JP2010104360A - Method for judging gene polymorph - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for judging gene polymorph, capable of accurately judging the mating type of polymorph part without concerning to the DNA amount. <P>SOLUTION: The method for judging the mating type of the gene polymorph comprises performing invader reaction generating a first fluorescence derived from a first oligonucleotide and a second fluorescence derived from a second oligonucleotide by mixing the target DNA with the first oligonucleotide complementary to the target sequence containing a first allele composing the gene polymorph of the target DNA and the second oligonucleotide complementary to the target sequence containing a second allele forming the gene polymorph and controlling the temperature, approximating the curves representing temporal changes of the first and second fluorescence intensities at the predetermined judging section after the reaction start are approximated by quadratic equations, respectively, and using the ratio of the secondary coefficient of the first and second secondary approximation formulae as the judging index, or performing secondary differentiation of each of the curves representing temporal changes of the first and second fluorescence intensities and using the ratio of the maximum values of the first and second secondary differentiation values as the judging index. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for determining a genetic polymorphism of genomic DNA, and more particularly to a method for determining a genetic polymorphism using an invader reaction.

  A gene polymorphism is an individual difference in the base sequence of DNA constituting a gene, and is generally defined to occur at a frequency of 1% or more of a population. In addition to single nucleotide polymorphism (SNP: single nucleotide polymorphism) in which only one base of a DNA base sequence is mutated, the gene polymorphism is a microsatellite polymorphism that is the difference in the number of repeating units of about 2 to 4 bases ( microsatellite polymorphism), base deficiency, insertion. It is known that some gene polymorphisms affect the susceptibility to illness and drug metabolism. The base of the SNP site is used for diagnosing the disease morbidity, the effect of the administered drug, and predicting side effects. Is being determined.

One method for determining genetic polymorphism is to use an invader reaction. In the invader reaction, for example, when judging the single nucleotide polymorphism junction type, two types of oligonucleotides (allele oligos) that recognize the nucleotide sequence of the site where the single nucleotide polymorphism occurs (SNP site) are subject to determination at the SNP site. Constructs an SNP comprising an allele oligo hybridized to a gene and an oligonucleotide (invader oligo) that invades between the gene to be judged, an enzyme that recognizes and cleaves the structure where the oligonucleotide overlaps (invasion structure) (Crybase: registered trademark) An invader reaction reagent containing two types of fret probes (FRET ^™ probes) containing different fluorescent substances corresponding to two types of alleles is used.

  When the invader reaction reagent is mixed with DNA containing the SNP to be determined and the invader reaction is performed, a fluorescence signal is generated depending on the presence or absence of the SNP corresponding to each allele oligo. By detecting the intensity of the fluorescence signal with a fluorescence detector, it is possible to determine the presence or absence of an SNP corresponding to each allele oligo and whether the SNP is a homozygote or a heterozygote (Patent Document 1). , See Patent Document 2).

Japanese Patent Laid-Open No. 2002-300894 WO 2006/106867 A1

  The reaction reagent contains an excessive allele oligo or fret probe, and the invader reaction is repeated by the excessive allele oligo or the like, thereby amplifying the fluorescence signal. Therefore, the fluorescence signal intensity detected by the fluorescence detector gradually increases from the start of the reaction and eventually reaches a plateau. For this reason, conventionally, the SNP is determined from the fluorescence signal intensity when a specific time has elapsed from the start of the reaction, the time until the fluorescence signal intensity reaches the plateau, the fluorescence signal intensity at that time, and the like.

However, the fluorescence signal intensity at a specific time varies depending on various factors such as a difference in DNA amount and invader reaction efficiency. In addition, the arrival of the fluorescence signal intensity at the plateau is determined from the change in signal intensity per unit time (the slope of the signal intensity curve). Is likely to occur. If the invader reaction time is increased, the plateau arrival time can be accurately determined regardless of the amount of DNA, but in this case, the time required for determination of SNP is increased.
In addition, fluorescent signals corresponding to alleles not present in the DNA to be judged should not be detected originally, but the intensity of fluorescent signals corresponding to alleles not present in the DNA to be judged may increase due to non-specific reactions. is there. In such a case, if the SNP determination is performed based on the fluorescence signal intensity at a specific time, there is a high possibility of erroneous determination.

