TM (Roche, Germany), 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 measured value was calculated by dividing the BCR-ABL value of the sample obtained in Example 1-2-2 by the ABL value of the sample obtained in Example 1-2-3 (see Table 1).

Comparison of the measured values calculated by the BCR-ABL value and ABL value of each experimental group sample Probe BCR-ABL Value (Replication) ABL value (replicate) Measured value (BCR-ABL / ABL) 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 382 423 368 Not measurable Not measurable 352 600 380 200 346 100 Not measurable Uncalculated Uncalculated 0.00108 0.00111 0.00106 Uncalculated Experimental group 1-2 Experiment group A Experiment group B Experiment group C Experiment group D Experiment group E Experiment group F Not measurable Not measurable 31 42 32 Not measurable Not measurable Not measurable 30860 34350 31020 Not measurable Uncalculated Uncalculated 0.00100 0.00122 0.00103 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 0.3 0.4 0.3 Not measurable Not measurable Not possible 250 290 245 Not measurable Uncalculated Uncalculated 0.00120 0.00138 0.00122 Uncalculated Experimental Group 2-1 Experiment group A Experiment group B Experiment group C Experiment group D Experiment group E Experiment group F Not measured Not measured 823420 1052 480 925 500 Not measured Not measurable Not measurable 372600 420 200 396 100 Not measurable Uncalculated Uncalculated 2.20 2.50 2.34 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 Not measurable 83200 103666 90324 Not measurable Not measurable Not measurable 38012 41650 39900 Not measurable Uncalculated Uncalculated 2.18 2.49 2.26 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 820 1076.8 878 Not measurable Not measurable Not measurable 321 400.8 357 Not measurable Uncalculated Uncalculated 2.55 2.69 2.46 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 value of the diagnosed patients was that the BCR-ABL fusion gene was expressed at a higher level than the 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 BCR-ABL Quantification Kit Applicable to Real-Time Polymerase Chain Reactor and Measurement of Recombination Degree of BCR-ABL Fusion Gene Using the Same (II)

Example 2-1 Preparation of BCR-ABL Quantification Kit Applicable to ABI PRISM ^™ 7000/7700

PCR reaction mixture 1.225 ml, 200 µl BCR-ABL probe and primer mixture, 200 µl ABL probe and primer mixture, 160 µl BCR-ABL standard plasmid DNA, 160 µl ABL standard plasmid DNA, 200 µl BCR-ABL negative control, ABL negative A BCR-ABL 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 BCR-ABL probe and primer mixture, ABL probe and primer mixture, BCR-ABL standard plasmid DNA, ABL standard plasmid DNA, BCR-ABL, 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 as used.

Example 2-2 Determination of Recombination Degree of BCR-ABL Fusion Genes Using BCR-ABL Quantitative Kit Applicable to ABI PRISM ^™ 7000/7700

The experimental group 2-2 and the experimental group 2-3 prepared in Example 1-2-1 were used as samples to be measured, and the degree of recombination of the BCR-ABL fusion gene was calculated and analyzed.

Example 2-2-1 : Measurement of BCR-ABL Value of Samples

Into the PCR tube, 12.5 μl of the PCR reaction mixture prepared in Example 2-1, 4 μl of the BCR-ABL probe and primer mixture and 4 μl of sterile water were dispensed, followed by 2 × 10 ⁶ , 2 × 10 ⁴ , 2 × 10 ^3, and 2 × 10 ² . Samples were prepared by dispensing 4 µl each of BCR-ABL standard plasmid DNA having replication number, cDNA of Experimental group 2-2 and cDNA of Experimental group 2-3, and 25µl of BCR-ABL negative control group in separate tubes. Each BCR-ABL PCR sample was prepared. Subsequently, each prepared sample was applied to ABI PRISM ^™ 7000 (Applied Biosystems, USA), which is a real-time polymerase chain reaction device, and PCR was performed to obtain a BCR-ABL standard PCR product and a BCR-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 the BCR-ABL standard PCR product and the amount of the BCR-ABL sample PCR product were obtained, respectively, and the relative amounts of the BCR-ABL sample PCR product were calculated based on the amount of the BCR-ABL standard PCR product. By this, the BCR-ABL value of the sample was obtained.

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 2-2 and cDNA of Experimental group 2-3, and aliquoting 25µl of ABL negative control group into separate tubes. Samples were prepared. Subsequently, each prepared sample was applied to an ABI PRISM ^™ 7000, which is 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 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 measured value was calculated by dividing the BCR-ABL value of the sample obtained in Example 2-2-1 by the ABL value of the sample obtained in Example 2-2-2.

Comparison of the measured values calculated by the BCR-ABL value and ABL value of each experimental group Experimental group Amount of RNA BCR-ABL Value (Replication) ABL value (replicate) Measured value (BCR-ABL / ABL) Experiment Group 2-2 Experiment Group 2-3 100ng 1ng 74626 699.4 98209 945.1 0.76 0.74

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

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

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

Example 3-1 Preparation of BCR-ABL Quantification Kit Applicable to iCycler ^™

PCR reaction mixture 1.225 ml, 200 µl BCR-ABL probe and primer mixture, 200 µl ABL probe and primer mixture, 160 µl BCR-ABL standard plasmid DNA, 160 µl ABL standard plasmid DNA, 200 µl BCR-ABL negative control, ABL negative A BCR-ABL 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. BCR-ABL probe and primer mixture, ABL probe and primer mixture, BCR-ABL standard plasmid DNA, ABL standard plasmid DNA, BCR-ABL 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 Determination of Recombination Degree of BCR-ABL Fusion Genes Using BCR-ABL Quantification Kit Applicable to iCycler ^™

The experimental group 2-2 and the experimental group 2-3 prepared in Example 1-2-1 were used as samples to be measured, and the degree of recombination of the BCR-ABL fusion gene was calculated and analyzed.

