WO2005062047A1

WO2005062047A1 - Method of analyzing biological samples, liquid reaction mixture and analytical device

Info

Publication number: WO2005062047A1
Application number: PCT/JP2003/016572
Authority: WO
Inventors: Takayuki Kanda; Shinichi Fukuzono
Original assignee: Hitachi High-Technologies Corporation
Priority date: 2003-12-24
Filing date: 2003-12-24
Publication date: 2005-07-07
Also published as: JPWO2005062047A1; JP4227139B2

Abstract

Biological samples are analyzed by synthesizing two or more types of biological samples, attaching labeling agents having different wavelengths for individual types to the thus synthesized biological samples, and then detecting the labeling agents. In this analysis, the detection intensities of the labeling agents of the biological samples are controlled depending on the types. Among the thus detected ones, the detection intensity of a labeling agent the wavelength range of which exerts a larger effect on the other labeling agent is weakened. In a specific embodiment, two or more types of biological samples are synthesized and labeling agents having different wavelengths for individual types or media for the binding to labeling agents are attached to materials to be used in the synthesis (materials for the synthesis). Thus, the biological samples are labeled during the synthesis or thereafter. Then the amount (attachment level) of the labeling agent or a medium for the binding to the labeling agent attached to at least one of the materials for the synthesis is controlled to a level less than the labeling agent or a medium for the binding to the labeling agent attached to the other material for the synthesis, thereby labeling the biological samples. According to this constitution, biological samples (nucleic acids, etc.) having a plural types of labeling agents can be simultaneously analyzed at a high quantitative accuracy as well as a high quantitative accuracy while preventing overlap of wavelength regions of labeling agents each other.

Description

Biological sample analysis method, reaction solution, and analyzer

The present invention relates to a method for analyzing a biological sample, a reaction solution, and an analyzer used for diagnosis, inspection, and research in the fields of biochemistry, molecular biology, and medical care.

Rice field

Background technology ''

As a technique for detecting trace amounts of biological samples, mainly nucleic acids and proteins, target, synthesized or purified biological samples are labeled with fluorescent, luminescent, or radioactive isotopes for qualitative and quantitative determination. And so on.

As a nucleic acid labeling method, a technique of labeling a target nucleic acid synthesized by a PCR (polymerase chain reaction) method with a fluorescent substance, a luminescent substance and a radioisotope during or after the synthesis is well known.

For example, an amplification reaction is performed using a primer to which biotin-digoxigenin (DIG) is added, and the amplified (synthesized) nucleic acid product is labeled with a fluorescent substance, a luminescent substance, and a radioisotope via biotin-digoxigenin (DIG). It is known to add a radioactive isotope or to add a radioactive isotope after treating a synthesized nucleic acid with an alkaline phosphatase.

Among the methods for detecting a biological sample to which a label has been added as described above, a detection method using a fluorescent substance and a luminescent substance has been developed in recent years, and has become a simple method, for example, a kit for performing detection has been sold. It is used for the diagnosis of infectious diseases, genetic diseases, and cancer as a highly sensitive analysis method for biological samples.

Furthermore, the development of chemicals used as labels and the improvement of analyzers have enabled the detection of very small amounts of labeled biological samples. Also, (1) Each chemical substance used as a label has its own wavelength band. If labels of different wavelengths are added to several types of biological samples for each type, multiple types of biological samples can be detected simultaneously by one reaction by simultaneously measuring their wavelength spectra.

Japanese Unexamined Patent Publication No. Hei 9-213 495-49 relates to a technique for detecting a base sequence of a nucleic acid. A technique has been disclosed in which a sample is labeled with a plurality of types of fluorescent dyes, the sample is separated (for example, separation by gel electrophoresis), and then a plurality of target nucleic acids are simultaneously detected.

Also, (2) Japanese Patent Application Laid-Open No. 5-118991 discloses that a mixture (combination) of two kinds of phosphors is used as a label, and the four kinds of phosphors are changed by changing the mixing ratio of these phosphors. There is disclosed a method for detecting a base sequence which generates labels and discriminates four kinds of nucleic acids (base sequences) using these labels. Here, the labels (mixing ratio of the first fluorescent substance and the second fluorescent substance) added to the four kinds of nucleic acids are separated after separating the nucleic acids by type (for example, separation by gel electrophoresis). By calculating the intensity ratio between the first and second phosphors excited by nucleic acids (this intensity ratio is determined by detecting the fluorescence intensity of the first and second phosphors with two detectors). Is found). Four types of nucleic acids are distinguished based on the intensity ratio. The mixing ratio of the fluorescent substance labeled on the nucleic acid is determined by the primer (or terminator) modified with the first fluorescent substance and the primer (or terminator) modified with the second fluorescent substance during nucleic acid generation by the PCR method. It is changed by adjusting the mixing ratio.

