JP2002272463A - Method for judging form of monobasic polymorphism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply judging the form of a monobasic substitution in a short time. SOLUTION: This method for judging the form of monobasic polymorphism comprises a process for preparing a polynucleotide which is a polynucleotide obtained by hybridizing a target polynucleotide having a monobasic polymorphism part at, n-position with a primer and in which a nucleotide located at 3' end of the primer is hybridized with a nucleotide at (n+1) position of the target polynucleotide, a process for carrying out an elongation reaction using a labeled modified nucleotide which is a modified nucleotide complementary to a nucleotide species, which exists at the polymorphism part and is to be detected, and modified to stop a further elongation reaction, a process for assaying a position change in a minute space of the labeling with time and a process for analyzing the assayed result of the previous process using a fluorescent correlation analysis method and determining whether the labeled nucleotide is taken in the polynucleotide or not to judge the form of a monobasic polymorphism.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for determining the type of a single nucleotide polymorphism.

[0002]

2. Description of the Related Art In order to efficiently identify a disease susceptibility gene or a gene relating to a drug response, a highly reliable marker (Genetic Landmark) is required. Therefore, restriction fragment length polymorphism (RFLP) is required.
And VNTR (Variable Number of T)
Markers such as andem repeats and microsatellite markers have been used. However, since the distribution of each marker is uneven or the number of markers is small, it has been a limit to narrow down the gene region to about several Mb. In this respect, a single nucleotide polymorphism (Single N
nucleotide Polymorphism (hereinafter, referred to as SNP) has a region of a candidate gene of 30 kb.
Various techniques have been developed because they can be narrowed down to the extent.

As a method for detecting such a single nucleotide polymorphism, there is a method by primer extension (US Pat.
013,431). In this method, after a target gene is converted into a single strand, the target gene is solid-phased on a solid phase surface, for example, via a biotin-avidin reaction, and complemented over a region from the 3 ′ side immediately adjacent to a portion having a single base mutation. Is hybridized. Thereafter, a base analog labeled with a fluorescent dye or the like (extension reaction is stopped, for example, d
dNTP), and the base corresponding to the mutation is extended by one base from the 3 ′ side of the primer. Thereafter, washing is performed, and a signal from the label is detected to identify the nucleotide sequence of the mutated portion.

[0004] Further, using this primer extension method,
Methods using mass spectroscopy detection have also been devised. Tang et al., PROBE (Primer
A method called Oligo Base Extension has been developed (Proc. Natl. Aca).
d. Sci. USA, 96: 10016-1002.
0). In this method, a DNA having a length of about 100 bp including an SNP site was amplified by PCR, purified by desalting, and chemically modified at the 5 ′ end, such as biotinylation or thiolation, to form lattice-like wells. Fix to silicon chip. After fixation and washing, 3 types of dNTPs and 1
A primer extension reaction is performed using various kinds of ddNTPs. After the reaction, the chip is washed and the TOF MAS
The sample was crystallized by adding a matrix solution for S, and the final MALDI (Matrix Assisted L
asser Desorption Ionizatio
n) By mass spectroscopy, the base species extended by extension, that is, the base species of a single base mutation portion is identified. These primer extension reactions directly detect the base immediately adjacent to the 3′-terminal side of the primer, and are an effective method for detecting mutation of one base. Further, M
The method using the ALDI TOF has an effect that a label of a base type is unnecessary. However, in these techniques, in performing a primer extension reaction, a target nucleic acid molecule is immobilized on a solid phase surface, and further, the hybridization and extension reaction of the solid-phased nucleic acid molecule and the primer are performed. There is a problem that the efficiency is remarkably reduced and the operation is complicated.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method which is simple in operation and can determine the type of single nucleotide substitution in a short time.

