JP4454377B2 - Method for detecting mutation of thiopurine methyltransferase and nucleic acid probe and kit therefor - Google Patents

Description

  The present invention relates to a method for detecting a mutation in thiopurine methyltransferase (TPMT) and a kit therefor.

  TPMT is an enzyme responsible for metabolic inactivation of drugs such as 6-mercaptopurine (6MP) and azathioprine used for the treatment of leukemia and the like.

  If there is a mutation (A719G mutation) in which the 719th A is substituted for G in the gene of this enzyme, the enzyme activity decreases. Therefore, in patients with mutant homozygotes, the metabolic efficiency of various drugs changes, increasing the risk of side effects during drug administration.

  As a method for detecting A719G mutation, a restriction enzyme that performs PCR using a primer designed to amplify the portion containing the 719th base of the TPMT gene, and the presence or absence of cleavage is determined by the presence or absence of the mutation at the 719th base. There is known a method (PCR-RFLP) of detecting whether or not it has been cleaved by electrophoresis and then cleaved by electrophoresis.

  Since PCR amplifies billions of times from several molecules of template, even a small amount of amplified product can cause false positives and false negatives. Since PCR-RFLP needs to take out the amplification product after PCR reaction and perform restriction enzyme treatment, the amplification product may be mixed into the next reaction system. Therefore, false positive and false negative results may be obtained. In addition, after PCR is completed, treatment with a restriction enzyme is performed, and then electrophoresis is performed. Therefore, it takes a very long time for detection. Moreover, since the operation is complicated, automation is difficult.

  As a method for detecting A719G mutation, PCR is performed using a primer having a base complementary to the 719th base of the TPMT gene at the 3 'end, and then it is detected whether it has been amplified (ASP-PCR) Is known (Patent Document 2).

  In this method, since the amplification reaction must be performed for each of A and G, a double amount of reagent is required. In addition, when the amount of template used is different, it is difficult to set reaction conditions such as different reaction conditions, and the reliability of the results is low.

On the other hand, a method is generally known in which a region containing a mutation is amplified by PCR, a melting curve analysis is performed using a nucleic acid probe labeled with a fluorescent dye, and the mutation is analyzed based on the result of the melting curve analysis. (Non-patent document 1, Patent document 1).
Clinical Chemistry, 2000, 46, 5, p. 631-635 JP 2002-119291 A JP 2001-17185 A

An object of the present invention is to identify a quenching probe effective for detecting the A719G mutation, and to provide a method for detecting the A719G mutation and a kit therefor.

  In the literature on the method using a probe, regarding the probe design, when a quenching probe labeled with a fluorescent dye at the end is hybridized to a target nucleic acid, at least one probe-nucleic acid hybrid has at least one base pair at the end. There is only teaching to design to form two G and C pairs. The inventors of the present invention designed a quenching probe that satisfies the above conditions for the A719G mutation and tried to detect it. However, a quenching probe that enables easy detection was not obtained.

The inventor has found that the A719G mutation can be detected by melting curve analysis using a quenching probe by designing a quenching probe based on a specific region containing the A719G mutation, and the present invention has been completed.
The present invention provides the following.

(1) A nucleic acid probe whose end is labeled with a fluorescent dye and whose fluorescence decreases when hybridized, and satisfies any of the following (a) to (c). (a) The base sequence shown in SEQ ID NO: 2 has a sequence complementary to a base sequence having a length of 6 to 50 bases starting from base number 181 and the 3 ′ end is labeled with a fluorescent dye.
(b) The base sequence shown in SEQ ID NO: 2 has a sequence complementary to a base sequence having a length of 11 to 50 bases starting from base number 176, and the 3 'end is labeled with a fluorescent dye.
(c) The base sequence shown in SEQ ID NO: 2 has a sequence complementary to a base sequence having a length of 19 to 50 bases starting from base number 168, and the 3 'end is labeled with a fluorescent dye.

  (2) The nucleic acid probe according to (1), wherein the nucleic acid probe has the base sequence shown in any one of SEQ ID NOs: 5 to 7.

  (3) Using a nucleic acid probe labeled with a fluorescent dye, the nucleic acid having a single nucleotide polymorphism site is subjected to melting curve analysis by measuring the fluorescence of the fluorescent dye, and the mutation is based on the result of the melting curve analysis. Wherein the single nucleotide polymorphism is a mutation at position 719 of the TPMT gene, and the nucleic acid probe is the nucleic acid probe of (1) or (2).

