JPH05252998A - Method for nmeasuring dna amplified by pcr - Google Patents

Abstract

PURPOSE:To measure a DNA fragment amplified by PCR(Polymerase Chain Reaction) by a sandwich hybridization method using a solid phase in excellent handleability and improved sensitivity. CONSTITUTION:When PCR is carried out in a method for measuring a DNA amplified by PCR, 5' end of one of two kinds of primers is phosphorylated and the prepared product amplified by PCR is treated with gamma-exonuclease to form a single stranded DNA suitable for sandwich hybridization. The single stranded DNA is caught on a solid phase and hybridized with a labeled probe to measure its labeling activity. Since the single stranded DNA is caught on the solid phase, disturbance by re-annealing can be avoided and measuring sensitivity and accuracy are improved.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for measuring PCR amplified DNA. More specifically, PCR (polymerase Chain
Reaction) method for measuring (quantitatively or qualitatively) the DNA amplified.

[0002]

2. Description of the Related Art Conventionally, as clinical examination methods for infectious diseases caused by viruses, bacteria and the like, methods such as detection of the presence of serum antibodies and isolation of pathogens from patients have been carried out. However, in the former case, it may take a long time from infection to antibody production, and the time of infection may not be determined. Further, in the latter, since the amount of pathogen is generally small, it is necessary to repeat subculture in cultured cells to an amount that can be detected, and there is a problem that measurement takes a long time. Due to such problems, a method of directly diagnosing by amplifying and detecting the pathogen genome by the PCR method has been proposed. According to this method, it is possible to make a reliable diagnosis in a short time. As is well known, PCR is a method for amplifying a target DNA fragment in a DNA sample, which has an appropriate length starting from the 3'end of the region to be amplified of the DNA sample (double-stranded DNA). Nucleotide sequence (2
It is carried out by repeating a heat denaturation step, an annealing step and an extension reaction step by using two kinds of primers and a DNA polymerase having a base sequence complementary to 0 base sequence).

In order to quantify the product DNA specifically amplified by PCR, the PCR amplification product is subjected to agarose gel electrophoresis as it is and stained with ethidium bromide to determine the relative intensity of the signal of the specific band. Quantitative method: After agarose gel electrophoresis, the gel is alkali-denatured as it is, and the DNA is transferred to the membrane as a single strand DNA, and then RI labeled DNA probe having a complementary and specific sequence to the amplified PCR product is used. A method in which a hybrid is formed, and its RI activity is relatively quantified by exposing it to an X-ray film based on the intensity of the signal; the PCR amplification product is directly subjected to membrane blot or slot blot to a probe similar to the Southern blot method. Using the same procedure to form hybrids, A method of relative quantification by comparing the known amount of control signals by the strength of the photosensitive signal by beam. Further, in recent years, a DNA probe chemically modified with an enzyme instead of RI is used to form a hybrid with a single-stranded PCR product blotted on a membrane, and then a chromogenic substrate or a fluorescent substrate is used as an enzyme substrate. There is a way to quantify the signal.

More recently, a quantification method using a solid phase has also been proposed, in which streptavidin-agarose (or silica gel) is used as a solid phase and a pair of a primer having a biotin-modified 5 ′ end and a primer modified with a fluorescent substance is used. A method in which the amplified PCR product is contacted with a solid phase and bound to the solid phase by forming a streptavidin-biotin complex, and the fluorescence is measured [Axel Landgraf et al.
Analytical biochemistory, 193, 231-235 (1991)]; DNA-binding protein (GCN4) is immobilized on a solid phase, the 5'end of one primer is biotinylated, and the binding sequence for GCN4 is attached to the 5'end of the other primer. PCR is carried out using the one into which DNA is introduced, and the above solid phase is brought into contact with the PCR amplification product, and DNA (double-stranded) is mediated via GCN4.
There is known a method in which the enzyme activity is measured, followed by contacting with an avidin-enzyme (peroxidase etc.) conjugate prepared in advance to allow binding via a biotin-avidin bond, and then measuring the enzyme activity.

