JP4517175B2 - NAT2 * 7 mutation detection method and nucleic acid probe and kit therefor - Google Patents

Description

  The present invention relates to a method for detecting a mutation in a gene of N-acetyltransferase 2 (NAT2) and a kit therefor.

NAT2 is an enzyme that metabolizes isoniazid, an antituberculous drug. If there is a mutation that replaces G at position 857 in the cDNA of the NAT2 gene with A (G857A mutation), the 197th Arg of NAT2 is mutated to Gln, and the protein structure becomes abnormal . Therefore, if this mutant allele is present in a heterozygous manner with a homozygous or other enzyme-inactivating mutant allele, the metabolic efficiency of the drug decreases and the risk of side effects and the like increases (Non-patent Document 2). A gene having a G857A mutation is called a NAT2 * 7 allele.

  When the G857A mutation is present, the restriction enzyme recognition site disappears, so amplification is performed to include the mutation part by PCR, the restriction enzyme is cleaved, and then it is detected whether it has been cleaved by electrophoresis ( PCR-RFLP) can be used for detection.

  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 the G857A mutation, PCR is performed using a primer having a base complementary to the 857th base of the NAT2 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, the amplification reaction must be carried out one by one for each of G and A, so that 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 Mutation Research, 1997, 376, p. 61-70 JP 2002-119291 A JP 2001-17185 A

An object of the present invention is to identify a quenching probe effective for detecting the G857A mutation, and to provide a method for detecting the G857A mutation and a kit therefor.

  In the literature regarding the method using a probe, when a quenching probe labeled with a fluorescent dye is hybridized to a target nucleic acid, at least one base pair of a probe-nucleic acid hybrid is present at the terminal. There is only the teaching of forming a pair of G and C, or designing G so that G exists in the base sequence of the target nucleic acid 1 to 3 bases away from the end (Patent Document 1, paragraph number 0048). The inventors of the present invention designed a quenching probe that satisfies the above conditions and attempted detection for the G857A mutation, but could not obtain a quenching probe that could be easily detected.

The present inventor has found that the G857A mutation can be detected by melting curve analysis using a quenching probe by designing a quenching probe based on a specific region containing the G857A mutation, thereby completing the present invention.
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 (e): (a) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a base sequence having a length of 20 to 30 bases starting from base number 301, and the 3 'end is labeled with a fluorescent dye.
(b) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a base sequence having a length of 20 to 30 bases starting from base number 300, and the 3 'end is labeled with a fluorescent dye.
(c) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a base sequence having a length of 18 to 28 bases starting from base number 299, and the 3 'end is labeled with a fluorescent dye.
(d) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a 20-30 base long base sequence ending with base number 328, and the 5 'end is labeled with a fluorescent dye.
(e) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a 19-29 base long base sequence ending with base number 322, and the 5 '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 10.

  (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 857 in the cDNA of the NAT2 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 (e): ) Kit for the method.
(a) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a base sequence having a length of 20 to 30 bases starting from base number 301, and the 3 'end is labeled with a fluorescent dye.
(b) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a base sequence having a length of 20 to 30 bases starting from base number 300, and the 3 'end is labeled with a fluorescent dye.
(c) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a base sequence having a length of 18 to 28 bases starting from base number 299, and the 3 'end is labeled with a fluorescent dye.
(d) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a 20-30 base long base sequence ending with base number 328, and the 5 'end is labeled with a fluorescent dye.
(e) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a 19-29 base long base sequence ending with base number 322, and the 5 '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 10.

  (9) The kit according to (7) or (8), further comprising a primer for amplifying a region containing the mutation at position 857 in the cDNA of NAT2 gene by a method using DNA polymerase.

  According to the present invention, a quenching probe effective for detecting the G857A mutation is provided, and further, a method for detecting the G857A mutation using the same and a kit for the same are provided. Since the NAT2 * 7 allele contains the G857A mutation, the quenching probes and methods and kits of the invention can be used to detect the NAT2 * 7 allele.

  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.

<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 (e) Any one of the above is satisfied.
(a) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a base sequence having a length of 20 to 30 bases starting from base number 301, and the 3 'end is labeled with a fluorescent dye.
(b) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a base sequence having a length of 20 to 30 bases starting from base number 300, and the 3 'end is labeled with a fluorescent dye.
(c) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a base sequence having a length of 18 to 28 bases starting from base number 299, and the 3 'end is labeled with a fluorescent dye.
(d) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a 20-30 base long base sequence ending with base number 328, and the 5 'end is labeled with a fluorescent dye.
(e) The base sequence shown in SEQ ID NO: 1 has a base sequence complementary to a 19-29 base long base sequence ending with base number 322, and the 5 '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. Moreover, that a nucleic acid probe has a base sequence means that it consists of the base sequence.