  The problem to be solved by the present invention is to provide a gene polymorphism determination method capable of accurately determining the polymorphic site junction type regardless of the amount of DNA to be determined.

The first form of the genetic polymorphism determination method according to the present invention, which has been made to solve the above-mentioned problems, is to complement a target DNA with a target sequence comprising a first allele constituting the genetic polymorphism of the target DNA. Derived from the first oligonucleotide by controlling the temperature by mixing with a second oligonucleotide complementary to the target sequence containing the target first oligonucleotide and the second allele constituting the gene polymorphism Performing an invader reaction for generating first fluorescence and second fluorescence derived from the second oligonucleotide, and determining the allele junction type of the gene polymorphism from the first and / or second fluorescence intensity A method,
Curves representing temporal changes in the first and second fluorescence intensities in a predetermined determination section after the start of the invader reaction are approximated by a quadratic expression, respectively, and the second order of the first and second quadratic approximate expressions. It is characterized in that a genetic polymorphism junction type is determined using a coefficient ratio as a determination index.

The second form of the gene polymorphism determination method of the present invention is the first oligonucleotide complementary to the target sequence containing the first allele constituting the gene polymorphism of the target DNA and the target oligonucleotide By mixing with a second oligonucleotide complementary to a target sequence containing a second allele that constitutes a gene polymorphism and controlling the temperature, the first fluorescence derived from the first oligonucleotide and the second Performing an invader reaction that generates second fluorescence derived from the oligonucleotide of the above, and determining the allelic junction type of the gene polymorphism from the first and / or second fluorescence intensity,
The curves representing the time-dependent changes in the first and second fluorescence intensities in the predetermined determination section after the start of the invader reaction are respectively second-order differentiated, and the ratio of the maximum values of the first and second second-order derivative values. It is characterized in that a genetic polymorphism junction type is determined using as a determination index.

In this case, the determination index of the target DNA may be normalized with the determination index of the internal standard DNA, and the genetic polymorphism mating type may be determined based on the normalized determination index. Here, the internal standard DNA refers to a DNA having a polymorphic site that is a polymorphic site different from the polymorphic site to be determined and is heterozygous.
In addition, it is preferable that the end of the determination interval is a time when the first-order differential value of the curve representing the temporal change in the first and second fluorescence intensities is maximized.

By the way, the invader reaction is performed by mixing an invader reaction reagent and a sample containing a target DNA, and maintaining this mixed solution at an optimum temperature for the invader reaction. The fluorescence intensity has temperature dependence, and generally the fluorescence intensity decreases as the temperature increases. For this reason, conventionally, a point at which the slope is equal to or less than a preset threshold value is obtained from a fluorescence intensity curve including the fluorescence intensity before reaching the optimum temperature of the invader reaction, and is used as the starting point of the determination section.
However, since the invader reaction actually starts when it approaches the optimum temperature, the change in the fluorescence intensity near the reaction start point also depends on the amount of target DNA in the mixture. The amount of target DNA in the mixture varies depending on the target DNA concentration in the sample, the invader reaction reagent, the accuracy of sample dispensing, and the like. For this reason, it is difficult to simply determine the start point of the determination section from the comparison between the slope of the fluorescence intensity curve and the threshold value.
Therefore, in the gene polymorphism determination method of the present invention, the time when the reaction system of the invader reaction that generates the first and second fluorescence intensities reaches a predetermined reaction temperature is set as the beginning of the determination section. Here, the predetermined reaction temperature refers to a temperature at which the invader reaction actually starts, and can be set to an optimum temperature for the invader reaction or an appropriate temperature in the vicinity of the optimum temperature. According to such a method, it is possible to accurately determine the start period of the determination interval regardless of the target DNA concentration, dispensing accuracy, and the like.