Of the above conventional techniques, the latter, ie, the method of (2), is suitable for a qualitative analysis method such as nucleotide sequencing.

In order to enable simultaneous quantitative analysis of a plurality of types of biological samples (for example, target nucleic acids), it is effective to use the former, that is, the method of (1). For example, a method in which labels of different wavelengths (for example, phosphors) are added for each type of target sample and the fluorescence intensity (spectrum) of the separated target sample is detected is suitable. This method also allows for qualitative analysis. However, in the method (1), since the fluorescent substance, luminous substance, etc. used for the label have a wavelength band having a slope-like spread centered on the peak wavelength instead of a single wavelength, a plurality of detections are performed. When the wavelengths (labels) of the samples are close, a part of the wavelength band overlaps. Thereby, other labels may influence the strength of the detected label, and the accuracy of the quantification may be reduced.

In order to solve such a problem, it is only necessary to select a label so that the wavelength bands of the detection samples do not overlap. However, such a label does not easily exist, and the selection is limited. Also, during detection, correction by calculation, etc., is required to minimize the effect of overlapping wavelengths.

By solving these problems, the present invention makes it possible to use many types of labels (fluorescent, luminescent, or radioactive isotopes). Provided are an analysis method, a reaction solution, and an analyzer capable of suppressing the overlap of the wavelength bands and performing quantitative analysis with high accuracy as well as qualitative analysis. Disclosure of the invention

Basically, the present invention provides a method for synthesizing biological samples by synthesizing two or more biological samples, adding labels having different wavelengths to the synthesized biological samples, and detecting those labels. To analyze. In this analysis, the detection intensity of the label in the biological sample is adjusted for each type of the biological sample, and in particular, among the detected labels, the detection intensity of the wavelength band having a greater influence on the other label is reduced. . In this way, even if different labels (different biological samples) are mixed in the reaction solution for synthesizing the biological sample, when the labels are detected simultaneously, the influence of the overlapping of the labels is almost eliminated. It is possible.

As a specific embodiment, the following labeling method is proposed.

Synthesizing or synthesizing biological samples by synthesizing two or more biological samples and adding a label with a different wavelength for each type or a label binding medium to the substance used for the synthesis (substance for synthesis) Labeling later. And at least The amount of label or label binding medium added to one type of synthesis substance (addition rate) should be less than the amount of label or label binding medium added to another type of synthesis substance. Adjust to label the biological sample.

When multiple types of biological samples are detected with different detection wavelengths (labels) for each type, the overlap of the wavelength bands indicates the ratio of the amount of label (addition rate) added to one sample to the other. It can be made smaller by making it smaller than that of the sample. In other words, when the labeling rate of one sample is reduced, the detection intensity (for example, the waveform in the wavelength band of fluorescence, light emission, etc.) of the detection wavelength band of the reduced sample is weakened, and the slope of the waveform is also reduced. As a result, the degree of influence on the detection wavelength region of the other sample (label) is reduced.

The present invention is effective as a specific method for suppressing the overlapping of the long bands as described above.

In this case, as the marker for decreasing the amount (decreasing the addition rate), the one that has a greater effect on one of the detection wavelengths (maximum wavelength: spectrum) is selected from the partially overlapping markers.

When the biological sample is a nucleic acid, when quantifying two or more types of nucleic acids, before measuring the intensity of the label (fluorescent substance, luminescent substance or radioisotope having a wavelength band) of each nucleic acid, The nucleic acids are separated for each type by electrophoresis or the like. Then, after the measurement of the label, the peak height, area, band luminance, and the like of the obtained detection waveform are directly used for analysis. Brief Description of Drawings