In addition, a sandwich hybridization method using a solid phase such as an ELISA plate (in the present specification, the sandwich hybridization method using a solid phase means that DNA amplified by PCR is converted into single-stranded DNA and is solidified). Trapped on the phase and said single-stranded D
Single-stranded DN by contacting with a probe having a base sequence complementary to at least part of the base sequence of NA and having a label
It also means a method of hybridizing A with a probe and measuring the DNA captured on the solid phase by using a label). For this type of measurement, for example, single-stranded D
After the NA was set, single-stranded DNA was adsorbed on the solid phase under a high salt concentration, and then, a probe having a base sequence complementary to the single-stranded DNA and biotin, and avidin-
Enzyme (peroxidase etc.) conjugates are brought into contact one after another,
Then, a method is known in which the amplified DNA is quantified by measuring the enzyme activity.

[0006]

SUMMARY OF THE INVENTION In the above method,
DNA with ethidium bromide that has been used since the early days
Quantification by staining intensity of agarose gel electrophoresis must be carried out once, and the operation is very complicated, and the signal intensity of a specific electrophoresed DNA band is known to be of a known amount and of almost the same molecular weight. Since it was performed by comparison with the migration signal of DNA, it could be determined only by its relative strength, and fine quantification was impossible. In the Southern blotting method, the DNA is transferred from the electrophoretic gel to the membrane and hybridized with the RI-modified DNA probe to the membrane, so the operation is very complicated and the blotting efficiency is easily affected. Further, the obtained signal can be quantified only by comparison with the signal of a known control DNA, and a fine quantification is impossible like the ethidium bromide staining method. In comparison, the dot blot and slot blot methods do not require electrophoresis but are simple, but the binding signal (non-specific) of the DNA probe other than the target PCR product cannot be determined. In addition, a method of binding to the solid phase by forming a complex with biotin on the primer side using streptavidin-agarose solid phase, and measuring the fluorescence of the PCR product is a biotin modified primer and fluorescent substance modified primer pair. A product that has been non-specifically PCR-amplified also binds to the streptavidin-agarose solid phase and cannot retain its specificity depending on the primer selection. In addition, biotin and DNA binding protein (GCN4)
In the method of using two kinds of primers having a binding sequence bound to the above, it is necessary to prepare these two kinds of primers, and the operation is complicated. Further, in the sandwich hybridization method, the method of adsorbing the single-stranded DNA generated by heat denaturation on the solid phase is difficult to solve
Re-annealing easily occurs to double-stranded DNA, PCR amplification products that are not adsorbed on the solid phase increase, and there is a problem in quantification. The present invention has been made to solve the above-mentioned problems of the prior art, and an object of the present invention is to provide a method for measuring PCR-amplified DNA that has high measurement accuracy and excellent operability.

[0007]

In order to solve the above problems, the inventors of the present invention have earnestly studied a method for measuring PCR-amplified DNA using a sandwich hybridization method which is excellent in operability, and as a result, PCR amplification products were obtained. The present invention has been completed by finding that the measurement accuracy can be remarkably improved by improving the step of preparing a single-stranded DNA from the above. That is, the method for measuring PCR-amplified DNA of the present invention is a sandwich hybridization method using a solid phase,
A method for measuring DNA amplified by PCR, wherein one of the two kinds of primers used is a primer having a phosphorylated 5'end, and PCR is carried out using the two kinds of primers. And the step of treating the generated PCR amplification product with λ-exonuclease to generate single-stranded DNA. In a preferred embodiment of the above sandwich hybridization method, a solid phase on which a probe having a base sequence complementary to a part of the base sequence of amplified DNA containing a non-phosphorylated primer chain is immobilized is used. And a λ-exonuclease-treated PCR amplification product are brought into contact with each other to capture the single-stranded DNA on the solid phase, and further to have a nucleotide sequence complementary to at least a part of the nucleotide sequence of the captured single-stranded DNA. And contacting the labeled probe with each other to hybridize the single-stranded DNA with the probe, and then measuring the single-stranded DNA captured with the label; The single-stranded DNA is captured on the solid phase by bringing the phase into contact with a PCR amplification product treated with λ-exonuclease, and further, at least a part of the base sequence of the captured single-stranded DNA After hybridization and single-stranded DNA and the probe are specific have nucleotide sequences were and contacting a labeled probe, a method of measuring the single-stranded DNA that has been captured using the labels.