  According to the gene polymorphism determination method of the present invention, even when the amount of target DNA is small and the rise of the invader reaction is poor, the mating type of the gene polymorphism can be determined based on the determination index reflecting the DNA amount. Further, the ratio of the secondary coefficients obtained from the curves representing the time-dependent changes in the first and second fluorescence intensities corresponding to the target sequences including the first and second alleles constituting the gene polymorphism of the target DNA, or Since the ratio of the maximum value of the secondary differential values is used as the determination index, the influence of the change in the fluorescence intensity due to the nonspecific reaction on the determination index can be reduced. Therefore, more accurate determination can be performed.

Schematic of the invader reaction according to the present invention. Schematic of the Invader Plus method. The figure which shows the time-dependent change of the fluorescence intensity by two types of label | marker fluorescence. The figure which shows the time-dependent change of the 1st derivative value of the fluorescence signal intensity curve of the allele 1 with the time-dependent change of the fluorescence signal intensity. The figure which shows the time-dependent change of the second derivative value of the fluorescence signal intensity curve of allele 1 with the time-dependent change of the fluorescence signal intensity. The figure which shows the time-dependent change of the 1st derivative value of the fluorescence signal intensity curve of the allele 2 with the time-dependent change of the fluorescence signal intensity. The figure which shows the time-dependent change of the second derivative of the fluorescence signal intensity curve of the allele 2 with the time-dependent change of the fluorescence signal intensity. The table | surface which shows the relationship between the determination parameter | index R (i) with respect to a determination result, and a threshold value. The figure which shows the relationship between the junction type of VKORC1 1173 C> T which is one of the warfarin related SNPs, and the determination index. The figure which shows the relationship between the joining type | mold of CYP2C9 * 3 which is one of warfarin related SNP, and a determination parameter | index. The figure which shows the example of a measurement of the time-dependent change of the fluorescence signal intensity after the start of the invader reaction in the case of performing the type determination of the gene polymorphism of the target DNA by the invader plus method. The figure which shows the other example of a measurement of the time-dependent change of the fluorescence signal intensity after the start of the invader reaction in the case of carrying out type determination of the gene polymorphism of the target DNA by the invader plus method.

Hereinafter, embodiments of the present invention will be described.
In the genetic polymorphism determination method of the present invention, an invader reaction is used. A method for determining a gene polymorphism using an invader reaction is disclosed in detail in JP-A-2002-300894, JP-A-2005-160481, and the like.

  The invader reaction will be described by taking the genetic polymorphism at the 1173 position in the intron 1 region of VKORC1, which has been reported to have a significant effect on the sensitivity of the oral anticoagulant warfarin as an example. FIG. 1 shows a schematic diagram of the invader reaction, and the explanation will be made based on this figure.

  The 1173th position in the intron 1 region of VKORC1 is C / C homozygous in the wild type, whereas C / T heterozygous and T / T homozygous are known as mutant types. . Therefore, the alleles composing the 1173rd gene polymorphism are C and T. In the invader reaction, the first oligonucleotide 41a complementary to the target sequence 31a containing the allele C of the target DNA 11 (hereinafter sometimes referred to as the first allele oligo) and the target sequence 31b containing the allele T of the target DNA 21 are complementary. The second oligonucleotide 41b (hereinafter sometimes referred to as the second allele oligo) selectively hybridizes 5 'downstream including the polymorphic sites of the target DNAs 11 and 21. Subsequently, invader oligos 51a and 51b having a non-specific single base at the polymorphic site hybridize to the 3 ′ upstream side including the polymorphic site of the target DNA. The formed triple chain structure is formed. Flap sequences 42a and 42b are designed to further protrude from the 5 ′ ends of the first oligonucleotide and the second oligonucleotide, respectively. The flap sequence is a non-complementary sequence with respect to genomic DNA, and is determined to be a different sequence for each allele oligo. Here, Chrybase, a kind of endonuclease, recognizes the triple-stranded structure formed at this polymorphic site and cleaves and releases the flap sequences 42a and 42b.

