JP2002291500A - Method for detecting mutation of nucleic acid using nucleic acid melt curve and nucleic acid dissociation curve - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for quickly, easily and inexpensively detecting the mutation of a nucleic acid. SOLUTION: This method for easily detecting the mutation of a nucleic acid in a short time comprises heating a double-stranded DNA or an amplified product thereof and analyzing the difference of the temperature (Tm value) at which it is dissociated into a single-stranded DNA and the characteristics of the difference. In this method, by preparing the Tm value and the pattern of wild type (normal type) in advance, it is possible to detect not only the mutation type but also estimate a nonspecific reaction or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for heating a double-stranded DNA and dissociating the double-stranded DNA into single strands based on the difference in temperature and the characteristics thereof.
The present invention relates to a DNA discrimination method for estimating a mutation of a single-stranded DNA.

[0002]

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

[0003] As a method for detecting a mutation in a base sequence, the 3 'end of a PCR primer is intentionally placed at a portion where a base mutation is present, and a PCR-based method is used to determine whether or not PCR is performed.
ASP (Allele Specific Prime)
r) method or a PCR-RFLP (Res) in which a mutated portion is amplified by PCR and discriminated based on whether it can be cleaved with a restriction enzyme.
trickion Fragment Length
(Polymorphism) method.

[0004] In addition, PCR-SSCP (Single), which performs electrophoresis without intentionally denaturing a PCR product and detects a change in electrophoretic mobility caused by a difference in three-dimensional structure caused by the mutation.
Strand Conformation Poly
Morphism) method, and a Taqman probe method in which a mutated portion is further detected with a fluorescent-labeled probe are also widely used.

In recent years, in addition to the above-mentioned methods, a method of directly detecting all nucleotide sequences, a method of detecting a mutation in a nucleotide sequence of a gene by using a method such as a DNA chip method and the like has also been carried out.

[0006] Variations in the length of the base sequence have been similarly performed by various methods for detecting microsatellite.
The PCR-RFLP method, the PCR-SSCP method, the sequencing method and the like are known as typical methods.

[0007]

All of the nucleic acid mutation detection methods that have been carried out so far require skilled techniques, and the detection requires a considerable amount of time and cost. The only method that uses the real-time quantitative PCR method is the TaqMan probe method, which enables rapid detection of nucleic acid mutations.However, fluorescent labeled probes are expensive, cannot cope with unknown mutations, There are drawbacks such as the need for advanced technology for design.

[0008] As described above, each of the methods is a technology and
At present, it is not a detection method that satisfies all of cost and time, that is, a method for detecting a mutation of a nucleic acid in a short time and at low cost without requiring advanced technology has been developed.

[0009]

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

To explain the principle of the present invention, when a temperature is applied to a double-stranded DNA PCR product in a specimen, each of them dissociates into single-stranded DNA. This rapidly dissociating temperature point is defined as Tm (Melting Temperature).
The properties such as dissociation temperature and speed are different in the base sequence of double-stranded DNA and the binding strength between bases adenine (A) and thymine (T) and between guanine (G) and cytosine (C). This depends on the content of each base A, G, C, T. In addition, the temperature and speed of dissociation also depend on the length of the PCR product, and thus vary depending on the difference in the number of microsatellite repeats. Therefore, if the Tm value or Tm pattern of the wild type (normal type) is prepared in advance, the presence of the mutant type (abnormal type) can be detected by comparing the Tm value or Tm pattern of the sample DNA. It is possible.

The principle of the invention will be described with reference to the drawings as shown in FIGS. FIG. 1 is a nucleic acid dissolution curve in which temperature is plotted on the horizontal axis and fluorescence intensity is plotted on the vertical axis. For example, when temperature is applied to three types of PCR products A, B and C, 2
The fluorescence intensity gradually decreases due to the dissociation of single-stranded DNA into single-stranded DNA at a certain temperature, but the pattern of this decrease depends on the GC content, length, three-dimensional structure, etc. in the PCR product. Defined and each has its own curve. Here, the temperature at which the dissociation proceeds rapidly is called the dissociation temperature (Tm value).

The nucleic acid dissociation curve shown in FIG. 2 is obtained by differentiating FIG. In this figure, the Tm value is displayed as a peak, so that the difference between the curves becomes more apparent. Since each PCR product has its own dissociation curve, the principle of the present invention is to detect a nucleic acid mutation by comparing it with a dissociation curve of a normal nucleic acid which has been experimentally prepared in advance.

[0013] The present invention provides a method for preparing a genomic DN from a collected specimen.
A is extracted and a DNA dissolution curve or a DNA dissociation curve,
Or a direct PCR buffer containing a component that destroys a sample, a component that suppresses an enzyme reaction inhibitor, and the like in addition to a method for obtaining a Tm value by preparing a dissolution curve or a dissociation curve of a nucleic acid amplification product by performing nucleic acid amplification The present invention is also effective in a method of performing nucleic acid amplification by directly performing PCR without extracting DNA from a sample by using such a method.