FIG. 1 is an example of a flowchart of a biological sample analysis according to an embodiment of the present invention. Fig. 2 shows the conventional method, in which the addition rates of the labels X and Y for the primers A and B, and also for the nucleic acids A 'and B' are almost the same, and the fluorescence intensity of the nucleic acids A 'and B' is about the same. 5 shows the fluorescence wavelength-fluorescence intensity characteristics in the case of the above. FIG. 3 shows the labeling of the primers A and B and the nucleic acids A ′ and B ′ according to the present embodiment. The addition ratio of X and Y is 1 to 10 and shows the fluorescence wavelength-fluorescence intensity characteristics when A 'is 1/10 of B'. Fig. 4 shows the detection wavelength vs. fluorescence intensity characteristics of the conventional method. The addition rates of the labels X and Y for the primers A and B are almost the same, but the original production amount of the nucleic acid B 'is shown. Is much less than A "". FIG. 5 is a diagram showing a relationship between the detected wavelength and the fluorescence intensity when the fluorescence intensity of the nucleic acid A ′ in FIG. 4 is reduced according to the present invention. FIG. 6 is a diagram showing the detection results of the conventional method when a PCR product amplified using ROX-303F and FAM-304F as FORWORD primers is used. FIG. 7 is a view showing detection results when PCR products amplified using ROX-303F and 304FMIX as FORWORD primers are used. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited only to these examples.

FIG. 1 shows an example of a flowchart of a biological sample analysis according to the embodiment of the present invention. Here, two kinds of nucleic acids will be described as an example of a biological sample.

After extraction of the genomic DNA containing the target type III (target nucleic acid) and the set of primers (Steps S1, S2), the primer solution used for PCR is mixed with the solution containing the sample and the thermal cycler is used. And perform PCR (step S 3). Specific examples of genomic DNA extraction and primer set will be described later. In addition, the description of the primer set will be described using a schematic diagram in the PCR sequence of step S3.

In this example, when performing synthesis (replication) of the target nucleic acid by PCR, two sites a and b sandwiching the target site of the double-stranded DNA are selected (see step S 3 (2)). A site targeted by a single chain (eg, RNA) may be selected. In the double-stranded DNA, only one target nucleic acid is schematically illustrated in step S3 (1), but genomic DNA other than the target is actually contained in the reaction solution. The target nucleic acid is a pair of target nucleic acids (alleles) from the father and the mother, respectively. Exists.

In this example, the two strands of type I DNA were separated one by one by denaturation [Step S3 (2)], and the nucleic acid of the nucleic acid was placed at two sites a and b sandwiching the target sites of these chains. Anneal primers (oligomers) A and B, which are the starting points for replication.

The nucleotide sequences of the primers A and B are, of course, complementary to the corresponding replication origins a and b.

The reaction solution contains primers to which labels (fluorescent, luminescent, radioactive isotopes) X and Y of different wavelengths or label binding substances (modifying groups or antigens) are added. Here, the label and the substance for binding the label are collectively referred to as labels X and Y. Label X modifies primer A (this primer is AX) and label B modifies primer Y (this primer is BY).

The reaction solution also contains unlabeled primers A and B. Total amount of primer A (sum of primer AX with label X and primer A without label) and total amount of primer B (primer BY with label Y and primer B without label) And the same amount). However, the rate of addition of label X added to primer A and the rate of addition of label Y added to primer B are different. When X and Y are fluorophores, luminophores, and radioisotopes, the addition rate (quantity ratio) of the label X, Y to the primer is determined by the fluorescence intensity, luminescence intensity, or radioactivity ratio.

The ratio between the primer AX and the primer BY is, for example, 1:10. In addition, the quantitative ratio of the labeled nucleic acid to the unlabeled nucleic acid of each type, that is, the quantitative ratio of AX to A, 8 丫 to 8, is 1: 1 to 1:50, preferably 1 to 5, respectively. : 5-1: Adjusted in the range of 10

This adjustment is made in advance by a primer solution for each type in a primer set process (specific examples will be described later).

In this example, in the repetition of the thermal cycle, the target nucleic acid is replicated through the initializing and amplification in step S3 (2). In annealing, the target nucleus One of the primers AX and A binds to the acid replication origin a, and one of the primers BY and B binds to the replication origin b.

The replicated target nucleic acids include labeled nucleic acids A'X (target product based on primer AX) and B'Y (target nucleic acid based on primer BY), and unlabeled nucleic acids A 'and B'. Is included. In this example, the ratio of A'X to B'Y is almost the same as the ratio of primers AX and BY, and is approximately 1 to 10.

As described above, by adjusting the mixing ratio between the labeled primer AX and the unlabeled primer A, and the labeled BY and the unlabeled primer B, the labeled nucleic acid primers AX and BY (with different types of labeled primers) can be used. Primers), and thus the ratio of labeled nucleic acid products A'X, B'Y. As in this example, by adjusting the labeled primer (or modifying group or antibody) AX to be smaller than the labeled primer BY, the labeled nucleic acids A'X and B ' Less than Y [Step S 3 (5)].