  Next, each of the flap sequences 42a and 42b hybridizes to two types of FRET probes 61a and 61b having complementary sequences to each other, and a triple-stranded structure is formed by the flap sequences and the FRET probe. This triple-stranded structure is recognized again by CLIBASE, and the fluorescent substance bound to the FRET probe is cleaved and released. The fluorescent substance is separated from the quenching substance Q that has been close to the fluorescent substance and emits fluorescence. The FRET probes 61a and 61b include fluorescent substances that emit different fluorescence wavelengths, for example, FAM for the allele C (denoted as F in the figure) and RED for the allele T (denoted as R in the figure). Are bound, and as a result of cleavage and release by the chestnut base, a first fluorescence signal and a second fluorescence signal are obtained. Thus, in the gene polymorphism determination method using the invader reaction, the gene polymorphism is determined based on the values of the first fluorescence signal intensity derived from the first allele and the second fluorescence signal intensity derived from the second allele. The joining type of the first and / or second alleles is determined. This invader reaction is started by controlling the temperature of the reaction system at a temperature (60 to 65 ° C.) at which the chestnut has enzyme activity, for example, 63 ° C.

  A reagent suitable for the invader reaction is commercially available as an invader assay kit (manufactured by Third Wave Technology). As will be described later, when an invader reaction is performed after a genomic DNA prepared as a determination target is subjected to a gene amplification reaction such as PCR, the gene amplification reaction and the invader reaction may be performed in the same reaction system. A technique combining the invader method and the gene amplification reaction in this way is called an invader plus method, and a reagent suitable for this technique is commercially available as an Invader Plus kit (manufactured by Third Wave Technology).

FIG. 2 shows a schematic diagram of the Invader Plus method. Invader Plus is a method for adding DNA samples 10 and 20, a reagent suitable for PCR reaction, and the above-mentioned invader reaction reagent to a reaction container in a predetermined amount, and adjusting the temperature of the mixed solution to adjust the temperature of the PCR reaction. The treatment is carried out at the optimum temperature for the cycle and invader reaction.
PCR reaction reagents consist of a primer pair (forward primer 71a, 72a and reverse primer 71b, 72b) designed to sandwich the polymorphic site of DNA sample 10,20, DNA synthase such as Taq polymerase, and 4 types of It contains at least deoxyribonucleotide triphosphate (dNTP) and salts.

  The temperature cycle of the PCR reaction includes three steps of a denaturation step, a primer attachment (annealing) step, and a primer extension step, or two steps of a denaturation step and a primer attachment / extension step that simultaneously performs primer attachment and primer extension, The conditions are such that the oligonucleotides for invader reaction (the first and second oligonucleotides 41a and 41b and the invader oligos 51a and 51b) are not annealed. By repeating the temperature cycle of the PCR reaction a predetermined number of times, the region sandwiched between the primer pairs 71a, 71b and 72a, 72b in the DNA sample is amplified in the reaction vessel.

  After repeating the temperature cycle of the PCR reaction a predetermined number of times, the polymerase is inactivated by heating the reaction vessel at a high temperature (for example, 99 ° C.) (heat kill treatment). Thereafter, the invader reaction is carried out by maintaining the optimum temperature of the invader reaction (for example, 63 ° C.) for a certain period of time. At this time, the above-described invader reaction is performed using the PCR products derived from the DNA samples 10 and 20 as the target DNAs 11 and 21, respectively.