[0006]

In order to solve the above-mentioned problems, the present invention provides a method for determining the type of a single nucleotide polymorphism, wherein the method comprises the step of n-position (n is an integer of 1 or more). A polynucleotide obtained by hybridizing a primer to a target polynucleotide having a site, wherein the nucleotide located at the 3 ′ end of the primer is hybridized with the nucleotide at the (n + 1) position of the target polynucleotide. Preparing a soybean polynucleotide; a modified nucleotide complementary to the nucleotide species to be detected among the nucleotide species that may be present at the single nucleotide polymorphism site, and labeled with a traceable label; The modified nucleotide modified to stop further extension reaction is mixed with the polynucleotide prepared in the above step, and the extension reaction is performed. Performing the step of measuring the change in position of the label in the micro space over time; analyzing the measurement result of the previous step using a fluorescence correlation analysis method, and a labeled nucleotide complementary to the nucleotide to be detected, Determining the type of the single nucleotide polymorphism by determining whether or not the polynucleotide has been incorporated into the polynucleotide in the extension reaction step.

[0007] In the present specification, "single nucleotide polymorphism" refers to
Genetic polymorphism based on single nucleotide mutation is meant.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for determining the type of a single nucleotide polymorphism.

In the present specification, “determining the type of a single nucleotide polymorphism” refers to estimating or determining the type of nucleotide present at a site where a single nucleotide mutation has occurred (a single nucleotide polymorphism site). That means. Therefore, using the method of the present invention, for example, when it is known that any of adenine, guanine, or cytosine is present at a single nucleotide polymorphism site, the nucleotide at that site is determined to be adenine. Or it is not adenine.

The target polynucleotide having a single nucleotide polymorphism site is a single-stranded polynucleotide in the following examples, but is not limited to a single-stranded polynucleotide. For example, a double-stranded polynucleotide may be used. May be used.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are not intended to limit the scope of the present invention in any way.

Example 1 In this example, referring to FIG. 1, a single nucleotide polymorphism site is detected by using a labeled modified nucleotide complementary to one type of nucleotide to be detected. A method for determining whether or not a nucleotide species to be present exists will be described.

In order to carry out the method of the present embodiment, first, the primer 12 is hybridized so that the primer 12 is adjacent to the single nucleotide polymorphism site 13 in the target single-stranded polynucleotide 11. Prepare 14.

As shown in FIG.
The nucleotide present at the 3 ′ end of 2 is hybridized with the nucleotide adjacent to the 3 ′ side of the nucleotide (adenine (A) in the figure) existing at the single nucleotide polymorphism site 13.

To prepare the partially double-stranded polynucleotide 14, typically, the target single-stranded polynucleotide 1
1 may be hybridized with the primer 12.

After preparing the partially double-stranded polynucleotide 14, a modification that is complementary to the nucleotide species to be detected among the nucleotide species that may be present at the single nucleotide polymorphism site and that is labeled with a traceable label Nucleotide 15 is mixed with partially double-stranded polynucleotide 3. In FIG. 1, unlabeled modified nucleotides 16 are shown in addition to the labeled modified nucleotides 15, but the unlabeled modified nucleotides 16 need not always be added.

The label can be any label that can be tracked, but the preferred label can be a luminescent label, especially a fluorescent label.

FIG. 1 illustrates a case where the nucleotide species to be detected is adenine, so that the nucleobase contained in the labeled modified nucleotide is thymine.

After the addition of the labeled modified nucleotide 15, an extension reaction is performed. To perform the extension reaction, an enzyme,
For example, a polymerase such as a DNA polymerase may be used.

The labeled modified nucleotide 15 is modified so that further extension reaction is stopped. "Modified to stop further extension reaction" means that in a nucleic acid extension reaction, modified nucleotide 15 itself is added to the primer to extend the primer, but is modified so that subsequent extension reaction does not occur. Means that. Therefore, when the modified nucleotide 15 is used, the extension reaction is stopped only by extension of one base. Such modified nucleotides can be, but are not limited to, dideoxynucleotides that are widely used in fields such as nucleic acid sequencing.