The two nucleic acids A '(including A'X) and B' (including B'Y) generated by the PCR method are separated by electrophoresis (step S4). That is, each single-stranded DNA (A ′, B ′) has a complementary base sequence and has a higher-order structure specific to each base sequence. Secondary structural polymorphism: single-strand conformation polymorpmsm: SSCJ, separated at different positions by electrophoresis, and single-stranded when a single base in the DNA fragment is replaced by mutation, etc. The higher order structure of DNA changes, and when such a change occurs, its migration position also changes, and the mutation in the gene is detected by comparing such a change in the migration position with, for example, a reference one. This detection is used, for example, in the LOH method (Loss of heterozygosity) described below.

The migration position can be detected by detecting the maximum wavelength of the target nucleic acid label using a spectroscope and analyzing the detected spectrum. In this case, the qualitative and quantitative determination of the target nucleic acid can be obtained by spectral analysis (steps S5 and S6). The ratio of labeled primers AX and BY is such that the labels X and Y are fluorescent, luminescent, and radioactive. In the case of radioisotopes, it is determined by their fluorescence intensity or radioactivity ratio. Also,

When X and Y are modifying groups or antigens, the determination is made based on the fluorescent intensity or the radioactivity ratio of the fluorescent substance, luminescent substance or radioisotope added to X or Y.

When X and Y are a modifying group or an antigen, a reaction of adding a fluorescent substance, a luminescent substance, and a radioisotope to the modifying group or the antigen is performed after the PCR reaction. For example, when the modifying group is biotin, the treatment of binding to biotin using a mixture of avidin or streptavidin to which a fluorescent substance, a luminescent substance, and a radioactive isotope has been added, and avidin or streptavidin not having been added is performed. Do. In the case of an antigen such as digoxigenin (DIG), a mixture of an antibody to which an enzyme such as alkaline phosphatase (AP) and horseradish peroxidase (HRP) has been added and a mixture of an antibody to which no enzyme has been added are used. After binding the antibody to the antigen, treatment is performed using a chromogenic substrate.

As the fluorescent substance bound to the primer,

CyDve, Carboxyfluorescein (FAM), fluorescein-5-isothiocyanate (FITC), hexac loroflviorescein (HEX) rhodamine (ROX),

Carboxytetramethylrhodamine (TAMRA)> tetrachlorofluorescein (TET)> souffle Horodamine 101 acid chloride (trade name: Texas Red®). As a luminous body, for example,

3- (2'spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) -phenyl-l, 2-dioxetane (trade name: AMPPD®), CSPDTM, MDP ™, CDP ™, and the like.

Radioactive isotopes include, for example, 32 P, 131 1, 35 S, 45 Ca, ³ H, and the like.

The quantitative ratio of the fluorescent substance, the luminescent substance, the radioisotope or the modifying group or the antigen to be used as the label of the nucleic acid is set according to the chemical substance to be used as the label.

Here, according to FIGS. 2 and 3, nucleic acids A ′ (= A′X) and B ′ (= B′Y), which replicate (amplify) the above two sites a and b, respectively, are fluorescent substances. The form of the measured spectrum when labeled with 5-carboxypluorescein (5_FAM) and 5-carboxy-X-i'hodamine (5'ROX) will be described. In addition, nucleic acid A ', B' The case where quantification is performed simultaneously through each measurement spectrum will be described as an example.

Fig. 2 shows the conventional method, in which the addition rates of the labels X and プライマー for the primers A and B and thus the nucleic acids A 'and Β' are similar, and the fluorescence intensity of the nucleic acids Α 'and Β' is similar. 4 shows a fluorescence wavelength-fluorescence intensity characteristic when measured. FIG. 3 relates to the present example, in which the addition rate of the labels X and Y for the primers I and B ′ and the nucleic acids A ′ and B ′ is 1 to 10 and A ′ is 110 for B and B ′. It shows the fluorescence wavelength-fluorescence intensity characteristics when there is.