Example 3-2-1 : Measurement of BCR-ABL Value of Samples

10 μl of the PCR reaction mixture prepared in Example 3-1, 4 μl of the BCR-ABL probe and primer mixture and 4 μl of sterile water were dispensed into a PCR tube, and then 2 × 10 ⁶ , 2 × 10 ⁴ , 2 × 10 ^3, and 2 × 10 ² were used. Dispense 2 μl of BCR-ABL standard plasmid DNA having replication number, cDNA of Experimental group 2-2 and cDNA of Experimental group 2-3 to prepare each sample, and 20µl of BCR-ABL negative control group in a separate tube. Dispense and prepare each BCR-ABL PCR sample. 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 a BCR-ABL standard PCR product and a BCR-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 the BCR-ABL standard PCR product and the amount of the BCR-ABL sample PCR product were obtained, respectively, and the relative amounts of the BCR-ABL sample PCR product were calculated based on the amount of the BCR-ABL standard PCR product. By this, the BCR-ABL value of the sample was obtained.

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 2-2 and cDNA of experimental group 2-3, and 20 μl of ABL negative control group in separate tubes. Samples were prepared. Subsequently, each prepared sample was applied to iCycler ^™ , a real-time polymerase chain reaction device, and PCR was performed to obtain a BCR-ABL standard PCR product and a BCR-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 the ABL standard PCR product and the amount of the ABL sample PCR product were measured, respectively, and the relative amount of the ABL sample PCR product was calculated based on the amount of the ABL standard PCR product. It was.

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

The measured value was computed by dividing the BCR-ABL value of the sample calculated | required by the previous Example 3-2-1 by the ABL value calculated | required by the previous Example 3-2-2.

Comparison of the measured values calculated by the BCR-ABL value and ABL value of each experimental group Experimental group RNA amount BCR-ABL Value (Replication) ABL value (replicate) Measured value (BCR-ABL / ABL) Experiment Group 2-2 Experiment Group 2-3 100ng 1ng 37400 301.5 33300 360 1.12 0.84

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 BCR-ABL 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 ACR of the BCR-ABL quantitative kit of the present invention, it was confirmed that the amount of the BCR-ABL 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 a BCR-ABL fusion gene, a probe and primer mixture capable of detecting an ABL gene, a BCR-ABL standard plasmid DNA, ABL Provided are methods for quantifying BCR-ABL fusion genes using BCR-ABL quantitative kits and electrical kits comprising standard plasmid DNA, BCR-ABL negative controls and ABL negative controls. When the BCR-ABL quantitative kit of the present invention is used, the degree of recombination of the BCR-ABL fusion gene which causes chronic myelogenous leukemia can be more easily and accurately quantified. It can be widely used.

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Claims (8)

(Iii) a PCR reaction mixture; (Ii) two sense primers (SEQ ID NOS: 1 and 2), antisense primers (SEQ ID NO: 3) and 6'carboxylates at the 5 'end, 6-carboxyfluorescein, hexachloro, specific for the BCR-ABL fusion gene Labeled with -6-carboxyfluorescein or hexachloro-6-carboxyfluorescein and 6-carboxy of thymine in the 10-20th base from the 5 'end. BCR-ABL probe and primer mixtures labeled with 6-carboxytetramethylrhodamine, consisting of a probe phosphorylated at the 3 'end (SEQ ID NO: 4); (Iii) ABL gene specific, sense primer (SEQ ID NO: 5), antisense primer (SEQ ID NO: 6) and 6'carboxycarboxyescein at the 5 'end, hexachloro-6-carboxyfluorescein ( labeled with hexachloro-6-carboxyfluorescein or tetrachloro-6-carboxyfluorescein and 6-carboxytetramethylrhodamine in the 10th to 20th bases from the 5 'end. ABL probe and primer mixture, labeled A) consisting of a probe phosphorylated at the 3 'end (SEQ ID NO: 7); (Iii) BCR-ABL standard plasmid DNA comprising cDNA of the BCR-ABL fusion gene (SEQ ID NO: 10); (Iii) ABL standard plasmid DNA comprising the cDNA of the ABL gene (SEQ ID NO: 13); (Iii) a BCR-ABL negative control group comprising an electrical PCR reaction mixture and a BCR-ABL probe and primer mixture; And, (Iii) A BCR-ABL 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 BCR-ABL 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 BCR-ABL 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. BCR-ABL Quantitative Kit. delete The method of claim 1, BCR-ABL standard plasmid DNA comprises cDNA of BCR-ABL gene of SEQ ID NO: 10 and has a genetic map of FIG. BCR-ABL Quantitative Kit. The method of claim 1, ABL standard plasmid DNA comprises cDNA of the ABL gene of SEQ ID NO: 13 and has a genetic map of FIG. BCR-ABL Quantitative Kit. (Iii) the BCR-ABL standard plasmid DNA and the sample described in claim 1 are mixed with the BCR-ABL probe and primer mixture and the PCR reaction mixture, respectively, and then subjected to PCR to perform the BCR-ABL standard PCR product and the BCR-ABL 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 BCR-ABL value of the sample by measuring the amount of the BCR-ABL sample PCR product based on the amount of the BCR-ABL 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, (Iii) dividing the BCR-ABL value of the sample obtained in the preceding steps (iii) and (iv) by the ABL value to obtain the measured value, and present in the sample using the BCR-ABL quantitative kit of claim 1; Method for quantifying the degree of recombination of the BCR-ABL fusion gene.

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