The present invention has the above-mentioned constitution, and of the two kinds of primers used in the PCR reaction, one of the two primers is phosphorylated at the 5'end (that is, phosphate is added), and these primers are added. PCR amplification was carried out with, and the resulting PCR amplification product (double-stranded DNA) was digested with λ-exonuclease (EC 3.1.11.3) to decompose only the DNA strand extended from the phosphate-added primer. The use of double-stranded DNA eliminates the possibility of re-annealing. Hereinafter, the measuring method of the present invention will be described with reference to the accompanying drawings. FIG. 1 is a schematic process diagram schematically showing an example of the method of the present invention. An example of measuring PCR-amplified DNA by using an enzyme as a probe label and measuring absorbance change or fluorescence intensity due to enzymatic reaction Show. In the figure, P surrounded by a circle represents a phosphate group, and E surrounded by a circle represents an enzyme. Amplification by PCR can be performed according to a conventional method, and is not limited to the following description.

First, the two kinds of primers used are prepared. The primer is a DNA fragment having a base sequence complementary to a base sequence (usually about 20 bases) of an appropriate length starting from 3 ′ of the region to be amplified of the DNA sample,
Corresponding to the (+) chain and the (-) chain of DNA, two types are chemically synthesized according to a conventional method. In the present invention, one of the above two types of primers has a phosphate added to its 5 ′ end. The addition of phosphate at the 5'end can be performed by a conventional method. For example, the primer can be added to ATP in an appropriate buffer.
And a polynucleotide kinase. The primer thus obtained, in which the 5'end is not phosphorylated (hereinafter referred to as primer 1)
And a primer having a phosphorylated 5 ′ end (hereinafter, referred to as primer 2) is measured by the following procedure.

The sample double-stranded DNA is denatured,
Dissociate into two single-stranded DNAs (step a). This step is generally performed by heating at about 90 to 95 ° C. for about 20 seconds to 5 minutes. At this time, unnecessary heating is not preferable because it deactivates the DNA polymerase described later.

DNA which has become a single strand by the above denaturation
In the presence of the above-mentioned two kinds of primers (primer 1 and primer 2), the temperature is lowered to anneal the two kinds of primers with single-stranded DNA complementary to each (step b). This step is usually 40 ~
It is performed at about 65 ° C. for about 20 seconds to 2 minutes.

Then, in the presence of four kinds of deoxyribonucleotide triphosphates (dNTPs), a DNA polymerase is allowed to act, and a primer extension reaction is carried out according to the base sequence of the template DNA, whereby a new complementary DNA chain containing the primer is obtained. Are respectively synthesized (step c). As the DNA polymerase used in this step, Taq DNA polymerase, which is a thermostable DNA polymerase, is preferably used, and it is possible to prevent inactivation during heat denaturation and increase the annealing temperature. The reaction conditions are usually 7
It is performed at 0 to 75 ° C. for about 1.5 to 3 minutes.

The two double-stranded DNAs thus obtained are subjected to the above-mentioned cycles of denaturation, annealing and extension to amplify the DNA (step d). The reaction conditions of this step include the following conditions. Denaturation of DNA 90-95 ° C, 30-6
About 0 seconds Annealing of primer 40 to 65 ° C, 20 to 5
About 0 seconds DNA synthesis (extension) 70-75 ° C, 90-1
About 50 seconds The number of cycles is not particularly limited and can be appropriately adjusted depending on the initial concentration of the sample DNA, etc.
It is set to about 50 cycles. Although the amount of amplified DNA increases as the number of cycles increases, the amount of non-specific products due to misannealing and the like also increases, which may reduce the measurement accuracy. By such amplification, a large amount of double-stranded DNA in which the DNA extended from Primer 1 and the DNA extended from Primer 2 are complementary bound to each other is obtained, and a PCR amplification product is obtained.