Since the extension reaction is extended by one base and then stopped, the partial double-stranded polynucleotide 14 is converted only when the nucleotide species to be detected is present at the single nucleotide polymorphism site of the target single-stranded nucleotide 11. Constituent primer 12
A labeled modified nucleotide 15 is added to the 3 ′ end of the compound. On the other hand, when there is no nucleotide species to be detected at the single nucleotide polymorphism site of the target single-stranded nucleotide 11, the modified nucleotide 15 labeled on the partially double-stranded polynucleotide 14 is not introduced. In the case of FIG. 1, since the target single-stranded nucleotide 11 contains adenine, which is a nucleotide to be detected, it is shown that the labeled dideoxythymidine was introduced into the partially double-stranded polynucleotide 14. ing.

Subsequent to the extension reaction, the change in the position of the label in the micro space incorporated or not incorporated in the partially double-stranded polynucleotide 14 is measured over time.

In the present specification, the term “micro space” means a volume of 10 ⁻²¹ L (corresponding to the volume of a 1 nm square cube) to 10 ⁻³ L, typically 10 ⁻¹⁸ L to 10 ^−9.
L, most typically refers to the space of ¹⁰ -15 ^{L~10 -12} L.

The shape of the minute space can be any shape such as a sphere, a cone, a cube, a rectangular parallelepiped, and the like.

In order to measure the change in the position of the marker in such a minute space, a confocal microscope is typically used. Confocal microscopes themselves are known in the art.

The detection using the confocal microscope 21 is performed as shown in FIG. 1. Irradiate laser light as excitation light from the laser generator 22; 2. After passing through the filter 23, the laser light is focused, and one point in the sample is irradiated with the laser light by the dichroic mirror 24; 3. Excite the fluorescent substance in the sample with laser light to emit light; By passing through the pinhole 25, only the fluorescence emitted from the fluorescent substance in the focal point of the sample is converted to a photomultiplier tube (P
MT) 26; This is performed by analyzing the amplified fluorescence by the data processing device 27 and displaying the result on the display device 28.

As a light source of the confocal microscope, for example, an argon ion laser can be used, but a krypton argon ion laser, a helium neon laser, and a helium cadmium laser having different wavelengths according to the kind of the fluorescent substance can also be used.

Since such a detection method detects only the fluorescence emitted from the label existing in the minute space,
The background is extremely low and the sensitivity is significantly higher than that of ordinary fluorescence detection.

"Measurement of changes in the position of a marker over time" is generally performed in milliseconds to minutes, most commonly in seconds, and can be completed in a very short time.

After the measurement is completed, the measurement result is analyzed,
It is determined whether a labeled nucleotide complementary to the nucleotide to be detected has been introduced into the target polynucleotide.

The analysis of the measurement results is preferably performed by a fluorescence correlation analysis method. Here, the “fluorescence correlation analysis method” refers to measuring the intensity of the fluorescent light emitted from an average number of fluorescent substances (one in some cases) existing in a minute space for a predetermined time, and then measuring the intensity of the fluorescent light derived from Brownian motion. It refers to a method of obtaining the autocorrelation function of fluctuation and acquiring various data on the fluorescent substance by analyzing the function. Fluorescence correlation analysis (hereinafter referred to as FCS (fluorescence correla)
itself, which is known in the art, and is described in detail in JP-A-11-502.
608, etc.

The following operation is performed to analyze the measurement result using the FCS.

[0033] 1. A small space is irradiated with a laser beam. 2. The intensity of the fluorescence emitted from the fluorescent substance existing in the minute space is measured with time, and the data shown in FIG. 3 is obtained. 3. The expected value of the product of the fluorescence intensity I (t) and I (t + τ) at two different time points is calculated, and the autocorrelation function G (τ) = <I
(T) I (t + τ)> is obtained. 4. Equation 1:

(Equation 1) (Where, N: average number of fluorescent molecules τ _small = wo ^2/4 D _small : translational diffusion time of small-sized nucleic acid τ _large = wo ^2/4 D _large : translational diffusion time of _large -sized nucleic acid y = repetition S = wo / zo (where wo is the diameter of the detection region, 2zo is the region length, D
_small and D _large are the translational diffusion constants of small and large nucleic acids, respectively))
And 3. Analyze the autocorrelation function obtained in.