  In the present invention, the target DNA to be subjected to genetic polymorphism determination can be genomic DNA or cDNA synthesized from mRNA. Genomic DNA can be selected from animals including humans, plants, and viruses. Genomic DNA and mRNA may be genomic DNA obtained by appropriately extracting and purifying blood-derived samples, biological samples such as urine, hair, etc., and derived from living organisms that have not been extracted or purified. It may be genomic DNA or mRNA present in the sample. The cDNA synthesized from these genomic DNA and mRNA may be subjected to amplification treatment by a known amplification reaction such as polymerase chain reaction (PCR) method or ligase chain reaction (LCR) method. . When multiple polymorphisms existing in genomic DNA or mRNA are to be judged, amplification is performed by multiplex PCR using multiple pairs of primers designed to amplify the region containing the polymorphism. Can be subjected to reaction.

As a reaction vessel for performing the invader reaction, a normal PCR reaction tube, a tube in which a plurality of reaction tubes are connected, a microtiter plate, or the like can be used. In a plurality of reaction vessels, there are individually allele oligos, invader oligos, and fret probes designed to correspond to the SNP to be determined.
The invader reaction, PCR reaction, and fluorescence detection described above may be performed by separate devices, or a single device that combines the functions of a thermal cycler and a fluorescence detector (for example, a microplate reader with a temperature control function). Etc.).
Furthermore, as a reaction vessel used for the determination of gene polymorphism by the above-described Invader Plus method, a general-purpose single tube or connection tube for PCR, a microplate or the like can be used, and a large number of wells are formed on a substrate. A microchip formed (see, for example, Japanese Patent Application Laid-Open No. 2003-070456, WO / 2008/053751) can also be used.

Next, the genetic polymorphism determination method of the present invention will be described based on examples.
[Example 1]
In this example, a target DNA having an SNP at position 1173 of the VKORC1 gene (VKORC1 1173C> T) was prepared and an invader reaction was performed to determine the polymorphism of the target DNA. Allele 1 was C and allele 2 was T.

  FIG. 3 shows the change over time of the fluorescence signal intensity obtained by the invader reaction. In FIG. 3, the solid line shows the change over time in the fluorescence signal intensity derived from allele 1, and the broken line shows the change over time in the fluorescence signal intensity derived from allele 2. The left vertical axis and right vertical axis in FIG. 3 indicate the fluorescence signal intensities (arbitrary units) derived from allele 1 and allele 2, respectively, and the horizontal axis is set to the temperature necessary for the invader reaction after the start of the invader reaction. The elapsed time (reaction time: second) is shown.

  From FIG. 3, it can be seen that the fluorescence signal intensity derived from allele 1 rapidly increases in the initial stage from the start of the reaction, and reaches a plateau after about 400 seconds have elapsed from the start of the reaction. On the other hand, it can be seen that the fluorescence intensity derived from allele 2 gradually increases as the reaction time elapses.

In this example, with respect to the temporal change in fluorescence intensity for allele 1 and allele 2 shown in FIG. 3, a curve representing the temporal change in fluorescence intensity in a predetermined determination section according to the following procedures (1) to (4) ( Hereinafter, the maximum value of the secondary differential value of “fluorescence signal intensity curve” is obtained, and the SNP junction type determination is performed based on the ratio of the maximum values of allele 1 and allele 2.
(1) For each of the fluorescence signal intensity curves derived from allele 1 and allele 2, data of the section D (i, j) from the rise of the fluorescence signal to the maximum slope (ie, first derivative value) is extracted. . This section D (i, j) corresponds to the determination section of the present invention. “I” represents the type of SNP, which is 1173rd in VKORC1 in this example. “J” represents the type of the allele, and j = 1, 2 here.
(2) The maximum value M (i, j) (j = 1,2) of the secondary differential value of the fluorescence signal intensity curve in the section D (i, j) is obtained.
(3) As a determination index, a ratio R (i) between the maximum values M (i, 1) and M (i, 2) obtained in step (2) is obtained (R (i) = log (M (i, 1 ) / M (i, 2))).
(4) The determination index R (i) obtained in step (3) is compared with four preset threshold values, and SNP junction type determination is performed.

