JP2002325581A - Method for detecting unknown or known nucleic acid mutation using nucleic acid melt curve and nucleic acid dissociation curve, and exhibition method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting an unknown and known nucleic acid mutation rapidly, easily and at a low cost, and an exhibition method thereof. SOLUTION: Problems are solved by a method for detecting a nucleic acid mutation through a difference and a feature of a temperature (Tm value) in which a double-stranded nucleic acid or an amplification product thereof is heated to melt into a single-stranded nucleic acid. The unknown and known nucleic acid mutation can be detected rapidly, easily and at a low cost by comparing and collating a Tm value, a Tm pattern and a change thereof of a sample with an obtained existing Tm value, a Tm pattern and a change thereof accumulated as a data base.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a nucleic acid discrimination method for estimating an unknown or known nucleic acid mutation of a double-stranded nucleic acid based on a difference in temperature at which a double-stranded nucleic acid is heated and dissociated into a single strand and its characteristics. .

[0002]

2. Description of the Related Art Various methods for detecting unknown or known nucleic acid mutations in double-stranded nucleic acid have been devised and put into practical use. The mutation of the nucleic acid mainly relates to a mutation of a base in the base sequence or a mutation of the length thereof, and has been distinguished by detecting them.

[0003] As a method for detecting an unknown nucleic acid base sequence variation, a portion of a target base sequence is amplified by PCR, and the amplified product is directly electrophoresed with polyacrylamide without denaturation. PCR-S for detecting mutations from
SCP (PCR-SingleStrand Conf)
operation polymorphism) method. In this method, a change in the three-dimensional structure of the amplification product due to the mutation is detected as a difference in electrophoretic mobility. In addition, a method for detecting an unknown mutation based on a sequencing technique such as a shotgun method, a primer walking method, and a cloning method is also widely used.

[0004] As a known method for detecting a nucleic acid base sequence mutation, a nucleic acid is appropriately cleaved with a restriction enzyme, and the 3 'end of a PCR primer is intentionally placed at a mutated portion.
PCR-ASP (PCR-
Allele Specific Primer) method,
The mutated portion is amplified by PCR, and the PCR-RFLP (PCR-Rest
rightion Fragment Length P
(Polymorphism) method, the Taqman probe method in which a mutated portion is further detected with a fluorescent label probe, and the latest technology such as the Invader method.

Recently, SNPs (Single Nuc)
Leotide Polymorphism) has been determined to be able to provide optimal medical care to individuals, and a method for detecting mutations in the base sequence of a gene using a DNA chip method or the like has been developed, particularly as a method for detecting known base mutations. However, there is a need for a more efficient method for detecting mutations in terms of time and economy as compared with those methods.

[0006]

However, all of the nucleic acid mutation detection methods performed so far require a skilled technique, and the detection requires a considerable amount of time and cost. In particular, as for the unknown mutation detection method, only a method that can be performed by a person skilled in advanced technology using expensive equipment and applying considerable time and money burden has been developed.

[0007] At present, the only method capable of detecting both known and unknown mutations is the direct sequence method, but this method is technically, temporally, and financially expensive especially for the executor among the above methods. It imposes a heavy burden.

[0008] At present, genetic information is required in various fields, and in particular, since the concept of SNPs was established, known SNPs have been known.
Similar to the detection of s, the development of a method for efficiently detecting unknown SNPs, that is, a method for detecting a nucleic acid mutation in a short time and at a low cost without requiring advanced technology is awaited.

[0009]

Means for Solving the Problems In view of such circumstances, the present inventors have conducted intensive studies and as a result, have found that the difference between the temperature points at which a double-stranded nucleic acid or its amplified product is heated and dissociated into single-stranded, and the characteristics thereof,
The invention of a nucleic acid mutation detection method for easily and inexpensively estimating unknown or known mutations of the present strand nucleic acid in a short time has been attained.