[0014] Ampdirect from Shimadzu Corporation is useful as a direct PCR buffer when nucleic acid amplification is performed by direct PCR without extracting DNA from the sample, but the present invention is not limited to this.

[0015]

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.

[0016]

[Table 1]

A total of 50 μl including the sample DNA of the sample
The reaction solution shown in Table 1 was thoroughly stirred, and a total volume of 50 μl was transferred to a real-time PC manufactured by ABI.
The PCR is started by setting the R quantifier GeneAmp5700. The PCR protocol was 98 ° C / 1 second, 55
C./10 seconds and 72.degree. C./10 seconds are performed for 40 cycles.

After completion of the PCR, the DNA dissolution curve (Melt
Curve) and DN obtained by differentiating the curve.
A dissociation curve (Dissociation Curve)
These are compared with a wild-type DNA dissolution curve and a DNA dissociation curve, which have been experimentally prepared in advance, to detect the presence or absence of a mutation in the sample DNA. In addition, real-time PC
The R quantifier is not limited, and any product may be used. In addition, the PCR protocol is appropriately changed and performed according to the primers used.

[0019]

Example 1 Human Glyceraldehyde-3-
Phosphate Dehydrogenase (G
Assuming that 3PDH) was a wild type and mouse G3PDH was a mutant type, the Tm values and Tm patterns of both were compared.

Human and mouse genomic DNAs were extracted according to a conventional method using Isogen-LS of Nippon Gene Co., Ltd. as an extraction reagent, and the reaction solutions were prepared according to the prescription shown in Table 1. Primer 1: Primer 1: 5'-ACCACAGTCCATGCCC
ATCAC-3 '(base sequence 1), Primer 2: 5'-TCCACCACCCTGTTG
CTGTA-3 '(base sequence 2), using a real-time PCR quantifier Ge manufactured by ABI
98 ° C./1 second, 55 ° C./10 seconds, 72
PCR was performed according to a protocol of 40 cycles at / 10 ° C. The examination was performed six times for each sample, and the obtained results are shown in Table 2.

[0021]

[Table 2]

Table 2 shows that the normal human (wild-type) Tm
One peak is observed at around 90.5 ° C., but two peaks are observed in the Tm value of the mouse (mutant type), and the peak temperatures are clearly different from those of the human (wild type). This indicates that this nucleic acid is mutated.

[0023]

[Example 2] Human G3PDH was used as a wild type, whereas an excessive amount of magnesium chloride (12 µl: final 6 mM) was used.
Was used as a non-specific nucleic acid amplification model, and the Tm values and Tm patterns of both were compared. However,
In the non-specific nucleic acid amplification model, the total amount was adjusted with water with respect to the increased amount of magnesium chloride.

After extracting human genomic DNA 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, and 1 μg of each genomic DNA and the above-mentioned nucleotide sequences 1 and 2 were prepared. ABI real-time PCR quantifier Ge using primers
98 ° C./1 second, 55 ° C./10 seconds, 72
PCR was performed according to a protocol of 40 cycles at / 10 ° C. The examination was performed six times for each sample, and the obtained results are shown in Table 3.

[0025]

[Table 3]

From the results in Table 3, magnesium chloride was subjected to PCR.
In a model in which non-specific nucleic acid amplification was performed by adding an excess amount to the reaction solution, a nucleic acid dissociation curve clearly different from that of a normal human (wild type) Tm value was shown, and mutations could be easily detected.

[0027]

Example 3 Human G3PDH was used as a wild type, and an excess amount of primer (10 μl each: final 100 pmo)
l) was added to the reaction solution, and the resulting mixture was used as a primer dimer formation model to compare the Tm values and Tm patterns of the two. Table 4 shows the results of performing real-time PCR in the same manner as in Example 2.

[0028]

[Table 4]

From Table 4, when the primer is excessively added to the PCR reaction solution to form a primer dimer, a peak is observed at a temperature different from the Tm value in a normal case. This difference is due to the difference in the binding force between A = T and G = C in DNA, and the primer dimer formation model dissociates at a lower temperature due to the lower G = C content. From this, the mutation can be easily detected.

[0030]

According to the present method, the Tm value or T
By simply comparing the patterns of m, it is possible to easily detect both known nucleic acid mutations, base substitution mutations and base number mutations in a short time and at low cost. Furthermore, unknown nucleic acid mutations can be estimated by grouping patterns. In addition, when there is any problem in the nucleic acid amplification process, such as when a foreign gene is mixed, when a dimer is formed on the primer, when nonspecific nucleic acid amplification is performed, the Tm value or Because a change is seen in the pattern of Tm,
It is also possible to determine whether nucleic acid amplification has been performed normally.
Table 5 shows a comparison of the present invention with the conventional method.

[0031]

[Table 5]

As shown in Table 5, it is clear that the method of the present invention is superior to the conventional method such as the PCR-ASP method in the detection time and the detection cost, and does not require special techniques.