The maximum fluorescence wavelength of each fluorescent substance, that is, the detection wavelength, is 518 nm for 5-FAM and 604 nm for 5-ROX. When nucleic acids labeled with these two fluorescent substances are detected in such amounts that they have the same level of fluorescence intensity, there is a wavelength band that overlaps the two, as shown in Fig. 2, and the detection wavelength of 604 nm is the detection wavelength of 5-ROX. Also includes the 5-FAM wavelength band. Therefore, in the quantification of B ', as can be seen from Fig. 2, approximately 30% of the fluorescence intensity at the detection wavelength of 5-FAM, which is the label of A', is added, and the detection wavelength of B ' Is detected as the fluorescence intensity. In such a case, it is necessary to perform a process of removing the fluorescence intensity of A 'from the fluorescence intensity at the detection wavelength of B' by correcting by calculation or the like.

On the other hand, as in this example, for example, in a reaction for amplifying a nucleic acid to be detected, a primer to which 5-FAM has been added in advance is compared with a primer to which 5-ROX has been added at a detection wavelength. When the site A is amplified so that the fluorescence intensity becomes 110, the fluorescence intensity of A ′ also becomes approximately 110 (this fluorescence intensity is indicated by A〃 in FIG. 3). Here, if A ′ and B ′ are detected simultaneously, the fluorescence intensity at 604 mn will be close to the actual fluorescence intensity of Β ′ without correction, as shown in FIG. In other words, by making the fluorescence intensity of A '(Α〃) sufficiently smaller than Β' within the measurable range, the slope on the longer wavelength side of 小さく becomes smaller, and the detection wavelength of B 'becomes smaller. Has almost no effect.

In the present embodiment, as shown in FIGS. 2 and 3, labeling of the target nucleic acid A ′, Is a compound having a spectrum with a sharp slope on the short wavelength side and a gentle slope on the long wavelength side based on the maximum wavelength of the detection spectrum. In this case, the label with the shorter wavelength affects the label with the longer wavelength, so the label (modification group, label binding for antigen, etc.) added to multiple types of primers (synthesis materials) is used. (Including the medium) make the shorter wavelength A 'less than the longer wavelength B'.

FIG. 4 also shows a detection wavelength-fluorescence intensity characteristic according to the conventional method. In this example, although the addition rates of the labels X and Y for the primers A and B are almost the same, the original production amount of the nucleic acid B 'labeled with 5-ROX is the nucleic acid A' labeled with 5-FAM. This is the case when the fluorescence intensity at the detection wavelength of B 'is 10% of A'. Also in this case, a part of the wavelength band of A 'is included in the detected wavelength (maximum wavelength) of B', and it becomes difficult to detect B '. In this case as well, if the labeling rate of primer A is made smaller than the labeling rate of primer B in the same manner as described above, the fluorescence intensity on the A 'side is reduced as shown in Fig. 5. In addition, the detection wavelength band of A 'hardly affects the detection wavelength (maximum wavelength) of B', and the actual fluorescence intensity of B 'can be approximated.

When analyzing (analyzing) two types of target nucleic acids, the analyzer does not correct any of the data and either performs the analysis operation as it is, or when performing the quantitative operation, Data on the addition rate can be entered, and the quantification of each biological sample is calculated based on the addition rate data and the measurement data.

In this way, by changing the ratio of the amount of the labeling substance according to the biological sample, detection using the labeling substance having overlapping wavelength bands becomes possible, and more types of nucleic acids can be detected. It can be quantified at the same time.

Hereinafter, specific examples of the present invention will be described.

(Example 1)

1. Extraction process of type I DNA from specimen (Step S 1)

Samples include human peripheral blood (whole blood), biopsy tissue fragments, and urine. The extraction of genomic DNA described here is performed by a well-known method. For example, in the case of human peripheral blood, QIAGEN DNA Blood Mini Kit may be used. Specifically, follow the standard protocol of the kit, but add 200 L of human whole blood and 20 L of QIAGEN Protease to a 1.5 mL tube. Next, add 200 L of Buffer AL, mix well using a voltex, and incubate for 56 to 10 minutes. Thereafter, the liquid is collected by centrifuging the microtube, adding 200 L of ethanol, and sufficiently stirring again using a voltex. The whole amount of the mixture is injected into a spin column in a collection tube, centrifuged at 800 rpm for 1 minute, the solution is passed through the column, and genomic DNA is captured in the column. Then transfer the same spin column to a new collection tube, open the lid, inject 500 L of Buffer AW into the spin column, and centrifuge at 800 rpm for 1 minute. Transfer the spin column to a new collection tube again, then inject 500 liters of Buffer AW250, centrifuge at 1,400 rpm for 3 minutes, and wash the column. Transfer the spin column to a new collection tube, inject 100 L of Buffer AE, centrifuge at 800 rpm for 1 minute, elute the captured genomic DNA, and obtain a genomic DNA solution. The amount of nucleic acid in the eluate is quantified by measuring the eluted genomic DNA using an absorptiometer.