Next, the PCR-enhanced byproduct obtained above is treated with λ-exonuclease (step e). λ-exonuclease is DNA with phosphate added to the 5'end
Since it has an action of sequentially cleaving the DNA from the end, the DNA extended from the primer 2 is decomposed, and only the DNA extended from the primer 1 having no phosphate added to the end (hereinafter referred to as the primer 1 side DNA) remains. This step is carried out in the presence of λ-exonuclease in an appropriate buffer under conditions of about 30 to 40 ° C. and about 5 to 20 minutes.

The primer 1 side DNA obtained above is
DNA probe-immobilized solid phase (1) on which DNA having a nucleotide sequence complementary to a part of the nucleotide sequence of the DNA is immobilized
And the DNA on the primer 1 side is captured on the solid phase (step f). Furthermore, an enzyme-labeled DNA probe (2) having a base sequence complementary to at least a part of the base sequence of the primer 1-side DNA and having a label (enzyme in this example) (2)
Are brought into contact with each other to hybridize the probe with the DNA on the primer 1 side (step g). In this way, the primer 1-side DNA is captured on the solid phase, and the complex (3) in which the enzyme-labeled DNA probe (2) is hybridized to the primer 1-side DNA is obtained.

Next, the complex (3) is washed and the free enzyme-labeled DNA probe (2) is removed, and then an enzyme substrate is added to carry out an enzyme reaction, and the change in absorbance due to the enzyme reaction or the fluorescence intensity is measured. (Step h). Based on this value, the PCR-amplified DNA can be quantified by determining the amount of the primer 1-side DNA captured on the solid phase from a calibration curve prepared in advance.

FIG. 2 is a schematic process diagram schematically showing another example of the measuring method of the present invention. Instead of the enzyme-labeled DNA probe (2) used in the above-mentioned example, binding as a label is carried out. An example of using a DNA probe in which one substance of a substance pair having properties is bound will be shown. Biotin-avidin (or streptavidin) as a substance pair having binding properties
Pair, concanavalin A-saccharide (eg glucose etc.)
An example is a pair. In the example of FIG. 2, a biotin-labeled DNA probe (4) which is a DNA probe bound with biotin is used. More specifically, after performing steps a to f in the method of FIG. 1 in the same manner, in step g, a biotin-labeled DNA probe (4) is used in place of the enzyme-labeled DNA probe (2) to obtain a solid phase. A primer (1) side DNA is captured on the top, and a complex (5) is obtained in which the biotin labeled DNA probe (4) is hybridized to the primer 1 side DNA. By bringing the avidin-enzyme conjugate (6) into contact with the complex (5), the enzyme is captured on the solid phase via the biotin-avidin bond, and the complex (7) is obtained. The PCR-amplified DNA can be quantified by adding an enzyme substrate to the thus obtained complex (7) and measuring the change in absorbance or the fluorescence intensity due to the enzymatic reaction as described above.

As the solid phase of the DNA probe-immobilized solid phase (1) used in the measurement method shown in FIGS. 1 and 2, the solid phase conventionally used in the ELISA method can be used,
For example, agarose, sepharose, plastic, glass, etc. are exemplified, and usually a microtiter plate is preferably used. As a method for immobilizing the DNA probe on the solid phase, an adsorption method can be used as well, but preferably a method in which the solid phase and the DNA probe are bound by a covalent bond is mentioned. As this covalent bond method,
Various covalent bond formation methods conventionally used in the ELISA method and the like can be adopted, and for example, solid phase and D
There may be mentioned a method in which a reactive group (for example, an amino group, a thiol group, a carboxyl group, etc.) is introduced into both NA probes and a space between them is covalently bonded using an appropriate bifunctional reagent. Examples of the bifunctional reagent used in this method include dialdehydes such as glutaraldehyde,
Examples include diisocyanates such as hexamethylene diisocyanate, disuccinimides such as Disuccinimidyl suberate, and compounds having a maleimide group and a succinimide group such as N- (ε-Maleimidocaproyloxy) succinimide.

