JP3322894B2 - Nucleic acid sample fluorescence detection method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for detecting a nucleic acid sample, and more particularly, to a novel nucleic acid sample capable of accurately and simply measuring a specific nucleic acid indispensable for the analysis of genes such as organisms and viruses. The present invention relates to a fluorescence detection method.

[0002]

2. Description of the Related Art As one method for preparing a nucleic acid sample, Japanese Patent Application Laid-Open
PCR (Polymerase Chain) described in
Reaction) method is known. This method uses double-stranded DNA
Amplifies a specific nucleic acid sequence site using two types of primers, each of which is an oligonucleotide complementary to each single strand.

That is, first, a double-stranded D serving as a sample
NA is prepared, two types of primers are hybridized to each single-stranded DNA, and DNA polymerase is allowed to act to generate an extended strand from each primer. Further, the above operation is repeated a plurality of times on the obtained double-stranded extension product, whereby a DNA fragment sandwiched between the primers in the double-stranded DNA and having a region to be detected is obtained. It can be obtained by amplification.

The DNA obtained by PCR is separated by molecular weight separation by electrophoresis using a gel such as polyacrylamide or agarose as a carrier for separation, and then stained with a dye such as ethidium bromide and visualized by fluorescence. Can be detected. Alternatively, if the sequence of the obtained DNA can be expected, the PCR product DNA is transferred to a membrane such as nylon, and then a probe labeled with a radioisotope and having a sequence complementary to the expected sequence is hybridized.
Analyzes can be made by so-called blotting operations that take autoradiography.

[0005] In this PCR method, various DNA regions can be specifically amplified by selecting a base sequence of a primer. For this reason, applications such as detection of genomes of pathogenic bacteria, viruses, and the like by the PCR method and diagnosis of infectious diseases have been made.

[0006]

The above-mentioned PCR method uses a single-stranded DNA for each of two or more different double-stranded DNAs.
By simultaneously using a plurality of primers having specificity for NA, different DNA regions can be simultaneously amplified in the same reaction vessel. However, the following problems occur when separating and separating a plurality of amplified DNAs obtained in such a reaction system.

(1) When a plurality of DNAs as PCR products have similar molecular weights, separation cannot be performed by separation means based on molecular weights represented by electrophoresis, and as a result, rapid and reliable separation is difficult. There are many. (2) Even if the molecular weights of a plurality of DNAs that are PCR products are different from each other, it is possible to determine from which nucleic acid sample region each DNA is derived without information on the length or base sequence of the target DNA. Difficult to identify.

[0008] Even when such information is known, an operation such as blotting for the identification usually takes a lot of trouble and time. (3) The amplification reaction by PCR usually involves the production of unintended DNA.
The product must be separated, and the problem (2) occurs in such separation.

Further, as a method widely applicable to gene diagnosis and the like, an RFLP (restriction fragment le) comprising analyzing an arbitrary DNA fragment treated with a restriction enzyme is disclosed.
ngth polymorphism) method (Yoshida, Metabolism, 22, 855 (1985), etc.) has been attracting attention, but in this method, when multiple types of restriction enzymes are simultaneously acted on, It is difficult to identify whether the connection has been cut. Also, when a plurality of short oligonucleotides are used as primers as a reaction corresponding to the simultaneous action of a plurality of types of restriction enzymes, it is necessary to identify from which primers each of a large number of products is an extension product. Is not easy.

In the case where primers having specificity for two or more different DNAs are used in the PCR method, if the above-mentioned problems can be solved, a target region in the genome of a plurality of pathogenic bacteria, viruses, etc. can be quickly identified. -It is possible to reliably detect and identify, which can greatly contribute to speeding up and ensuring diagnosis of infectious diseases. Furthermore, if the above-mentioned drawbacks of the RFLP method can be solved, it will be possible to greatly contribute to the advancement of diagnostic techniques for genetic diseases and the like.

[0011]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, at least two kinds of primers for PCR amplification having different sequences are labeled with different fluorescent labels, and the PCR is carried out. It has been found that the above-mentioned problem can be solved by performing a fluorescence analysis after amplification by the above method.