[0033]

[Sequence list] SEQUENCE LISTING <110> Joji Oshima <120> Nucleic acid mutation detection method using nucleic acid dissolution curve and nucleic acid dissociation curve <130> OJ010329 <141> 2001-3-29 <160> 2 <210> 1 <211 > 20 <212> DNA <213> Artificial Sequence <400> 1 accacagtcc atgccatcac 20 <210> 2 <211> DNA <213> Artificial Sequence <400> 2

[Brief description of the drawings]

FIG. 1 is an example of a nucleic acid dissolution curve represented by a relationship between a fluorescence intensity and a temperature.

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

Claims (12)

[Claims] 1. A method for detecting mutation of a nucleic acid, comprising extracting genomic DNA from a sample, amplifying the extracted genomic DNA by real-time quantitative PCR, and experimentally preparing a PCR product dissolution curve using wild-type genomic DNA. Melt Curve),
Alternatively, a nucleic acid mutation detection method characterized by estimating a mutation in genomic DNA from a comparison with a PCR product dissociation curve (Dissociation Curve) obtained by differentiating the curve. 2. A method for detecting mutation of a nucleic acid, comprising the steps of: amplifying genomic DNA from a sample by a nucleic acid amplification method;
Nucleic acid amplification product dissolution curve (Melt Curve) that has been experimentally prepared in advance using, or a nucleic acid amplification product dissociation curve (Dissocia) obtained by differentiating the curve.
A method for detecting a nucleic acid mutation, comprising estimating a genomic DNA mutation from a comparison with the T.C. 3. A method for detecting a mutation of a nucleic acid, wherein genomic DNA is directly extracted from a sample, and a DNA melting curve (M
elt Curve) or a DNA dissociation curve (Dissociation Cu) obtained by differentiating the curve.
a method for detecting a mutation in a nucleic acid, comprising estimating a mutation in genomic DNA from comparison with rve). 4. A method for directly amplifying wild-type genome D by real-time quantitative PCR using a direct PCR buffer or the like without extracting genomic DNA from the sample.
A PCR product dissolution curve (Melt Curve) experimentally prepared in advance using NA, or a PCR product dissociation curve (Dissocia) obtained by differentiating the curve.
A method for detecting a nucleic acid mutation, comprising estimating a genomic DNA mutation from a comparison with the T.C. 5. A method for dissolving a nucleic acid amplification product which has been amplified by a nucleic acid amplification method using a direct buffer or the like without extracting genomic DNA from the sample, and which has been experimentally prepared in advance using wild-type genomic DNA. Curve (Melt Cu
rve) or a nucleic acid amplification product dissociation curve (Dissociation Curv) obtained by differentiating the curve.
A method for detecting a nucleic acid mutation, comprising estimating a mutation in genomic DNA from the comparison with e). 6. A DNA which has been experimentally prepared in advance using wild-type genomic DNA in combination with a direct buffer or the like without extracting genomic DNA from the sample.
Melt curve or DNA dissociation curve (Dissociat) obtained by differentiating the curve.
(ion Curve), and a method for detecting a mutation in a nucleic acid, comprising estimating a mutation in genomic DNA based on comparison with ionic curve. 7. The method according to any one of claims 1 to 6, wherein detection of mutants having different lengths, such as microsatellite, is performed using a DNA melting curve (MeltCurv).
e) or a nucleic acid mutation detection method comprising estimating a DNA mutation from a comparison with a DNA dissociation curve (Dissociation Curve) obtained by differentiating the curve. 8. The method according to any one of claims 1 to 6, wherein the detection of a mutant having a different base sequence is carried out by DN.
A dissolution curve (Melt Curve) or a DNA dissociation curve (Dissociia) obtained by differentiating the curve.
A method for detecting a nucleic acid mutation, comprising estimating a DNA mutation based on comparison with the T. curve. 9. The method according to any one of claims 1 to 8, wherein the detection of the mutation of the artificially synthesized DNA is determined by differentiating the DNA melting curve (Melt Curve) or the curve. The obtained DNA dissociation curve (Disso
(Circulation Curve). 10. A reverse transcription method (RT-PCR) from RNA using the method according to any one of claims 1 to 8 above.
Detection of DNA mutations artificially synthesized by the
A dissolution curve (Melt Curve) or a DNA dissociation curve (Dissociia) obtained by differentiating the curve.
a method for detecting a nucleic acid mutation, which is inferred from a comparison with T.C. 11. The use of the method according to any one of claims 1 to 8, wherein an unexpected nucleic acid amplification product resulting from a nonspecific reaction caused by an exogenous factor such as contaminating DNA is obtained. Detection was performed using a DNA melting curve (Melt Curve) or a DNA dissociation curve (Diss) obtained by differentiating the curve.
and a nucleic acid mutation detection method characterized by estimating from a comparison with S.C. 12. A method for forming a primer dimer and a template DNA using the method according to any one of claims 1 to 8.
Detection of unexpected nucleic acid amplification products resulting from non-specific reactions caused by endogenous factors, such as non-specific reactions,
DNA melting curve (Melt Curve) or a DNA 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.