  (4) The method according to (3), comprising amplifying a region containing a single nucleotide polymorphism site in a nucleic acid contained in a sample to obtain a nucleic acid having a single nucleotide polymorphism.

  (5) The method according to (4), wherein the amplification is performed by a method using a DNA polymerase.

  (6) The method of (5), wherein the amplification is performed in the presence of a nucleic acid probe.

(7) a nucleic acid probe whose end is labeled with a fluorescent dye and whose fluorescence decreases when hybridized, the nucleic acid probe satisfying any of the following (a) to (c): ) Kit for the method.
(a) The base sequence shown in SEQ ID NO: 2 has a sequence complementary to a base sequence having a length of 6 to 50 bases starting from base number 181 and the 3 ′ end is labeled with a fluorescent dye.
(b) The base sequence shown in SEQ ID NO: 2 has a sequence complementary to a base sequence having a length of 11 to 50 bases starting from base number 176, and the 3 'end is labeled with a fluorescent dye.
(c) The base sequence shown in SEQ ID NO: 2 has a sequence complementary to a base sequence having a length of 19 to 50 bases starting from base number 168, and the 3 'end is labeled with a fluorescent dye.

(8) The kit according to (7), wherein the nucleic acid probe has the base sequence shown in any one of SEQ ID NOs: 5 to 7.

  (9) The kit according to (7) or (8), further comprising a primer for amplifying a region containing a mutation at position 719 of the TPMT gene by a method using DNA polymerase.

  According to the present invention, a quenching probe effective for detecting the A719G mutation is provided, and further, a method for detecting the A719G mutation using the probe and a kit for the same are provided.

  Since Tm analysis is completed in tens of seconds, the time required for detection can be greatly reduced. According to a preferred embodiment of the present invention that combines nucleic acid amplification and Tm analysis in the presence of a probe, it is only necessary to analyze the Tm of the probe after amplification of the nucleic acid, so there is no need to handle the amplification product after the reaction is complete. Therefore, there is no worry of contamination by amplification products. Furthermore, since it can be detected by the same equipment as that required for amplification, it is not necessary to move the container. Therefore, automation is also easy. In addition, since only one reaction needs to be performed for typing one single nucleotide polymorphism, the amount of reagent used can be reduced.

<1> Probe of the Present Invention and Detection Method of the Present Invention The probe of the present invention is a nucleic acid probe whose end is labeled with a fluorescent dye and the fluorescence of the fluorescent dye decreases when hybridized, and includes the following (a) to (c) Any one of the above is satisfied.
(a) The base sequence shown in SEQ ID NO: 2 has a sequence complementary to a base sequence having a length of 6 to 50 bases starting from base number 181 and the 3 ′ end is labeled with a fluorescent dye.
(b) The base sequence shown in SEQ ID NO: 2 has a sequence complementary to a base sequence having a length of 11 to 50 bases starting from base number 176, and the 3 'end is labeled with a fluorescent dye.
(c) The base sequence shown in SEQ ID NO: 2 has a sequence complementary to a base sequence having a length of 19 to 50 bases starting from base number 168, and the 3 'end is labeled with a fluorescent dye.

  In this specification, a complementary base sequence means that it is complementary to the full length of the target base sequence.

  The probe of the present invention is the above in the nucleotide sequence shown in SEQ ID NO: 1 (sequence having a wild type (normal type) base in A719G mutation) or the nucleotide sequence shown in SEQ ID NO: 2 (sequence having a mutant type base in A719G mutation). Other than having the sequences specified in (a) to (c), it may be the same as the quenching probe described in Patent Document 1. Examples of the base sequence of the quenching probe used in the present invention include those shown in any of SEQ ID NOs: 5 to 7. As the fluorescent dye, those described in Patent Document 1 can be used, and specific examples include FAM (trademark), TAMRA (trademark), BODIPY (trademark) FL, and the like. The method for binding the fluorescent dye to the oligonucleotide can be performed according to a conventional method, for example, the method described in Patent Document 1.

  In the detection method of the present invention, a melting curve analysis is performed on a nucleic acid having a single nucleotide polymorphism site by measuring the fluorescence of the fluorescent dye using a nucleic acid probe labeled with the fluorescent dye. A method for detecting a mutation based on the single nucleotide polymorphism is an A719G mutation, and the nucleic acid probe is a probe of the present invention.