That is, according to the present invention, an oligonucleotide is bound to each of a plurality of regions of the same or different sample DNA as a template, the extended chains are extended by an enzymatic reaction, and the extended chains corresponding to the obtained regions are added. By repeating the above reaction by using the sample D again,
In a nucleic acid sample preparation method for amplifying DNAs in a plurality of regions of NA, the oligonucleotides are at least two types of oligonucleotides having sequences that are not identical to each other, and the at least two types of oligonucleotides are respectively different fluorescent dyes. By extending the extended strand by an enzymatic reaction, to prepare nucleic acid fragments corresponding to the plurality of regions labeled with different fluorescent dyes,
An object of the present invention is to provide a method for detecting a nucleic acid sample, comprising separating a molecular weight, exciting a fluorescent dye with light, and detecting fluorescence.

In the nucleic acid detection method of the present invention, it is necessary to label at least two of the at least two oligonucleotide primers corresponding to a plurality of regions of the same or different sample DNAs as templates with different fluorescent dyes. is there. The present invention can be roughly classified into two aspects depending on what is selected as an oligonucleotide primer.

First, DNA whose sequence is known to some extent
This is a case where only a specific region of DNA is detected in a sample. In such a case, the same or different sample D serving as a template
It is necessary to label at least two oligonucleotides among at least three oligonucleotides corresponding to a plurality of regions of NA with different dyes.

The oligonucleotide corresponding to the sample DNA often corresponds to a single-stranded DNA obtained by heat denaturation of a different double-stranded DNA, and in this case, the number of oligonucleotides is at least four. is there. However, the present invention is also applicable to a case where a plurality of regions of the same sample DNA are prepared, where the target regions partially overlap, and where at least three types of oligonucleotides are to be prepared.

The above-described oligonucleotide is preferably designed so as to have strict complementarity with at least three different sample DNAs to be detected, respectively, in different regions. It is permissible to include various bases. Although the length of the oligonucleotide is not particularly limited in principle, the length of the sample D
From the viewpoint of giving high specificity to a specific region of NA, it is preferable that the length be at least 10 mer or more.

Next, the present invention relates to an RFL using a restriction enzyme.
P (Restriction-Fragments Length Polymorphism) method
(Yoshida, Metabolism, 22, 855 (1985), etc.), i.e., changing the base sequence of the primer to the pattern of the reaction product of the primer with various nucleic acid samples whose structure is relatively unknown. It is supposed that the method is used for a method to be obtained in the above and used for genome analysis or the like.

In such a case, the oligonucleotide to be selected does not necessarily have to have high specificity with the sample DNA, and a mixture of any sequence having two or more different sequences may be selected as the oligonucleotide serving as a primer. it can. The oligonucleotides used may be very short oligomers of a few mer with very low specificity, but are usually from 5 to 14 mer, more preferably 8 mer.
Mer to 10 mer oligonucleotides are used.

The oligonucleotide used in the present invention can be chemically synthesized by a conventional method, and can also be obtained by cutting out an actual genome or the like using a restriction enzyme. Then, it is necessary to label at least two kinds of oligonucleotides among the oligonucleotides obtained as described above with different fluorescent dyes. The two oligonucleotides must have different base sequences from each other.

The type and combination of fluorescent dyes used are not particularly limited as long as they are combinations of dyes emitting fluorescence of different wavelengths. That is, as long as fluorescence of a certain wavelength is emitted by certain excitation light, selection is possible regardless of whether the wavelength of the excitation light is close or not. For example tetramethylrhodamine isothiazole oscillation Aneito (TRITC), chlorosulfonic acid derivative of sulforhodamine 101, fluoride Les cell <br/>In'isochi oscillation Aneito (FITC), 4-fluoro - 7 - nitrobenzofurazan (NBD-F) And the like. As particularly preferred, tetramethylrhodamine isothiazole O
Shi Aneito (TRITC), mention may be made of chlorosulfonic acid derivative of sulforhodamine 101.