  4 and 5 show a first derivative curve and a second derivative curve of the fluorescence signal intensity curve derived from allele 1. FIG. 6 and 7 show the first derivative curve and second derivative curve of the fluorescence signal intensity curve derived from allele 2. FIG. FIG. 8 shows the case classification of the relationship between the determination index R (i) and the four threshold values for the determination index, and the determination results in each case. The four threshold values are the homo minimum value, hetero maximum value, hetero minimum value of allele 1, and homo maximum value of allele 2. From FIG. 8, for example, a sample in which the value of the determination index R (i) is not less than the hetero-minimum value and not more than the hetero-maximum value is determined to be a heterozygous type of allele 1 and allele 2, and the determination index R (i ) Is determined to be a homozygous allele 1 sample. A sample in which the value of the determination index R (i) is between the hetero maximum value and the allele 1 homo minimum value or between the allele 2 homo maximum value and the hetero minimum value is determined to be undecidable. By providing such a determination impossible zone, data with low determination accuracy can be eliminated and erroneous determination can be prevented.

[Example 2]
In this example, the method described in Example 1 has two types of SNPs (VKORC1 1173C> T and CYP2C9 * 3) that greatly affect the strength of the oral anticoagulant warfarin. A determination index R (i) was obtained for 255 samples of genomic DNA with a known SNP mating type, and it was verified whether the mating type determination based on the obtained determination index R (i) was effective. As described above, the prepared genomic DNA is genomic DNA that is known to be any of the heterozygous type of allele 1, heterozygous type, and homozygous type of allele 2 for position 1173 and CYP2C9 * 3 of VKORC1. Here, alleles constituting each SNP are omitted and described as allele 1 and allele 2. FIG. 9 shows the result of position 1173 of VKORC1, and FIG. 10 shows the result of CYP2C9 * 3. 9 and 10, the vertical axis represents the value of the determination index, the horizontal axis represents the SNP junction type, and the non-determinable zone is described by the two strips in each figure. That is, the upper limit value of the upper band-like portion corresponds to the allele 1 homo minimum value shown in FIG. 8, and the lower limit value corresponds to the hetero maximum value. Further, the lower limit value of the lower belt-like portion corresponds to the allele 2 homo maximum value shown in FIG. 8, and the upper limit value corresponds to the hetero minimum value. FIG. 9 and FIG. 10 were prepared by plotting the intersection of the obtained determination index R (i) value and the junction type of each SNP based on the known information for each specimen.

  As shown in FIG. 9 and FIG. 10, the obtained determination index R (i) is approximately in the threshold range (that is, the determination result is any one of the allele 1 homozygous type, the heterozygous type, and the allele 2 homozygous type). It was confirmed that each of the prepared genomic DNAs was correctly conjugated. Therefore, it can be seen that the above-described determination method is effective for determining the junction type of the SNP site.

Moreover, in this example, since the base sequence of the SNP site is determined using the initial determination section of the invader reaction, the invader reaction can be completed in a short time.
Even when the amount of DNA in the sample is small and the rise of the invader reaction is poor, an accurate determination index can be obtained regardless of the amount of DNA because the fluorescence signal intensity curve in the determination interval at the initial stage of the reaction is used.
Since the ratio of the maximum value of the second derivative obtained from the fluorescence signal intensity curve corresponding to each of the two types of SNP base sequences is used as a judgment index, the influence of the increase in signal intensity due to nonspecific fluorescence signals is reduced. Therefore, a more accurate determination index can be obtained.