[0010] To explain the principle of the present invention, when a temperature is applied to double-stranded nucleic acids in a specimen, each of them dissociates into single-stranded nucleic acids. The temperature point at which this rapid dissociation occurs is defined as Tm (Mel
The properties such as dissociation temperature and speed are determined by the base sequence of the double-stranded nucleic acid and the bond strength between the bases adenine (A) and thymine (T) and the bond strength between guanine (G) and cytosine (C). Of the bases A, G, C, and T depending on the content of each base. In addition, the temperature and speed of dissociation also depend on the length of the double-stranded nucleic acid, and thus vary depending on the difference in the number of microsatellite repeats. Therefore, the Tm value or Tm pattern of the target double-stranded nucleic acid portion can be compared between different samples, and some unknown mutation can be estimated in a sample showing a different value or pattern. If the Tm value or Tm pattern of the normal type or the abnormal type is created in advance, the presence of the abnormal type can be detected as a known mutation by comparing and comparing the Tm value or the Tm pattern of the sample nucleic acid. Is possible.

The principle of the invention will be described with reference to the drawings as shown in FIGS. FIG. 1 is a double-stranded nucleic acid dissolution curve shown with temperature on the horizontal axis and fluorescence intensity on the vertical axis.
For example, when a temperature is applied to three kinds of PCR products of A, B, and C, the double-stranded nucleic acid dissociates into a single-stranded nucleic acid at a constant temperature, so that the fluorescence intensity gradually decreases. The decrease pattern is defined by the GC content in the double-stranded nucleic acid, the length, the three-dimensional structure, and the like, and each curve has its own curve. Here, the temperature at which the dissociation proceeds rapidly is called the dissociation temperature (Tm value).

FIG. 1 is a derivative of the double-stranded nucleic acid dissociation curve shown in FIG. In this figure, the Tm value is displayed as a peak, so that the difference between the curves becomes more apparent. Since each double-stranded nucleic acid has its own dissociation curve, the principle of the present invention is to detect a nucleic acid mutation by comparing with a dissociation curve of a normal nucleic acid which has been experimentally prepared in advance.

Further, in the present invention, a method for making the Tm value or Tm peak clearer by the method according to claim 11 has been devised. The principle will be described with reference to FIG. In FIG. 3, an antisense primer (2) and a sense primer (3) in the presence of a base mutation (1) in the nucleic acid,
When nucleic acid amplification is performed using the sense primer (4) and the sense primer (5), the PCR product between the antisense primer and the sense primer is
m, PCR product between sense primer and Tm
When the PCR product between the primer and the sense primer is Tm, the effect of the base mutation on each Tm value is Tm, Tm
, Tm (Tm <Tm <Tm).

In general, the Tm value and the Tm peak are less affected by the mutation of one base as the number of bases in the target range is larger, and are larger as the number of bases in the target range is smaller. . Therefore, as shown in FIG. 3, a plurality of PCR primer sets having different nucleotide numbers in the nucleic acid amplification product and having different numbers of nucleotides in the nucleic acid amplification product are arranged, and their Tm values or Tm peaks are compared. Indicates a continuous change. Since this continuous change shows a different pattern depending on the presence or absence of a mutation, a comparison with a normal continuous pattern is used for the purpose of detecting a known mutation, and a specific continuous pattern is used for the purpose of detecting an unknown mutation. By selecting the indicated ones, it is possible to confirm the base mutation.

That is, in the case of a known mutation, there is almost no change in Tm between the case where there is a mutation and the case where there is no mutation, but the Tm values of Tm and Tm continuously increase. In the case of an unknown mutation, the Tm value hardly changes at Tm, and the change in Tm value of Tm and Tm continuously increases, so that the presence of the unknown mutation can be estimated. In any case, the presence of the mutation becomes clearer than when one type of primer set is used.