  The detection method of the present invention can be carried out according to the usual methods of nucleic acid amplification and melting curve analysis (Tm analysis) other than amplifying a region containing the A719G mutation and using the probe of the present invention.

  As a method for nucleic acid amplification, a method using a polymerase is preferred, and examples thereof include PCR, ICAN, LAMP and the like. When amplification is performed by a method using a polymerase, amplification is preferably performed in the presence of the probe of the present invention. It is easy for those skilled in the art to adjust the amplification reaction conditions and the like according to the probe used. As a result, only the Tm of the probe is analyzed after amplification of the nucleic acid, so that it is not necessary to handle the amplification product after the reaction is completed. Therefore, there is no worry of contamination by amplification products. Moreover, since it can detect with the same apparatus as an apparatus required for amplification, it is not necessary to move a container. Therefore, automation is also easy.

  Hereinafter, the case where PCR is used will be further described as an example. The primer pair used for PCR can be set in the same manner as the primer pair setting method in ordinary PCR, except that the region where the probe of the present invention can hybridize is amplified. The length and Tm of the primer are usually 12 to 40 mer and 40 to 70 ° C., preferably 16 to 30 mer and 55 to 60 ° C. The length of each primer in the primer pair may not be the same, but the Tm of both primers is preferably substantially the same (usually, the difference is within 2 ° C.). The Tm value is a value calculated by the nearest base pair (Nearest Neighbor) method. Examples of primer pairs include those consisting of primers having the base sequences shown in SEQ ID NOs: 3 and 4.

  PCR is preferably performed in the presence of the probe of the present invention used in the present invention. Thus, Tm analysis can be performed without performing an operation for handling the amplification product after the amplification reaction is completed. It is easy for those skilled in the art to adjust the Tm of the primer and the PCR reaction conditions according to the probe to be used.

  A typical PCR reaction solution composition is as follows.

Moreover, if a typical temperature cycle is mentioned, it will be as follows and this temperature cycle will be repeated 25-40 times normally.
(1) Denaturation, 90-98 ° C, 1-60 seconds
(2) Annealing, 60-70 ° C, 10-60 seconds
(3) Elongation, 60-75 ° C, 10-180 seconds

  When annealing and elongation are performed in one step, the conditions are 60 to 70 ° C. and 10 to 180 seconds.

The Tm analysis can be performed according to a usual method except that the fluorescence of the fluorescent dye of the probe of the present invention is measured. The fluorescence can be measured by measuring light having an emission wavelength using excitation light having a wavelength corresponding to the fluorescent dye. The rate of temperature increase in Tm analysis is usually 0.1-1 ° C./second. The composition of the reaction solution for performing Tm analysis is not particularly limited as long as hybridization between the probe and a nucleic acid having a sequence complementary to the base sequence is possible, but usually the monovalent cation concentration is 1.5-5 mM, pH is 7-9. Since the reaction solution of the amplification method using DNA polymerase such as PCR normally satisfies this condition, the amplified reaction solution can be used as it is for Tm analysis.

  Detection of the A719G mutation based on the result of Tm analysis can be performed according to a usual method. The detection in the present invention includes not only detection of the presence or absence of mutation, but also quantification of mutant DNA and measurement of the ratio of normal DNA to mutant DNA.

<2> Kit of the Present Invention The kit of the present invention is a kit for use in the detection method of the present invention. This kit is a nucleic acid probe (quenching probe) whose end is labeled with a fluorescent dye and the fluorescence of the fluorescent dye decreases when it hybridizes, and a nucleic acid probe that satisfies any of the above (a) to (b) It is characterized by including.

  The quenching probe is as described above for the probe of the present invention.

  In addition to the quenching probe, the detection kit of the present invention may further contain reagents necessary for performing nucleic acid amplification in the detection method of the present invention, particularly primers for amplification using DNA polymerase.

  In the detection kit of the present invention, the quenching probe, primer and other reagents may be separately accommodated, or a part of them may be a mixture.

  Hereinafter, the present invention will be specifically described by way of examples.

  A base sequence (SEQ ID NO: 1 (wild type) or SEQ ID NO: 2 (mutant), base number 186 corresponds to position 719 of the TPMT gene) containing the site of the A → G mutation (A719G mutation) at position 719 of the TPMT gene Based on this, the primers shown in Table 2 were designed so that the portion containing the A719G mutation could be amplified. In Table 2, the position indicates the base number in the base sequence shown in SEQ ID NO: 1 or 2.