[Example 3]
In this example, the fluorescence signal intensity curves derived from allele 1 and allele 2 in the determination section are approximated by a quadratic expression (y = Ax2 + Bx + C), respectively, and the ratio of the quadratic coefficient is used as a determination index. Determine. The quadratic approximate expression of the fluorescence signal intensity curve can be obtained using a known method such as a least square method. In the present embodiment, a ratio R (i) = log (A (i, 1) /) using a second-order coefficient A (i, j) instead of the maximum value M (i, j) obtained in the first embodiment. Except for obtaining A (i, 2)), polymorphic joint type determination is performed in the same manner as in the first embodiment.
According to Jeff G. Hall et al. (PNAS 2000 vol.97 no.15 (8272-8277)), when the fluorescence signal intensity curve is approximated by a quadratic equation, the efficiency of the invader reaction, that is, the quadratic coefficient, The amount of DNA is included. Therefore, by using a second-order coefficient, a determination index reflecting the amount of DNA can be obtained.

[Example 4]
In this example, the determination index R (i) obtained in Example 1 or Example 3 is normalized by the determination index obtained for the internal standard DNA. The specific procedure is as follows.

(4-1) The ratio R (a) is determined for the internal standard DNA according to the procedure of steps (1) to (3) in Example 1. As described above, the internal standard DNA refers to DNA having a polymorphic site that is a polymorphic site different from the polymorphic site to be determined and is heterozygous. When the ratio R (a) is obtained for each of a plurality of internal standard DNAs, the average value is taken as the internal standard DNA ratio R (a).
(4-2) Normalize the target genomic DNA by dividing the ratio R (i) obtained by the procedure of steps (1) to (3) of Example 1 by the ratio R (a) of the internal standard DNA, and logarithmically Is determined as a determination index a (i) (a (i) = log (R (i) / R (a))).
The subsequent determination method is performed in the same manner as in step (4) of the first embodiment.

[Example 5]
This example shows the invader reaction temperature after starting the invader reaction according to the heating conditions of the heat kill process when the polymorphism of the target DNA is determined by the invader plus method, which is a method combining the PCR reaction and the invader reaction. It shows that the time to reach is different.
Similar to Examples 1 to 4, the target DNA was a DNA having an SNP at position 1173 of the VKORC1 gene (VKORC1 1173C> T).

In the invader plus method of this example, pre-denaturation treatment, PCR reaction, heat kill treatment, and invader reaction were performed in this order.
In the pre-denaturing treatment, the reaction system was heated at 95 ° C. for 10 seconds.
The PCR reaction included two steps, a denaturation step and a primer attachment / extension step, and the temperature cycle was 95 ° C for 5 seconds at the denaturation step and primer attachment / extension step at 68 ° C for 8 seconds. By repeating such a temperature cycle 28 times, the regions containing the target sequences 31a and 31b on the target DNAs 11 and 21 are amplified.
Then, after heat-killing which inactivates polymerase by heating the reaction system at a high temperature, an invader reaction was performed by maintaining the reaction system at 62 ° C. for 300 seconds.

FIG. 11 shows the fluorescence signal obtained by the invader reaction after the invader reaction was started (ie, after the heat kill process was completed) until 300 seconds passed when the heating condition of the heat kill treatment was 97 ° C. for 180 seconds. An example of measurement of intensity change with time is shown.
On the other hand, FIG. 12 shows the fluorescence obtained by the invader reaction after the elapse of 300 seconds from the start of the invader reaction (ie, after the end of the heat kill process) when the heating condition of the heat kill treatment is 99 ° C. for 120 seconds. An example of measurement of changes in signal intensity over time is shown.
In each measurement example, the fluorescence signal intensity was measured every 5 seconds, and the time when the temperature reached 62 ° C., which is the optimum temperature for the invader reaction, was recorded in the photometric data.