For data analysis by FCS, Evote
c A computer program “FCS” released from BioSystems can be used. The operating time for said steps 1-4 can be less than 10 seconds per sample.

The concept of such an analysis is shown in FIG.
Be clearer. That is, when the size is small, the speed of the Brownian motion is high, so that the frequency of I (t) is large. On the other hand, when the size is large, the speed of the Brownian motion is low, so that the frequency of I (t) is low.

Therefore, by using the equation (1), the size of the nucleic acid and the ratio of each nucleic acid can be estimated.

When the labeled modified nucleotide 15 is incorporated into the target single-stranded polynucleotide 11, the speed of the Brownian motion of the label decreases, whereas the modified nucleotide 15 is incorporated into the target single-stranded polynucleotide. Otherwise, the speed of the sign's Brownian motion is high. Therefore, by applying the fluorescence correlation analysis to the method of the present invention, it can be determined whether or not the modified nucleotide 15 has been incorporated into the target single-stranded polynucleotide.

In the graph of FIG. 1, a curve 17 shows a change with time of the autocorrelation function G (τ) when the labeled dideoxythymidine was not taken in, and a curve 18
Shows the change over time of the autocorrelation function G (τ) when labeled dideoxythymidine is incorporated.

As is clear from the graph, the shape of the curve differs between the case where labeled dideoxythymidine was incorporated and the case where it was not incorporated. It can be determined immediately whether or not it has been captured.

In the actual measurement, the target single-stranded polynucleotide 11 and the labeled modified nucleotide 15 are not present in a one-to-one ratio, and the labeled modified nucleotide 15 is not present.
Is usually greater than the target single-stranded polynucleotide 11. Therefore, in the middle of curves 17 and 18, curve 1
In many cases, a curve having a shape like 9 is obtained.

If the labeled modified nucleotides 15 are in excess relative to the target single-stranded polynucleotide 11, the amount of the modified nucleotides 15 incorporated into the target single-stranded polynucleotide 11 will be extremely small. For this reason, the curve obtained in such a case cannot be distinguished from the curve 17, and it is erroneously determined that the modified nucleotide 15 has not been incorporated into the target single-stranded polynucleotide 11. Therefore, it is desirable that the amount of the modified nucleotide 15 not incorporated into the target single-stranded polynucleotide 11 be less than half of the total amount of the modified nucleotide 15.

On the other hand, if the amount of the labeled modified nucleotides 15 is too small, the progress of the extension reaction will be extremely slow. Is preferred.

As described above, by using the method of this embodiment, it is possible to easily and quickly determine whether or not a nucleotide species to be detected exists at a single nucleotide polymorphism site.

In the present embodiment, the case where it is clear that the predetermined site is a single nucleotide polymorphism site has been described as an example. When it is unclear whether the site is a type site, it can also be used to check whether a single nucleotide polymorphism exists at the site.
That is, the present invention also provides a method for examining whether or not a single nucleotide polymorphism exists at a predetermined site in a single-stranded target polynucleotide.

When it is not known that the predetermined site is a single nucleotide polymorphism site, the site is extended using a modified nucleotide complementary to a nucleotide species different from the nucleotide species known to be present at the site. The reaction may be performed.
If a modified nucleotide complementary to a nucleotide species different from the nucleotide species known to be present at the site is added to the primer, the predetermined site is presumed to be a single nucleotide polymorphism site.

Example 2 In this example, referring to FIG. 4, which nucleotide species exists at the single nucleotide polymorphism site among a plurality of nucleotide species that can exist at the single nucleotide polymorphism site The method for determining is described.