[0016] The present invention provides a method for preparing a genomic DN from a collected specimen.
Samples are destroyed by a method other than the method of extracting A and preparing a double-stranded nucleic acid dissolution curve or a double-stranded nucleic acid dissociation curve, or a nucleic acid amplification product to prepare a dissolution curve or a dissociation curve and obtaining a Tm value. The present invention is also effective in a method for performing nucleic acid amplification by directly performing PCR without extracting DNA from a sample by using a direct PCR buffer or the like containing a component that suppresses an enzyme reaction inhibitor, or the like. It is.

When direct nucleic acid amplification is performed by direct PCR without extracting DNA from a specimen, Ampdirect manufactured by Shimadzu Corporation is used as a direct PCR buffer.
Is useful, but the present invention is not limited to this.

The present invention can be applied to a nucleic acid mutation detection and display technique in which a detected mutation is checked against known gene information obtained in advance, and the determination result is directly displayed in addition to the Tm value or Tm pattern. In addition, the present invention relates to ABO blood group, H
LA (Human Leukocyte Antige)
n) SNPs associated with typing and diabetic nephropathy (Dia
betic Nephropathy Related
Single Nucleide Polymo
(rphism) can also be used.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION Next, the nucleic acid mutation detection method of the present invention will be described in detail by using a known nucleic acid as a sample and formulating a PCR reaction solution shown in Table 1.

[0020]

[Table 1]

Put the sample DNA of the sample and add Yμl of distilled water.
The reaction solution shown in Table 1 which was formulated so that the total was 50 μl was thoroughly stirred, and the total volume of 50 μl was set in a real-time PCR quantifier GeneAmp5700 manufactured by ABI to start PCR. The PCR protocol is 95
40 cycles of 5 ° C./5 seconds, 55 ° C./10 seconds, and 72 ° C./20 seconds.

After completion of the PCR, a double-stranded nucleic acid dissolution curve (Me
lt Curve) and a double-stranded nucleic acid dissociation curve (Dissociation Cu) obtained by differentiating the curve.
rve), which can be compared between several samples to estimate any unknown mutations in samples showing different Tm values or Tm patterns.

When normal or abnormal Tm values or Tm patterns have been prepared in advance, the Tm
By comparing and comparing values or Tm patterns,
The presence of the abnormal form can be detected as a known mutation.

The real-time PCR quantifier is not limited to GeneAmp5700 manufactured by ABI, but any product may be used. Further, the fluorescent dye is not limited to SyGreen (Cyber Green) as long as it is an intercalator. The PCR protocol is appropriately changed according to the primers to be used.

[0025]

Example 1 Detection of an unknown mutation was performed using the human genome WO01.
1092 (Genbank: Accession Nu)
mber) using the method of the present invention to determine whether there is an unknown mutation in the gene sequence registered in
The value and Tm patterns were compared.

After extracting human genomic DNA from whole blood using Isogen-LS of Nippon Gene as an extraction reagent in accordance with a conventional method, a reaction solution was prepared according to the formulation shown in Table 1, 1 μg of each genomic DNA and primers having the following base sequence: Used set. Primer set 1, Sense primer: 5'-TTTTCCTCCCTGCC
CTTGTTTA-3 ′ (SEQ ID NO: 1), anti-antisense: 5′-AAAAGGGTTTAC
CAGGTTTG-3 ′ (SEQ ID NO: 2), Primer set 2, Sense primer: 5′-CTCTTTATCAATA
CTTCACA-3 '(SEQ ID NO: 3), Antisense primer: 5'-GGTTTGCTAG
AGTTAAAT-3 '(SEQ ID NO: 4), Primer set 3, Sense primer: 5'-ACACAAAAACTCA
AAAACCTT-3 '(SEQ ID NO: 5), Antisense primer: 5'-AGTAATACTA
CCGTTGATTCT-3 ′ (SEQ ID NO: 6),

The positional relationship between the primers is as shown in FIG. In the figure, a primer set 1 (7) and a primer set 2 (8) for a sample including the target search range (6)
Then, PCR is performed by arranging the primer set 3 (9).