As shown in FIG. 11, when the heating condition of the heat kill process was 97 ° C. and 180 seconds, the optimum temperature (62 ° C.) of the invader reaction was reached after 35 seconds from the start of the invader reaction. On the other hand, as shown in FIG. 12, when the heating condition of the heat kill process was 99 ° C. for 120 seconds, the optimum temperature for the invader reaction (62 ° C.) was reached after 30 seconds from the start of the invader reaction.
From this, it can be seen that the time from starting the invader reaction to reaching the optimum temperature differs depending on the heating temperature and heating time of the heat kill treatment.
Therefore, if the arrival point of the optimum temperature of the invader reaction is recorded and that arrival point is set as the start of the determination section, the influence of the target DNA concentration and the dispensing accuracy of the invader reaction solution on the start of the determination section is minimized. It is thought that it can be made smaller.
Note that the temperature for determining the start of the determination section is not limited to the optimum temperature for the invader reaction, and may be any suitable temperature in the vicinity of the optimum temperature.

  As another embodiment, in the first embodiment, the ratio itself (M (i, 1) / M (i, 2)) may be used as a determination index without taking the logarithm of the ratio of the secondary differential values. Further, the ratio of the determination index may be an inverse ratio of the ratio shown in the first embodiment. That is, the determination index R (i) = M (i, 2) / M (i, 1) may be used. These apply to Example 3 as well.

  The present invention can detect genomic DNA polymorphisms, particularly single nucleotide polymorphisms of animals and plants, including humans, and use the results to diagnose disease prevalence, types and effects of administered drugs, and side effects. It can be used for diagnosis of relationships, plant / animal breed determination, infectious disease diagnosis, and the like.

10,20… DNA sample
11,21… Target DNA
31a, 31b… Target sequence
41a, 41b ... first and second oligonucleotides
42a, 42b… Flap fragment
51a, 51b ... Invader Oligo
61a, 61b… FRET probe
71a, 72a… Forward primer
71b, 72b ... Reverse primer F, R ... Fluorescent substance Q ... Quenching substance

Claims (5)

A target DNA is complementary to a target sequence comprising a first oligonucleotide complementary to a target sequence comprising a first allele constituting the gene polymorphism of the target DNA and a second allele constituting the gene polymorphism. And an invader reaction that generates the first fluorescence derived from the first oligonucleotide and the second fluorescence derived from the second oligonucleotide by controlling the temperature by mixing with the second oligonucleotide. In the gene polymorphism determination method for determining the allele junction type of the gene polymorphism from the first and / or second fluorescence intensity,
Curves representing temporal changes in the first and second fluorescence intensities in a predetermined determination section after the start of the invader reaction are approximated by a quadratic expression, respectively, and the second order of the first and second quadratic approximate expressions. A genetic polymorphism determination method characterized by determining a polymorphism of a genetic polymorphism using a coefficient ratio as a determination index. A target DNA is complementary to a target sequence comprising a first oligonucleotide complementary to a target sequence comprising a first allele constituting the gene polymorphism of the target DNA and a second allele constituting the gene polymorphism. And an invader reaction that generates the first fluorescence derived from the first oligonucleotide and the second fluorescence derived from the second oligonucleotide by controlling the temperature by mixing with the second oligonucleotide. In the gene polymorphism determination method for determining the allele junction type of the gene polymorphism from the first and / or second fluorescence intensity,
The curves representing the time-dependent changes in the first and second fluorescence intensities in the predetermined determination section after the start of the invader reaction are respectively second-order differentiated, and the ratio of the maximum values of the first and second second-order derivative values. A genetic polymorphism determination method comprising determining a polymorphism of a gene polymorphism using as a determination index.   The genetic polymorphism according to claim 1 or 2, wherein the target DNA determination index is normalized with an internal standard DNA determination index, and the polymorphism of the gene polymorphism is determined based on the normalized determination index. Judgment method.   The time when the first derivative value of the curve representing the temporal change of the first and second fluorescence intensities is maximized is set as the end of the determination section, respectively. Genetic polymorphism determination method.   The gene according to any one of claims 1 to 4, wherein when the reaction system of the invader reaction that generates the first and second fluorescence intensities reaches a predetermined reaction temperature, the determination period starts. Polymorphism determination method.

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