The method of labeling the oligonucleotide with a fluorescent dye is not particularly limited. For example, a method of directly labeling a nucleic acid base with a fluorescent substance using an amino group-introducing reagent or a method disclosed in JP-A-61-44353 is disclosed. That is, the phosphoric acid bond at the fluorescent labeling site of the oligonucleotide is replaced with a phosphonic acid bond having a functional group, and the fluorescent group is realized by binding the functional group to the fluorescent substance. A method that can introduce a label into the label can be adopted.

It is not necessary that all nucleotide molecules of the oligonucleotide be fluorescently labeled. Next, using the oligonucleotides labeled with different fluorescent dyes obtained above, the polymerase chain reaction (polymerase cha
in reaction: PCR) method, that is, a plurality of labeled oligonucleotides are bound to a nucleic acid sample serving as a template, the extended strand is extended by a reaction using an enzyme, and the obtained extended strand is used as a template again for the above-described reaction. It is necessary to amplify the nucleic acid sample to be measured by a nucleic acid sample preparation method that amplifies the same nucleic acid sample.

The details of the amplification of a nucleic acid sample by the PCR method have already been reported in, for example, JP-A-62-217161. As the sample DNA to be amplified,
It can be arbitrarily selected. That is, all types of eukaryotes, regardless of their known or unknown sequence,
It can be selected for prokaryotic and viral DNA. It is also possible to select cDNA obtained by reverse transcription from eukaryotic or prokaryotic mRNA or RNA virus RNA.

Further, the present invention is not limited to the combination of DNAs having different lengths, and the analysis target is the same DNA.
The analysis method of the present invention can be applied to the above-mentioned plurality of regions. This reaction can be performed in a separate reaction vessel for each PCR primer used,
In consideration of the overall measurement efficiency, it is preferable to use a plurality of labeled oligonucleotides at once in the same reaction vessel.

This reaction can be performed in a test tube, but can also be performed using an automatic PCR reaction apparatus. Further, the present invention is characterized in that the amplified DNA strand is separated in molecular weight, and a fluorescent dye is excited by light to detect fluorescence. This molecular weight separation can be performed by a conventional method. That is, an electrophoresis method, a membrane separation method, or the like can be employed, but it is preferable to employ an electrophoresis method using an acrylamide gel from the viewpoint of simplifying the operation of the subsequent fluorescence detection step.

Next, it is necessary to excite a specific dye with light and to detect a specific DNA fragment labeled with the dye with fluorescence. The excitation light is not particularly limited as long as it can efficiently excite the labeled fluorescent dye. For example, when a sulfonic acid derivative of sulfolodamine 101 and TRITC are selected as the fluorescent dye, the wavelength is 543n.
Helium neon laser light in the vicinity of m is preferred.

In this fluorescence detection, a visual method using a device such as a transilluminator can be used for the gel, but a multicolor fluorescence analyzer or the like is used in order to ensure accurate and simple detection. It is preferable to use a fluorescence analyzer of the above. Cormorants <br/> Chi fluorescence analysis instruments that can be used in the present invention include the following multi-color fluorescence analysis apparatus as one preferred embodiment.

As such a fluorescence analyzer, for example, the fluorescence analyzers described in JP-A-2-269935, JP-A-2-269936 and JP-A-2-269937 can be suitably used. In each fluorescence analyzer, as a labeling dye,
For example, when selecting a fluorescent dye having a relatively close excitation wavelength such as Texas Red and TRITC, it is sufficient to use a laser light source having the same wavelength. However, when a large number of types of fluorescent dyes are selected, such as when detecting a large number of samples, and when a plurality of dyes having greatly different excitation wavelengths are selected, a plurality of laser light sources can be used. That is, it is also possible to use means for changing only the wavelength of the excitation light to excite only the target dye.

[0029]

According to the present invention, it has become possible to easily detect individual fragments of a PCR amplification product targeting a plurality of regions, which were conventionally difficult to detect. In particular, in the present invention, since a plurality of types of short oligonucleotides can be labeled and used with different dyes, it is possible to identify from which oligonucleotide each product is an extension product. In addition, the present invention is capable of distinguishing multiple products of the same or close length that are usually difficult to distinguish. As described above, since the origins of a plurality of products can be classified according to the label of the primer, detailed analysis with a greater amount of information based on a plurality of base sequence information is possible.