PCR is a real-time PCR manufactured by ABI
95 ° C./5 using a quantitative machine GeneAmp5700
Seconds, 55 ° C./10 seconds and 72 ° C./20 seconds were performed according to a protocol of 40 cycles. The examination was performed with three types of primer sets per sample, and a total of 35 samples were analyzed. Table 2 shows the obtained results.

[0029]

[Table 2]

In the table, regarding the notation of the Tm pattern,
A single-stranded nucleic acid dissociation curve having a single peak is designated as monomodal, and a peak having two peaks is designated as bimodal. or,
The case where the peak was displayed as a shoulder without a clear peak was described as notch formation.

From Table 2, it can be determined whether or not an unknown mutation exists in the gene sequence. When primer set 1 (7) including the entire search range (6) was used, 1 out of 35 samples was used.
Some mutation was presumed in the sample. Further, even in the primer set 2 (8) and the primer set 3 (9) in which the target search range (6) is narrowed, some mutation is estimated in the same sample.
Not only a change in the value but also a clear change in the Tm pattern was observed. Therefore, primer set 3 in the target search range (6)
The presence of the mutation was estimated in the range of (9), and a two-base mutation was confirmed in the sequence of this portion. This confirmed that primer set 1 (7) and primer set 2 (8) had a larger amount of base than primer set 3 (9), so that the effect of the two-base mutation on Tm was small. From the above results, when the primer set 3 (9) was used, it was possible to detect the base mutation in this portion as SNPs from the Tm value and the Tm pattern.

In the present embodiment, a direct PCR buffer for direct nucleic acid amplification by direct PCR without extracting DNA from a sample was manufactured by Shimadzu Corporation.
It is also possible to use mpdirect, in which case 1 μl of whole blood was used directly as a sample.

In this embodiment, the Tm value and Tm pattern obtained from an unknown sample are compared with the existing data by digitizing the Tm value and Tm pattern of each sample as existing data and installing the digitized data in a computer. Thus, a function for directly indicating the possibility of nucleic acid mutation has been added. Further, the present invention includes a nucleic acid mutation display method applying this principle.

[0034]

Example 2 Detection of a known mutation was performed using the human genome Homo.
Sapiens ABO Glycosyltran
sferase (ABO) Gene Exon 7: A
F182746-AF182756 (Genbank:
Accession Number), the known mutation is applied to the gene sequence registered in T
The ABO blood type was detected by comparing the m value and the Tm pattern.

After extracting human genomic DNA from whole blood using Isogen-LS of Nippon Gene as an extraction reagent according to a conventional method, a reaction solution was prepared according to the prescription shown in Table 1, 1 μg of each genomic DNA and primers of the following base sequence Used set. Primer set, sense primer: 5'-TGGAGACGGGCGGA
GAAGCAC-3 '(SEQ ID NO: 7), antisense primer: 5'-CCCCCAGGTA
GTAGAAATCG-3 ′ (SEQ ID NO: 8), PCR is a real-time PCR quantifier Gen manufactured by ABI
95 ° C / 5 seconds, 55 ° C / 1 using eAmp5700
0 seconds, 72 ° C./20 seconds were performed according to a protocol of 40 cycles. Examination of ABO blood type, A type, B type, AB
Six samples were analyzed for each of the four types of blood group, type O and type O. Table 3 shows the obtained results.

[0036]

[Table 3]

From the results in Table 3, each blood type could be easily discriminated from the Tm value and the characteristics of the Tm pattern by using this method.

In the present example, Amide manufactured by Shimadzu Corporation was used as a direct PCR buffer when nucleic acid amplification was performed directly by PCR without extracting DNA from the sample.
pdirect can be used. In this case, 1 μl of whole blood was directly used as a sample.