The enzyme-labeled DNA probe (2) and the biotin-labeled DNA probe (4) can also be prepared by a conventional method. For example, in the same manner as above, after introducing a reactive group into the DNA probe, a suitable DNA probe It can be prepared by binding an enzyme (or biotin) and a DNA probe using a bifunctional reagent. Furthermore, the avidin-enzyme conjugate (6) can also be prepared by a conventional method, for example, by using a reactive group possessed by avidin and the enzyme to combine avidin and the enzyme with an appropriate bifunctional reagent. can do. The enzyme used as a label is not particularly limited as long as it produces a fluorescent substance or a fluorescent substance by an enzymatic reaction, but is usually peroxidase, alkaline phosphatase, β
-Galactosidase and the like are used. When the enzyme is peroxidase or alkaline phosphatase, the enzyme activity is measured by adding the respective enzyme substrates o-phenylenediamine-hydrogen peroxide and p-nitrophenyl phosphate and measuring the absorbance of the product. The PCR amplified DNA is measured based on this value. When the enzyme is β-galactosidase, 4-methylumbelliferyl-β-D-galactoside is added as an enzyme substrate, and the fluorescence intensity of umbelliferone produced is measured,
The PCR amplified DNA is measured based on this value.

The measuring method of the present invention is not limited to the above-mentioned example, and can be carried out by appropriately changing it. For example, in the above example, the DNA probe-immobilized solid phase (1) in which the DNA probe is immobilized on the solid phase is used, but the primer 1-side DNA generated in step e is used in a high salt concentration (for example, 1. Alternatively, a method of adsorbing to a solid phase and immobilizing it under conditions such as 5 M calcium chloride or 0.5 M ammonium sulfate may be used. Further, as the primer 1, one having a substance having a binding property (for example, biotin) added to its 5 ′ end is used, and the avidin-labeled solid phase is used as the solid phase, and the DNA on the primer 1 side is avidin-biotin. It may be captured on the solid phase via a bond. Furthermore, a method of measuring RI activity by using a radioactive substance instead of the enzyme of the above example may be used.

The method for measuring PCR-proliferated DNA of the present invention comprises
It can be applied to various methods for measuring CR products, and is preferably used in the field of clinical examination and the like.
As an example in which the assay method of the present invention is applied to a clinical test, an example in which the detection of a pathogen is performed by the assay method shown in FIG. 1 will be described. Serum collected from a subject is used as a sample and the base sequence of the pathogen genome is used. Using the primer 1 and the primer 2 prepared based on the above, the enzyme activity is measured through steps a to h. When the pathogen genome is present in the sample, the DNA on the primer 1 side is captured on the solid phase to form the complex (3), and the enzyme activity is recognized. On the other hand, if the pathogen genome is not present in the sample, PC
Since the amplification of DNA by R does not occur, the complex (3)
Is not formed and no enzymatic activity is observed. From this measurement result, it is possible to diagnose whether or not the subject is infected with the pathogen, and further to quantify the pathogen amount by quantifying the enzyme activity. The sample can be applied to all DNA samples capable of PCR amplification. For example, as a virus, HBV (Hepatitis B virus),
HVC (Hepatitis C virus), HIV (Human immunodefic
iency virus), HSV-I (Herpes simplex virus I),
HSV-II (Herpes simplex virus II), HCMV (Huma
n cytomegalovirus), EBV (Epstein-barr virus), H
HV-6 (Human herpes virus 6), VZV (Varicella z
oster virus) and the like, and MRSA (M
ethicillin-resistant Staphyulococcus arueus) and the like.