The probe of the present invention is the same as that of Patent Document 1 except that the conditions (a) to (e) described above are satisfied in the base sequence shown in SEQ ID NO: 1 (sequence having a wild type (normal type) base in the G857A mutation). It may be the same as the quenching probe described in 1). A probe having an appropriate length can be selected in the range of the lengths described in the above (a) to (e) according to a normal probe setting method. 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 10. 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 a G857A 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 G857A mutation and using the probe of the present invention.

  As a method for nucleic acid amplification, a method using PCR polymerase is preferred, and examples thereof include PCR, ICAN, LAMP and the like. When amplification is performed by a method using PCR 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 G857A mutation based on the result of Tm analysis can be performed according to an ordinary 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 hybridized, and a nucleic acid probe that satisfies any of the above (a) to (e) 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.

  Nucleotide sequence (SEQ ID NO: 1 (wild type) or SEQ ID NO: 2 (mutant)) containing the site of G → A mutation (G857A mutation) at position 857 of the cDNA of NAT2 gene, base number 306 is position 857 of the cDNA of NAT2 gene The primers shown in Table 2 were designed so that a portion containing the G857A mutation could be amplified. In Table 2, the position indicates the base number in the cDNA of the NAT2 gene.

  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 letter in the base sequence indicates the G857A 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, probes 3FL-wt-R2-24, 3FL-wt-R1-24, 3T-wt-R3-23, 3FL-wt-R4-23, 5T-wt When -R5-24, 3T-wt-R8-22 and 5T-wt-R9-23 were used, changes in fluorescence intensity that could be analyzed by Tm analysis were observed. In addition, the arrangement | positioning with respect to the base sequence containing G857A variation | mutation of each probe is shown in FIG. Moreover, the arrangement | positioning with respect to the base sequence containing the G857A mutation of each probe in which the change of the fluorescence intensity which can be analyzed by Tm analysis was not recognized is shown in FIG. 1 and 2, the wild type sequence and the mutant type sequence are the base sequences of base numbers 290 to 329 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.

  As samples, DNA having mutant and normal sequences (mt / mt and wt / wt, respectively) and DNA having both mutant and normal sequences (wt / mt) were used, and probe 3FL-wt- Typing was performed using R2-24. The results are shown in FIG. Further, FIG. 4 shows the result of quantification by changing the amount of DNA (100 to 1000 copies). 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, and satisfies any of the following (a) to ( f ):
(A) It consists of the base sequence shown in SEQ ID NO: 5 and the 3 ′ end is labeled with a fluorescent dye.
(B) It consists of the base sequence shown in SEQ ID NO: 6, and the 3 ′ end is labeled with a fluorescent dye.
(C) It consists of the base sequence shown in SEQ ID NO: 7, and the 3 ′ end is labeled with a fluorescent dye.
(D) It consists of the base sequence shown in SEQ ID NO: 7, and the 5 ′ end is labeled with a fluorescent dye.
(E) It consists of the base sequence shown in SEQ ID NO: 8, and the 3 ′ end is labeled with a fluorescent dye.
(F) It consists of the base sequence shown in SEQ ID NO: 9, and the 5 ′ end is labeled with a fluorescent dye.
(G) Consisting of the base sequence shown in SEQ ID NO: 10, the 3 ′ end is labeled with a fluorescent dye. 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 857 in cDNA of the NAT2 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. 3. The nucleic acid probe according to claim 2 , wherein the nucleic acid probe is labeled with a fluorescent dye and the fluorescence of the fluorescent dye decreases when hybridized, and the nucleic acid probe satisfies any of the following (a) to ( f ): Kit for the method .
(A) It consists of the base sequence shown in SEQ ID NO: 5 and the 3 ′ end is labeled with a fluorescent dye.
(B) It consists of the base sequence shown in SEQ ID NO: 6, and the 3 ′ end is labeled with a fluorescent dye.
(C) It consists of the base sequence shown in SEQ ID NO: 7, and the 3 ′ end is labeled with a fluorescent dye.
(D) It consists of the base sequence shown in SEQ ID NO: 7, and the 5 ′ end is labeled with a fluorescent dye.
(E) It consists of the base sequence shown in SEQ ID NO: 8, and the 3 ′ end is labeled with a fluorescent dye.
(F) It consists of the base sequence shown in SEQ ID NO: 9, and the 5 ′ end is labeled with a fluorescent dye.
(G) Consisting of the base sequence shown in SEQ ID NO: 10, the 3 ′ end is labeled with a fluorescent dye. The kit according to claim 6 , further comprising a primer for amplifying a region containing a mutation at position 857 in the cDNA of the NAT2 gene by a method using DNA polymerase.

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