[0030]

DESCRIPTION OF THE PREFERRED EMBODIMENTS One example of the present invention will be described with reference to an embodiment.

[0031]

Example 1 (1) Preparation of Labeled Oligonucleotide The following sample DNA and oligonucleotide were used in Example 1. Sample DNA: plasmid pUC with 311 base insert
119: 1 ng Human leukocyte extracted DNA: 1 μg Oligonucleotide: Primer KM1 having base sequence of SEQ ID NO: 3 Primer KM2 having base sequence of SEQ ID NO: 4 Primer A having base sequence of SEQ ID NO: 5 Having base sequence of SEQ ID NO: 6 Primer B (a) Preparation of sample DNA a) Preparation of plasmid pUC119 having an insert of 311 bases The insert of 311 bases incorporated into the plasmid pUC119 was a C8- nucleotide consisting of 269 bases of hepatitis C virus RNA.
2 areas (Nucleic Acid Res. Vol.18, (1990) pp1685-pp
1689).

The DNA consisting of 311 bases was prepared from blood of a carrier of hepatitis C by a conventional method. That is, C
Hepatitis B carrier blood obtained by AGPC (Analytical Bioch
emistry, vol.162, (1987) pp156-pp159), and after extracting total RNA including hepatitis C virus RNA,
CDNA was prepared from A using reverse transcriptase. This c
PCR from DNA using oligonucleotides shown in SEQ ID NO: 1 and SEQ ID NO: 2 as primers (JP-A-62-2
No. 17161), the desired DNA of 311 bases was obtained.

The 5 ′ end of the 311 base DNA was phosphorylated by a conventional method and incorporated into the SmaI site of pUC119 (Takara Shuzo) to prepare pUC119 having a 311 base insert used as a sample DNA. E.coli JM109
Transformed into competent cells (Takara Shuzo). this
When pUC119 having a 311 base insert is used as sample DNA, E.co.
li JM109, alkaline-SDS method (Birnboim, HC a
pUC119 having a 311 base insert was prepared by nd Doly, J., Nucleic Acids Res., 7, 1513 (1979).

B) Preparation of DNA extracted from human leukocytes DNA extracted from human leukocytes was prepared from blood of healthy individuals by a conventional method. That is, heparin blood is collected from a healthy person in a considerable amount, and distilled water is added thereto to destroy blood cells.
This was centrifuged at about 3000 rpm for 5 minutes. After repeating this operation twice more, the lower layer containing leukocytes was separated, and SDS
(Sodium Dodecyl Sulfate) was allowed to act on proteinase K , followed by phenol-chloroform extraction and ethanol precipitation for purification to prepare human leukocyte-extracted DNA.

(B) Preparation of Labeled Oligonucleotides Primers KM1 and KM2 specific to pUC119 after being labeled with a chlorosulfonic acid derivative of fluorescent dye sulfolodamine 101 (trade name, Texas Red, manufactured by Molecular Probes); TRITC ( Primers A and B specific to the V region of human mitochondria after labeling with Molecular Probe Co.) were used as PCR primers.

These oligonucleotides were synthesized by a DNA synthesizer (manufactured by ABI), and at the time of this synthesis, each fluorescent dye was labeled at the 5 'end of each oligonucleotide using an amino group-introducing reagent. The labeled oligonucleotide was purified by HPLC and subjected to the following detection. (2) Amplification of sample DNA The above nucleic acid sample mixture and PCR primers were each
l, 250 μM of each dNTP, 2.5 mM of MgCl ₂ , Tris-HC
l (pH 8.5) 10 mM, KCl 50 mM, Gelatin 0.001%
(w / v) (final concentration)
It was set to 00 μl. Heat resistant D
NA polymerase (Perkin-Elmer Cetus) 5
The unit was added, mineral oil for preventing evaporation was overlaid, and a thermal cycle of 94 ° C./30 seconds, 50 ° C./90 seconds, 72 ° C./90 seconds was repeated 30 times to perform PCR amplification. Excess salts and the like were removed from the PCR product by performing an ethanol precipitation operation, and a portion was subjected to fluorescence measurement after electrophoresis using a two-color detection type apparatus shown in FIG.