In the case of urine, urine collected in a centrifuge tube is first centrifuged at 100 O rpm for 5 minutes to collect only the sediment, and the sediment is re-dispersed by adding physiological saline, washed, and washed again. Centrifuge at 0 O rpm for 5 minutes to collect the sediment and use it as a sample. After that, digestion with the well-known proteinase K is performed, and genomic DNA is extracted by phenol / clonal form extraction. Tissue sections can be made similarly. Alternatively, a sample obtained by cryopreserving a urine test substance, a tissue piece, or white blood cells of blood at -80 ° C may be used.

2. Step of Preparing Primer Set for Amplifying Target Nucleic Acid Portion (Step 2) The following six kinds of polynucleotides were used as primers for PCR amplification. 5'TET-CAACAAAGCAAGATCCCTTC-3 '(ROX-303F primer)

5'-CAACAAAGCAAGATCCCTTC-3 '(303F Primer)

5'-TAGGTACCTGGAAACTCTTGGC-3 '(303R primer) 5'TET-GTGCACCTCTACACCCAGAC-3 '(TET-304F primer)

5'-GTGCACCTCTACACCCAGAC-3 '(304F Primer)

5'-TGTGCCCACACACATCTATC-3 '(304R primer one)

The ROX-303F primer and the 303F primer, and the TET304F primer and the 304F primer are polynucleotides having the same sequence.

The ROX-303F primer and the 303F primer hybridize with a part of the 303 gene region on chromosome 9. The TET-304F and 304F primers hybridize to a portion of the 304 gene on chromosome 9.

These are both used as FORWARD primers in the PCR amplification reaction. In addition, the ROX-303F primer is a labeled polynucleotide with the fluorescent substance ROX at its 5 'end, and the TE "304F primer is a labeled polynucleotide with the fluorescent substance TET bound to its 5' end, and is detected by fluorescence detection. it can.

The 303F and 304F primers are unlabeled polynucleotides. The binding of the fluorescent substance to the polynucleotide can be performed by a known method.

The 303R primer and the 304R primer hybridize with the 303 gene or a part of the 304 gene on chromosome 9, respectively, and are used as REVERSE primers in the PCR amplification reaction.

For the FORWARD primer solution for the PGR amplification reaction, the ROX-303F primer and 303F primer, and the TET "304F primer and 304F primer were prepared at a concentration ratio of 1:10, respectively, to give a total concentration of 100 M (each 303FMIX primer solution and 304FMIX primer solution.)

The REVERSE primer solution used was prepared by preparing the 303R primer and the 304R primer at a concentration of 100 M each.

3. Amplification reaction process (Step S 3)

In this step, a PCR amplification reaction is performed using the aforementioned primers.

0.1 g of genomic DNA to be 铸, 4 L of 1.25 mM dNTP mixed solution, 10 L of 10 XAmpliTaq Buffer, 0.125 L of AmpliTaa polymerase, described above Add 1 L of each of the 303FMIX primer solution, 304FMIX primer solution, 303R primer solution, and 304R primer solution, and add sterile water to a total volume of 25 / iL, and mix. The PCR reaction conditions were the same as the initial denaturation conditions at 95 ° C for 5 minutes, and the amplification temperature cycle was 30 times at 95 ° C for 30 seconds, at 57 for 30 seconds, and at 72 ° C for 30 seconds 30 times. Finally, the extension reaction is performed at 72 ° C for 7 minutes. After the reaction, amplification was confirmed by well-known gel electrophoresis.

4. Blunt 3 'end of PCR product

0.5 units of Klenow Fragment was added to 5 L of the PCR reaction solution, and reacted at 37 ° C for 30 minutes. After the reaction, 1 L of a 10 O mM EDTA solution was added.

5. Detection process (Step S5)

In this step, the generated DNA prepared in the above step is detected. Various detection methods' devices can be applied. In this embodiment, a case in which SSCP analysis is performed by a capillary electrophoresis device will be described.