In order to carry out the method of the present embodiment, first, a partially double-stranded polynucleotide hybridized so that the primer 42 is adjacent to the single nucleotide polymorphism site 43 in the target single-stranded polynucleotide 41 Prepare 44. The method for preparing the partially double-stranded polynucleotide 43 is described in Example 1.
It is as described in.

In the example of FIG. 4, the single nucleotide polymorphism site 43 has
Adenine (A) or guanine (G) may be present.

After preparing the partially double-stranded polynucleotide 44, the labeled nucleotides 45 and 46 complementary to each nucleotide species that may be present at the single nucleotide polymorphism site are mixed with the partially double-stranded polynucleotide 43. . In the example of FIG.
Labeled nucleotide 45 is dideoxythymidine and labeled nucleotide 46 is dideoxyguanosine.
In order to distinguish both labeled nucleotides, it is preferable to label the labeled nucleotide 45 and the labeled nucleotide 46 with different substances.

If the single nucleotide polymorphism site 43 in the target single-stranded polynucleotide 41 is adenine, a labeled nucleotide 45 is added to the end of the primer 42. If the single nucleotide polymorphism site 43 in the target single-stranded polynucleotide 41 is cytosine, a labeled nucleotide 46 is added to the end of the primer 42.

After the extension reaction, the change in the position of the label in the labeled nucleotide in the micro space is measured with time. The measurement method is as described in Example 1. When no labeled nucleotide was incorporated, a curve 47 was obtained, and when a labeled nucleotide was incorporated, a curve 48 was obtained.
Is obtained.

After the measurement, the measurement result is analyzed by fluorescence correlation spectroscopy. If each primer is modified with a different fluorescent label, it is possible to determine which primer has bound to the target polynucleotide by using a filter or the like suitable for each fluorescent label during measurement. The method for determining whether or not a given primer has bound to a target polynucleotide using fluorescence correlation spectroscopy is as described above in Example 1.

According to the method of this embodiment using a plurality of types of primers, the type of nucleotide present at the single nucleotide polymorphism site can be accurately determined.

[Brief description of the drawings]

FIG. 1 is a schematic view illustrating a method according to a first embodiment.

FIG. 2 is a diagram showing a configuration of a confocal microscope.

FIG. 3 is a diagram showing data used for measurement of fluorescence correlation spectroscopy.

FIG. 4 is a schematic view illustrating a method according to a second embodiment.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 11 Target single-stranded polynucleotide 12 Primer 13 Single nucleotide polymorphism site 14 Partial double-stranded polynucleotide 15 Labeled modified nucleotide 16 Unmodified modified nucleotide 17 Curve 18 Curve 19 Curve 21 Confocal microscope 22 Laser generator 23 Filter 24 Dichroic Mirror 25 Pinhole 26 Photomultiplier Tube 27 Data Processor 28 Display 41 Target Single-Stranded Polynucleotide 42 Primer 43 Single Nucleotide Polymorphism Site 44 Partial Double-Stranded Polynucleotide 45 Labeled Nucleotide 46 Labeled Nucleotide 47 Curve 48 Curve

Claims (1)

[Claims] 1. A method for determining the type of a single nucleotide polymorphism, comprising: a polynucleotide comprising a primer hybridized to a target polynucleotide having a single nucleotide polymorphism site at the n-th position (n is an integer of 1 or more). And 3 ′ of said primer
Preparing a polynucleotide that hybridizes such that the nucleotide located at the end is paired with the nucleotide at position (n + 1) of the target polynucleotide; and the type of nucleotide species that may be present at the single nucleotide polymorphism site. A modified nucleotide complementary to the nucleotide species to be detected, which is labeled with a traceable label and modified so that further extension reaction is stopped, is mixed with the polynucleotide prepared in the above step. Performing the extension reaction; measuring the position change of the label in the micro space over time; analyzing the measurement result of the previous step using a fluorescence correlation analysis method to complement the nucleotide to be detected. To determine whether or not a typical labeled nucleotide has been incorporated into the polynucleotide in the extension reaction step. By a step of determining a type of single nucleotide polymorphism; method provided with.