In FIG. 1, reference numeral 11 denotes a glass gel plate;
Is an acrylamide gel, 13 is a helium-neon laser as an excitation light source, 14 is a mirror, 15 is a prism, 16 and 17
Denotes a bandpass filter, 18 denotes a lens, 19 denotes a two-dimensional detector, and 20 denotes a data processing computer.
In two-color detection-type device, first, a molecular weight separation subjected to electrophoresis 1/10 ⁴ weight of the resulting PCR products in 100μl scale reaction, helium-neon laser beam (wavelength 543 nm) was irradiated to the gel in electrophoresis To excite the labeled Texas Red and TRITC, and to obtain the fluorescence amount of each maximum wavelength
(Texas Red: 615 nm, TRITC: 575 nm).

FIG. 2 shows the results. In FIG.
Shows the result of detection of Texas Red, and the lower part 22 shows the result of detection of TRITC. The vertical axis indicates the relative value of the fluorescence intensity, and the horizontal axis indicates the molecular weight. The molecular weight increases toward the right. It was considered that the detection peak 23 of Texas Red and the detection peak 24 of TRITC were due to unreacted Texas Red labeled primer KM1 and unreacted TRITC labeled primer B. And detection peak 25 and detection peak 26
Is the plasmid pUC1 with the insert at peak 25 due to the difference in the fluorescent dye labeled on each primer.
It was found that 434 bases corresponding to 19 and peak 26 correspond to a PCR product of 121 bases corresponding to the V region of human mitochondria.

In the first embodiment, the expected PC
Although the effect of the method of the present invention was examined using a product having a known length of the R product, even if the length of the PCR product is unknown, its origin can be known by the difference in the labeled dye. Furthermore, the origin of the by-products produced by the reaction can also be known from the differences in the constituent dyes.

[0040]

[Sequence list]

SEQ ID NO: 1 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other sequence Synthetic DNA primer Sequence origin: Hepatitis C virus Sequence: TCATCCTCCCTCCGCTCGAA
G SEQ ID NO: 2 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other sequences Synthetic DNA primers Origin of sequence: Hepatitis C virus Sequence: TTGGAGCCCGAACCGGATAG
T Sequence number: 3 Sequence length: 18 Sequence type: Nucleic acid number of strands: Single strand Topology: Linear Sequence type: Other sequence Synthetic DNA primer Sequence: TGTAAAACGACGGCCAGT Sequence number: 4 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other sequence Synthetic DNA primer Sequence: CAGGAAACAGCTATGACC SEQ ID NO: 5 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded topology: linear Sequence type: other sequence Synthetic DNA primer Sequence: ATGCTAAGTTAGCTTTTACA
G SEQ ID NO: 6 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other sequence Synthetic DNA primer Sequence: ACAGTTTCCATGCCATCATGT
C

[Brief description of the drawings]

FIG. 1 is a two-color detection type device used in Example 1.

FIG. 2 shows the results obtained in Example 1.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12Q 1/68 C12N 15/09 BIOSIS / WPI (DIALOG)

Claims (6)