As a capillary electrophoresis device, one equipped with 16 capillary arrays and capable of electrophoresis simultaneously was used. Commercially available instruments of comparable performance include, for example, ABI PRISM ™ 3100 Genetic Analyzer from Applied Biosystems. Using this device, a uniquely created capillary with an inner diameter of 75 m and a detection length of 36 cm is used to fill and detect a proprietary GeneScan polymer that has been independently concentrated and adjusted.

The reaction solution containing the resulting DNA subjected to the amplification reaction step and 3′-end blunting is mixed with 37 L of formamide containing an appropriate amount of a well-known labeled fragment marker (oligomer of known base length) at 94 ° C. Heat denaturation for 2 minutes. After that, use ice to prepare a sample solution for capillary electrophoresis. Dilute the reaction solution containing the generated DNA as necessary. The electrophoresis conditions were as follows: the control temperature of the capillary was 22.5, the sample injection conditions were 20 KV for 5 seconds, and the electrophoresis separation was 15 KV for 70 minutes. The signal strength to be analyzed is based on the electrophoresis data obtained as standard, and the baseline and amplitude are changed independently. 6. Analysis of LOH (Loss of heterozygosity) (Step S 6)

In FIGS. 6 and 7, the solid line is 303 and the dashed line is the peak of the generated DNA amplified from the 304 gene. Type III is genomic DNA, and the target nucleic acids are amplified from both the paternal and maternal genomes, so that genes 303 and 304 appear as two peaks each. Diagnosis can be performed by analyzing these two peaks. In Fig. 6, the peak intensity obtained from 304 and the peak intensity obtained from 303 are almost equal, but in Fig. 7, the peak intensity obtained from 304 is lower than the peak intensity obtained from 303. Of the two peaks, the height of the peak detected earlier in time is Al, and the height of the peak detected later is A2.

min. (Al, A2) / max. (Al, A2) · · · · · The ratio of LOH can be determined from the formula (1).

In Fig. 6, the calculation of (1) was performed after minimizing the effect of each peak using the conversion formula.

In Fig. 7, the calculation of (1) was directly performed using the obtained peak height. As a result, almost the same calculation results were obtained for both, as shown in Table 1.

Table 1 LOH analysis results of each PCR product

Although not performed in this analysis, diagnosis is actually performed in consideration of the ratio of normal genes (JP-A-9-201199, etc.). For example, the percentage of cancer cell-derived genes in cancer tissue is

{A1 / A2 (gene from normal tissue) -A1 / A2 (gene from cancer tissue)} / {A1 / A2 (gene from normal tissue)}

Calculate with In diagnosis, a certain percentage is set and the value is used as a criterion for judgment. In this case, the calculation results are affected by each other's peak (example For example, in consideration of 303 and 304) used in the embodiment, it is necessary to set a certain range around the set value as false positive or false negative.

In the above examples, a nucleic acid was exemplified as a biological sample. However, the present invention is not limited to this, and the present invention is not limited to this and can be applied to other biological samples having the problems presented in the present invention. It is possible. Industrial applicability

According to the present invention, a plurality of labeled nucleic acids and proteins can be detected and quantified with higher precision as compared with conventional methods. In particular, a great effect can be obtained when using fluorescent and luminescent materials whose wavelength bands overlap.

The amount of fluorescent, luminescent, and radioactive isotopes used for labeling is reduced, and the number of specimens per analysis can be increased, thereby reducing costs for producing and analyzing labeled biological samples.

Claims

The scope of the claims

1. In a method of analyzing two or more biological samples by synthesizing two or more biological samples, adding labels with different wavelengths to the synthesized biological samples, and detecting those labels,

The detection intensity of the label of the biological sample is adjusted for each type of the biological sample, and in particular, among the detected labels, the detection intensity of the one whose wavelength band has a large influence on the other label is weakened, whereby the biological intensity is reduced. A biological sample analysis method for analyzing a sample.

2. By synthesizing two or more types of biological samples and adding labels with different wavelengths to each type or adding a label binding medium to the substances used in the synthesis (hereinafter referred to as “synthetic substances”). In a method of analyzing a biological sample by labeling a biological sample during or after the synthesis and detecting those labels, a method of labeling or label-binding medium added to at least one type of synthetic substance is used. A method for analyzing a biological sample in which a biological sample is labeled by adjusting the amount so that the amount of a label added to another type of synthetic substance is smaller than that of a label binding medium.

3. In claim 2, the two or more kinds of biological samples are nucleic acids synthesized by a polymerase chain reaction (hereinafter, referred to as “PCR”) method, and the substance for synthesis is a primer. The method for analyzing a biological sample, wherein the label is a fluorescent substance, a luminescent substance, or a radioisotope, and the label binding medium is a modifying group or an antibody.