[0022]

The present invention will be described in more detail based on the following examples, but the invention is not intended to be limited to these examples. Example 1 An example applied to detection of a PCR product of HBV DNA is shown below. 1. Preparation of Reagent PCR Primer Based on the base sequence of HBV DNA, the base sequences of two kinds of primers were set as follows, and Applied Biosyste
It was synthesized using a DNA synthesizer (381A) manufactured by ms. Primer A 5'CGTGTTACACGGCGGGGG
TTTTTCTGG primer B 5'TGGAAGTAGAGGACAA
ACGGGGCAAC Primer B was added to T4 Polynu in the presence of ATP.
Phosphorylation was performed as follows by the action of cleotide Kinase. Primer B 10 μl of buffer solution in 100 pmol [100 mM MgCl ₂ · 100 mM 2-mercaptoethanol · 500 mM Tri
s-HCl (pH8.0)], 2 μl of 50 mM ATP, 1 μl of T4 polynucleo
After adding tide kinase, the total volume was adjusted to 100 µl with purified water and the reaction was carried out at 37 ° C for 60 minutes. The reaction was stopped by treating at 70 ° C for 10 minutes.

Preparation of DNA probe-immobilized solid phase As a solid phase probe, set as follows in the DNA region amplified between the above primers A and B, and
It was synthesized using a DNA synthesizer (381A) manufactured by ed Biosystems, and an amino group was added to its 5 ′ end using Aminolink II. 5 'NH ₂ -GATAAAACGCCGCAGACACAT
CCACGGATA A DNA with an amino group added is treated with DSS (disuccinimidyl suber
ate) using amino group-containing microtiter plate
DN by covalently bonding to (Nova Covalink)
An A probe-immobilized solid phase was prepared.

Preparation of Alkaline Phosphatase-Labeled DNA Probe As an enzyme-labeled probe for signal detection, a DNA synthesizer manufactured by Applied Biosystems was set as follows in the DNA region amplified between the above primers A and B.
It was synthesized using r (381A) and then aminolink II
An amino group was added to the 5'end. 5 'NH ₂ -AAAATTGAGAGAAGTCCACCA
CGAGTCTAG Amino group-added DNA was labeled with EMCS [N- (ε-Maleimidocapr
oyloxy) succinimide], and alkaline phosphatase was treated with 2-Iminothiolane.
A group was added. An alkaline phosphatase-labeled DNA probe was prepared by mixing and reacting both.

2. With Cetus Corp. Gene Amp ^TM PCR Kit as amplification PCR reagents HBV DNA by method PCR, was carried out PCR amplification. That is, 1 μl of the plasmid solution containing HBV DNA was added to 5 μl of 10 times concentrated Taq buffer solution.
l, 25 mM dNTP solution 0.2 μl, 1 unit of Taq DNA polymerase, 12 pmol of each of primers A and B were added, and sterilized water was added to make a total volume of 50 μl. The target DNA was amplified by carrying out 40 cycles of 120 seconds at 72 ° C.

Λ-Exonuclease Treatment of PCR Amplification Product The PCR amplification product was purified by phenol treatment and ethanol precipitation, and dissolved in purified water. Purified PCR amplification product solution
0.67M Glycine-NaOH (pH9.4) 1μl containing 0.025M MgCl ₂ 1μl
l, λ-exonuclease (Bethesda research laborat
ory) 0.5 unit, and make up to a total volume of 10 μl with purified water.
After reacting at 0 ° C for 10 minutes, it was subjected to a sandwich hybridization reaction.

Sandwich hybridization method 5 to 10 μl of a measurement sample per well of a DNA probe-labeled solid phase
, 100 ng / ml alkaline phosphatase labeled DN
90-95 μl of A probe was added to adjust the total volume to 100 μl, and 37
React at ℃ for 4 hours. After the reaction is complete, add 2% X well containing 1% SDS
Wash 3 times with SSC [0.03M Na-citrate (pH 7.0) containing 0.3M NaCl] and p-NPP substrate solution [10mM p-nitrophenyl phosphate in 50
Add 1M diethanolamine buffer (pH9.8) containing mM MgCl ₂ ,
Incubate at 37 ℃ for 4 hours. After the reaction, 100 μl of 1N NaOH was added to stop the reaction, and the absorbance at 405 nm was measured.