In this embodiment, the Tm value and Tm pattern of each blood group are digitized as existing data and installed in a computer, so that the Tm value and Tm pattern obtained from an unknown sample can be converted to existing data. A function was added to directly display the blood type by comparing it with.
Further, the present invention includes a nucleic acid mutation display method applying this principle.

[0040]

Example 3 Detection of a known mutation using a plurality of primers according to claim 11 is performed using the human genome WO011092.
(Genbank: Accession Number
r) using the present invention to determine whether or not an unknown mutation exists in the gene sequence registered in r).
Was performed by comparing the patterns.

After extracting human genomic DNA from whole blood using Isogen-LS of Nippon Gene as an extraction reagent in accordance with a conventional method, a reaction solution was prepared according to the prescription shown in Table 1, 1 μg of each genomic DNA and a primer having the following base sequence: Used set. Primer set, sense primer: 5'-ATATCCCAAAAAG
TTATGATT-3 ′ (SEQ ID NO: 9), Sense primer: 5′-CCCAAAAGTTTTAT
GATTGAAC-3 ′ (SEQ ID NO: 10), Sense primer: 5′-TACTTGGTGGCC
TCCGAAGA-3 ′ (SEQ ID NO: 11), Antisense primer: 5′-GGACCACAC
TTTTCAGT-3 ′ (SEQ ID NO: 12) The positional relationship between the primers is as shown in FIG. PCR is ABI real-time PCR quantifier G
95 ° C / 5 seconds, 55 ° C using eneAmp5700
/ 10 sec, 72 ° C./20 sec according to a protocol of 40 cycles. The analysis was performed using five normal samples and five mutant samples as samples. Table 4 shows the obtained results. In the table,
The combination of a sense primer and an antisense primer was referred to as a primer set, the sense primer as a primer set, and the sense primer as a primer set.

[0042]

[Table 4]

As shown in FIG. 3, the number of bases of the PCR product
The order of primer set> primer set> primer set showed that the effect of base mutation on each Tm value was in the order of primer set <primer set <primer set.
Further, both the Tm value and the Tm pattern change continuously.
Looking at this change showed the presence of the mutation more clearly. Even if the mutation is unknown, it is highly possible that some mutation is present if such a primer set is assembled and a specific continuous change is observed.Therefore, the present invention is a useful method for screening. found.

In the present embodiment, Amide manufactured by Shimadzu Corporation is used as a direct PCR buffer for directly amplifying nucleic acids by PCR without extracting DNA from a sample.
It is also possible to use pdirect. In this case, PCR was performed using 1 μl of whole blood directly as a sample.

In this embodiment, the Tm value and Tm
Patterns and their changes are digitized as existing data, and installed on a computer, Tm values and Tm patterns obtained from unknown specimens are compared with existing data to directly display the possibility of mutation. Added the function to perform The present invention includes a display method applying this principle.

[0046]

Industrial Applicability The present invention relates to a method for detecting an unknown or known nucleic acid base sequence mutation using a difference in temperature at which a double-stranded nucleic acid is heated and dissociated into a single-stranded nucleic acid, and a display method. The base mutation can be easily detected from the double-stranded nucleic acid dissolution curve of the sample and the pattern of the double-stranded nucleic acid dissociation curve obtained by differentiating the curve. By using the method of the present invention, it is possible to detect a base mutation without acquiring a high level of technology as compared with a conventional detection method and without using an expensive apparatus. Further, the present invention relates to ABO
Blood type, HLA (Human Leukocyte)
Antigen) Typing and diabetic nephropathy-related SN
Ps (Diabetical Nephropathy R)
eluted Single Nucleotide
Polymorphism) can also be used.