(57) [Claims] To 1. A plurality of regions of the sample DNA as a template that is not a restriction enzyme digestion, a plurality of flops having different nucleotide sequences
Hybridize each of the limers and hybridize
The extended chain of each of the primers is extended by an enzymatic reaction , and the extended chain obtained corresponding to each of the regions is again used as a template to repeat the extension reaction. in a nucleic acid sample fluorescence detection method of nucleic acid fragments are amplified respectively detecting a nucleic acid fragment, a plurality of lengths have any sequence of numbers mer said
Labeling primers with different fluorescent dyes, preparing nucleic acid fragments corresponding to the plurality of regions labeled with the different fluorescent dyes by the extension reaction, separating the nucleic acid fragments, and detecting fluorescence of the nucleic acid fragments A method for detecting fluorescence of a nucleic acid sample, comprising: 2. A plurality of primers having different base sequences are respectively hybridized to a plurality of regions of a sample DNA which has not been digested with a restriction enzyme and used as a template, and the extended strand of each of the hybridized primers is extended by an enzyme reaction. A nucleic acid for performing an extension reaction, repeating the extension reaction using the extended strand obtained corresponding to each of the regions as a template again as a template, amplifying nucleic acid fragments corresponding to the plurality of regions, and detecting the nucleic acid fragment, respectively. In the sample fluorescence detection method, the plurality of primers having an arbitrary sequence of 5 mer to 14 mer in length are labeled with different fluorescent dyes, and the nucleic acid fragments corresponding to the plurality of regions labeled with the different fluorescent dyes by the extension reaction Preparing the nucleic acid fragment, separating the nucleic acid fragment, and detecting the fluorescence of the nucleic acid fragment. 3. A plurality of primers having different base sequences are respectively hybridized to a plurality of regions of a sample DNA serving as a template which has not been digested with restriction enzymes, and the extended chains of the respective hybridized primers are extended by an enzyme reaction. A nucleic acid for performing an extension reaction, repeating the extension reaction using the extended strand obtained corresponding to each of the regions as a template again as a template, amplifying nucleic acid fragments corresponding to the plurality of regions, and detecting the nucleic acid fragment, respectively. In the sample fluorescence detection method, a plurality of primers having an arbitrary sequence of 8 mer to 10 mer in length are labeled with different fluorescent dyes, and nucleic acid fragments corresponding to the plurality of regions labeled with the different fluorescent dyes by the extension reaction Preparing the nucleic acid fragment, separating the nucleic acid fragment, and detecting the fluorescence of the nucleic acid fragment. 4. A plurality of primers having different base sequences are respectively hybridized to a plurality of regions of a sample DNA serving as a template which has not been digested with restriction enzymes, and the extended chains of the hybridized primers are extended by an enzyme reaction. In the nucleic acid sample preparation method for performing an extension reaction, repeating the extension reaction using the extended strand obtained corresponding to each of the regions as a template again as a template, and amplifying nucleic acid fragments corresponding to the plurality of regions, A plurality of primers having an arbitrary sequence of several mer are labeled with different fluorescent dyes, and a nucleic acid fragment corresponding to the plurality of regions labeled with the different fluorescent dyes is prepared by the extension reaction. Sample preparation method. 5. A plurality of plug having different nucleotide sequences into a plurality of regions of the sample DNA as a template that is not a restriction enzyme digestion
Hybridize each of the immers and hybridize
An extension reaction is performed to extend the extended chain of each of the primers by an enzymatic reaction, and the extension reaction is repeatedly performed using the extended chain obtained for each of the above regions as a template again to correspond to the plurality of regions. in a nucleic acid sample preparation method of amplifying a nucleic acid fragment, respectively, in length from 5mer
A nucleic acid sample preparation comprising: labeling a plurality of primers having an arbitrary 14-mer sequence with different fluorescent dyes; and preparing nucleic acid fragments corresponding to the plurality of regions labeled with the different fluorescent dyes by the extension reaction. Method. 6. A plurality of primers having different base sequences are hybridized to a plurality of regions of a sample DNA serving as a template which has not been digested with restriction enzymes, and the extended chains of the hybridized primers are extended by an enzyme reaction. In the nucleic acid sample preparation method for performing an extension reaction, repeating the extension reaction using the extended strand obtained corresponding to each of the regions as a template again as a template, and amplifying nucleic acid fragments corresponding to the plurality of regions, Is characterized in that a plurality of the primers having an arbitrary sequence of 8 mer to 10 mer are labeled with different fluorescent dyes, and a nucleic acid fragment corresponding to the plurality of regions labeled with the different fluorescent dyes is prepared by the extension reaction. Nucleic acid sample preparation method.