3. Results FIG. 3 shows that the PCR amplification products were treated with λ-exonuclease (◯), those not treated with λ-exonuclease (□), and those which were denatured by heating and quenching without λ-exonuclease treatment (●). The results of measurement by the above-mentioned sandwich hybridization method for the samples of the species are shown. As a result, no reaction was observed without λ-exonuclease treatment, and a slight reaction was observed with heat-quenched denaturation, but λ-
The most sensitive results were obtained with the exonuclease treated ones. It is considered that the reaction that has been denatured by heating and quenching probably interferes with the reannealing of the difficult DNA.

[0029]

INDUSTRIAL APPLICABILITY As described above, in the assay method of the present invention, one of the two primers used for PCR is 5 ′.
Since a product with a phosphate added to the end is used, single-stranded DNA is produced by treating the resulting PCR amplification product with λ-exonuclease. This single chain D
Since NA is measured by sandwich hyibidization, it is possible to measure with high sensitivity and high accuracy without being disturbed by re-annealing, and the measurement operation is simple.

[Brief description of drawings]

FIG. 1 is a schematic process diagram schematically showing an example of the measuring method of the present invention.

FIG. 2 is a schematic process diagram schematically showing another example of the measuring method of the present invention.

FIG. 3 is a diagram showing a result of measuring a PCR amplification product by a sandwich hybridization method. In the figure, ◯ indicates λ-exonuclease-treated, □ indicates λ-exonuclease-free, and ● indicates heat-quenched denaturation without λ-exonuclease treatment.

Claims (5)

[Claims] 1. A sandwich hybridization method using a solid phase for PCR (polymerase Chain Reacti
on) to measure the DNA amplified by
One of the two kinds of primers used is a primer having a phosphorylated 5'end, and PCR is performed using the two kinds of primers, and the generated PCR amplification product is
-A method for measuring PCR-amplified DNA, which comprises the step of treating with exonuclease to produce single-stranded DNA. 2. The sandwich hybridization method comprises an amplification D containing an unphosphorylated primer strand.
Using a solid phase on which a probe having a base sequence complementary to a part of the base sequence of NA is immobilized, the solid phase is brought into contact with a PCR amplification product treated with λ-exonuclease to form a single strand on the solid phase. Single-stranded DN that traps DNA and is further trapped
A probe having a nucleotide sequence complementary to at least a part of the nucleotide sequence of A is brought into contact with the probe to hybridize the probe with the single-stranded DNA, and then the probe is captured with a label 1. The method for measuring PCR-amplified DNA according to claim 1, which is a method for measuring double-stranded DNA. 3. The sandwich hybridization method comprises contacting a solid phase with a PCR amplification product treated with λ-exonuclease to capture single-stranded DNA on the solid phase under a condition of high salt concentration, and further capturing. A labeled probe having a base sequence complementary to at least a part of the base sequence of the single-stranded DNA is contacted to hybridize the probe with the single-stranded DNA, and then the label is used. The method for measuring PCR-amplified DNA according to claim 1, which is a method for measuring the captured single-stranded DNA. 4. The label of the labeled probe is an enzyme, the absorbance change or fluorescence intensity due to an enzymatic reaction between the enzyme and the enzyme substrate is measured, and single-stranded DN is based on the value.
The method for measuring PCR-amplified DNA according to claim 2 or 3, wherein A is measured. 5. A substance which has one of a pair of substances capable of binding the labeled probe, and has a substance capable of binding to a hybrid of a single-stranded DNA captured on a solid phase and the labeled probe. The other substance of the pair is brought into contact with the enzyme conjugate, the enzyme is captured through the substance pair having binding properties, and the change in absorbance or fluorescence intensity due to the enzyme reaction between the enzyme and the substrate is measured, and based on the value The method for measuring PCR-amplified DNA according to claim 2 or 3, wherein the single-stranded DNA is measured.