[0047]

[Sequence list]

SEQUENCE LISTING <110> Joji Oshima <120> Unknown or Known Nucleic Acid Mutation Detection and Display Method Using Nucleic Acid Dissolution Curve and Nucleic Acid Dissociation Curve <130> OJ010427 <141> 2001-4-27 <160> 12 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 tttcctcct gccccttgta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2ga tagaga tagaga tagaga tagag > 20 <212> DNA <213> Artificial Sequence <400> 3 ctttttca atactcaca 20 <210><211> 18 <212> DNA <213> Artificial Sequence <400> 4 ggtttgctag atgtaaat 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5a acaca acaca acaca acaca acaca > 20 <212> DNA <213> Artificial Sequence <400> 6 agtaactacta ccgtgattct 20 <210> 7 <211> 20 <212> DNA <213> ArtificialSequence <400> 7ggaggacg20ggg20g <212> DNA <213> Artificial Seq ence <400> 8 cccccaggta gtagaaatcg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 atcccaaa agtattatatt 20 <210><12> 11 <2> 10 <11> 2. 400> 10 cccaaaagtt atgattgaac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 tacttggtgg cctccgaaga 20 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <400> 12 ggacaccacac ttttca gt 18

[Brief description of the drawings]

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is an example of a double-stranded nucleic acid dissolution curve shown as a relationship between fluorescence intensity and temperature.

FIG. 2 is an example of a double-stranded nucleic acid dissociation curve obtained by differentiating a double-stranded nucleic acid dissolution curve.

FIG. 3 is a schematic diagram showing locations of nucleic acid mutations and arrangement of primers.

FIG. 4 is a schematic diagram showing the positional relationship of a primer set when detecting an unknown nucleic acid mutation.

[Explanation of symbols]

 1 base mutation 2 antisense primer 3-5 sense primer 6 target search range 7-9 primer set

Claims (16)