  Next, probes having C at the end shown in Table 3 were designed. In Table 3, the position indicates the base number in the base sequence shown in SEQ ID NO: 1 or 2. The capital letters in the base sequence indicate the A719G mutation site, and P at the 3 ′ end indicates phosphorylation. Labeling with BODIPY FL and TAMRA was performed according to a conventional method.

  Using purified human genome (extracted from whole blood by GFX Genomic Blood DNA Purification Kit direct method) as a sample, PCR and Tm analysis were performed using Smart Cycler System (Cephied) under the following conditions. The excitation and detection wavelengths in Tm analysis were 450-495 nm and 505-537 nm (BODIPY FL), respectively, or 527-555 nm and 565-605 nm (TAMRA), respectively.

  As a result of PCR and Tm analysis using each probe, when using probes 3T-mt-R1-25, 3T-mt-R2-27, 3FL-mt-R2-27 and 3T-mt-R3-30 Changes in fluorescence intensity that can be analyzed by Tm analysis were observed. In addition, the arrangement | positioning with respect to the base sequence containing the A719G mutation of each probe is shown in FIG. In addition, FIG. 2 shows the arrangement of the probe with no change in fluorescence intensity that can be analyzed by Tm analysis with respect to the base sequence containing the A719G mutation. 1 and 2, the wild type sequence and the mutant type sequence are the base sequences of base numbers 166 to 205 of the base sequences of SEQ ID NOs: 1 and 2, respectively. In the figure, F represents a fluorescent dye. Whether the probe can be used in Tm analysis is considered to depend on the position of C to which the fluorescent dye is bound, and the length of the probe is considered to be less important as long as the polymorphic site is included.

  Prepare DNA (wt / wt) with normal sequence and DNA with both mutant and normal sequence (wt / mt) as samples, and perform typing using probe 3FL-mt-R2-27 It was. The results are shown in FIG. Moreover, the result of having carried out fixed_quantity | quantitative_assay by changing the quantity of blood (0.01-1 microliter) is shown in FIG. In FIGS. 3 and 4, NC is the control. From these results, it can be seen that this quantification method has good reproducibility and high sensitivity. Similar results were obtained with other probes that showed a change in fluorescence intensity that could be analyzed by Tm analysis.

  3 and 4, the vertical axis represents the value of the inverse sign of the first derivative of fluorescence intensity (-dF / dt), and the horizontal axis represents temperature (° C).

The positions of quenching probes that can identify mutations are indicated. The position of the quenching probe indistinguishable from the mutation is indicated. The reproducibility of the method of Example 1 is shown. The sensitivity regarding the absolute amount of genomic DNA of the method of Example 1 is shown.

Claims (7)

A nucleic acid probe whose end is labeled with a fluorescent dye and whose fluorescence decreases when hybridized, comprising a base sequence shown in any of SEQ ID NOs: 5 to 7, and its 3 ′ end is labeled with a fluorescent dye and a nucleic acid probe. Using a nucleic acid probe labeled with a fluorescent dye, nucleic acid having a single nucleotide polymorphism site is used to perform melting curve analysis by measuring the fluorescence of the fluorescent dye, and to detect mutations based on the results of the melting curve analysis The method according to claim 1 , wherein the single nucleotide polymorphism is a mutation at position 719 of the thiopurine methyltransferase gene, and the nucleic acid probe is the nucleic acid probe according to claim 1 . The method according to claim 2 , comprising amplifying a region containing a single nucleotide polymorphism site in a nucleic acid contained in a sample to obtain a nucleic acid having a single nucleotide polymorphism. The method according to claim 3, wherein the amplification is performed by a method using a DNA polymerase. The method according to claim 4 , wherein the amplification is performed in the presence of a nucleic acid probe. A nucleic acid probe whose end is labeled with a fluorescent dye and whose fluorescence decreases when hybridized, comprising a base sequence shown in any of SEQ ID NOs: 5 to 7, and its 3 ′ end is labeled with a fluorescent dye and comprising a nucleic acid probe is a kit for the method of claim 2 wherein. The kit according to claim 6 , further comprising a primer for amplifying a region containing a mutation at position 719 of the thiopurine methyltransferase gene by a method using a DNA polymerase.

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