[Claims] 1. A method for detecting an unknown nucleic acid mutation, which comprises extracting genomic DNA from a sample, amplifying it by real-time quantitative PCR, and denaturing the amplified double-stranded nucleic acid into a single-stranded nucleic acid when heated. Nucleic acid dissolution curve (Mel
t Curve) or a double-stranded nucleic acid dissociation curve (Dissociation Cu
rve) for each sample to estimate an unknown mutation in genomic DNA. 2. A method for detecting an unknown nucleic acid mutation, wherein genomic DNA is amplified from a sample by a nucleic acid amplification method, and the temperature at which the amplified double-stranded nucleic acid is denatured into a single-stranded nucleic acid when heated is observed. It is characterized by comparing a double-stranded nucleic acid dissolution curve (Melt Curve) or a double-stranded nucleic acid dissociation curve (Dissociation Curve) obtained by differentiating the curve for each sample to estimate an unknown mutation of genomic DNA. Nucleic acid mutation detection method. 3. A method for detecting a mutation of an unknown nucleic acid, which comprises directly extracting genomic DNA from a sample and observing a temperature at which the double-stranded nucleic acid is denatured into a single-stranded nucleic acid when heated. Curve (Melt Curve) or a double-stranded nucleic acid dissociation curve (Dissocia obtained by differentiating the curve)
Tion Curve) for each sample
A method for detecting a nucleic acid mutation, comprising estimating an unknown mutation of NA. 4. A method in which a genomic DNA is extracted from the sample without using a direct PCR buffer or the like and directly amplified by a real-time quantitative PCR method, and then denatured to a single-stranded nucleic acid when the amplified double-stranded nucleic acid is heated. Double-stranded nucleic acid dissolution curve (Melt Curve) obtained by observing the temperature at which the double-stranded nucleic acid dissociates, or a double-stranded nucleic acid dissociation curve (D
(Issociation Curve) for each sample to estimate an unknown mutation in genomic DNA. 5. A method in which a genomic DNA is extracted from the sample without using a direct buffer or the like, and after amplification by a nucleic acid amplification method, the temperature at which the amplified double-stranded nucleic acid is denatured into a single-stranded nucleic acid when heated. The observed double-stranded nucleic acid dissolution curve (M
elt Curve) or a double-stranded nucleic acid dissociation curve (Dissociation) obtained by differentiating the curve.
Curve) for each sample to estimate an unknown mutation in genomic DNA. 6. A double-stranded nucleic acid dissolution curve obtained by observing a temperature at which a double-stranded nucleic acid is denatured into a single-stranded nucleic acid when a double-stranded nucleic acid is heated, without extracting genomic DNA from the sample, without using a direct buffer or the like. Melt Curve) or a double-stranded nucleic acid dissociation curve (Di) obtained by differentiating the curve.
A method for detecting a nucleic acid mutation, comprising: comparing an S.S.C. 7. A method for detecting a known nucleic acid mutation, which comprises using the method according to any one of claims 1 to 6 to dissolve the double-stranded nucleic acid of a mutant genomic DNA (Melt Cu
rve) or a double-stranded nucleic acid dissociation curve (Dissociation Curve) obtained by differentiating the curve.
A method for detecting a nucleic acid mutation, comprising: preparing an experimental mutation in advance, and estimating a known mutation in genomic DNA from the comparison. 8. A method for detecting an unknown or known nucleic acid mutation, wherein the method for detecting a mutation in an artificially synthesized DNA using the method according to any one of claims 1 to 7 is a double-stranded DNA. Nucleic acid dissolution curve (Melt Curve) or a double-stranded nucleic acid dissociation curve (Disso
(Circulation Curve). 9. A method for detecting an unknown or known nucleic acid mutation, wherein the method according to any one of claims 1 to 8 is used to perform artificial transcription from RNA by a reverse transcription method (such as an RT-PCR method). The detection of mutations in DNA that has been chemically synthesized is detected by a double-stranded nucleic acid dissolution curve (Melt Curve) or a double-stranded nucleic acid dissociation curve (Dissocia) obtained by differentiating the curve.
a method for detecting a nucleic acid mutation, which is inferred from a comparison with T.C. 10. A method for detecting an unknown or known nucleic acid mutation, which comprises detecting two unexpected nucleic acids such as contaminating DNAs and unknown genes using the method according to any one of claims 1 to 7. Strand nucleic acid dissolution curve (Melt Curve) or a double-stranded nucleic acid dissociation curve (D
A nucleic acid mutation detection method characterized by estimating from a comparison with an isolation curve. 11. A method for detecting an unknown or known nucleic acid mutation, wherein the method according to any one of claims 1 to 8 further comprises several kinds of one primer (for example, a sense primer) and the other Using one type of primer (eg, antisense primer), a double-stranded nucleic acid dissolution curve (Melt Curve) or a double-stranded nucleic acid dissociation curve (Dissociatio) obtained by differentiating the curve
n Curve). 12. The method according to any one of claims 1 to 11, wherein a single base mutation, base deletion, base insertion,
Nucleic acid mutations such as microsatellite mutations are analyzed by double-stranded nucleic acid dissolution curve (Melt Curve) or double-stranded nucleic acid dissociation curve (Dissoc) obtained by differentiating the curve.
a method for detecting a nucleic acid mutation, which is inferred from a comparison with a mutation curve (i. 13. A method for comparing a mutation detected using the method according to any one of claims 1 to 12 with known gene information obtained in advance, and directly displaying a result of the determination. Characteristic nucleic acid mutation detection display method. 14. A method for detecting and displaying nucleic acid mutations, wherein a result detected by using the method according to any one of claims 1 to 12 is used for determination of an ABO blood type. 15. The method of claim 1, wherein the result of detection using the method according to claim 1 is HLA (Human).
Leukocyte Antigen) A method for detecting and displaying nucleic acid mutations, which is used for determining typing. 16. A method for detecting diabetic nephropathy-related S using a result detected using the method according to any one of claims 1 to 12 above.
NPs (Diabetical Nephropathy)
Related Single Nucleotide
(Polymorphism), which is used for nucleic acid mutation detection and display.