4. The label according to claim 2, wherein the label is a labeled compound having a spectrum with a steep slope on the short wavelength side and a gentle slope on the long wavelength side with respect to the maximum wavelength, and a label added to a plurality of types of synthetic substances. The amount of the label binding medium is such that the amount of the label having a shorter wavelength or the label binding medium binding to the label is smaller than that of the longer wavelength label or the label binding medium binding to the label. Sample analysis method.

5. The amount of the label or label binding medium added to at least one type of synthetic substance and the label or label binding medium added to another type of synthetic substance according to claim 2. The ratio is set to 1: 2 to 1:50, preferably 1: 5 to 1:10. Analysis method of the biological sample to be determined.

6. In Claim 2, the two or more biological samples are nucleic acids synthesized by a polymerase chain reaction,

The reaction solution for synthesizing the biological sample contains, as the substance for synthesis, a primer modified with the label or the modifying group or antibody serving as the label binding medium, and the label or the modifying group or the antibody. Method for analyzing biological samples that contain no primers.

7. The method according to claim 2, wherein the two or more biological samples are nucleic acids synthesized by a polymerase chain reaction,

The reaction solution for synthesizing the biological sample contains, as the substance for synthesis, a primer modified with the label or the modifying group or antibody serving as the label binding medium, and the label or the modifying group or the antibody. A method for analyzing a biological sample in which the number of primers is adjusted to a ratio of 1: 1 to 1:50, preferably 1: 5 to 1:10 by type.

8. In Claim 2, the label is a phosphor,

This phosphor is CyDye, Carboxyfluorescein (FAM),

tluorescem-5 "isothiocyanate (FITC)> hexachlorofluorescem (HEX), rhodamine (ROX), carboxytetramethylrhodamine (TAMRA),

A method for analyzing a biological sample that is either tetrachlorofluorescein (TET) or sulforhodamine 101 acid chloride (trade name: Texas Red®).

9. In Claim 2, the marker is a luminous body,

This phosphor sample

Biological sample that is one of 3- (2'spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) -phenyl-l, 2-dioxetane (trade name: AMPPD®), CSPDTM, MDPTM, CDPTM Analysis method.

10. The method according to claim 2, wherein the label is a radioisotope modified on a biological sample, The radioisotope is any one of ³² P, 131 1, ³⁵ S, 45 _Ca , ³ H, and ¹⁴ C. A method for analyzing a biological sample.

1.1. A reaction solution containing a substance used for the synthesis of two or more biological samples (hereinafter referred to as “synthesis material”). The reaction solution contains a label or a label for identifying the type of biological sample. A synthesis substance to which a label binding medium is added, and a synthesis substance to which no label is added, wherein the label is a substance identified by wavelength,

A biological sample in which the rate of addition of a label or label binding medium of at least one type of synthetic substance is adjusted to be smaller than the rate of addition of a label or label binding medium of another type of synthetic substance. Reaction solution.

12. The method according to claim 11, wherein the reaction solution is a primer used for a PCR method, the label is a fluorescent substance, a luminescent substance, or a radioisotope, and the label binding medium is a modifying group or an antibody. Reaction liquid of a certain biological sample.

13. The label according to claim 11, wherein the label is a label compound having a spectrum with a steep slope on the short wavelength side and a gentle slope on the long wavelength side with respect to the maximum wavelength. The reaction solution of the shorter label or the biological sample that is the medium for binding the label.

1 4. The method according to claim 11, wherein a label or label binding medium added to at least one kind of synthetic substance, and a label or label binding medium added to another kind of synthetic substance. A reaction solution of a biological sample, wherein the quantitative ratio of 1: is set to 1: 2 to 1:50, preferably 1: 5 to 1:10.

15. A detector that measures the synthesized biological sample via a label attached to the biological sample, and has a function of analyzing one or more biological samples based on the spectrum measured by the detector. In a biological sample analyzer comprising a computing device,

When analyzing two or more types of mixed biological samples, the arithmetic unit determines the rate of addition of a label or a label-binding medium added to at least one type of biological sample for synthesis. When the rate of addition of the label or label binding medium added to the substance for synthesis of various biological samples is smaller than the rate of addition, the measurement data of those labels is supplemented. A biological sample analyzer that can be used in analytical calculations without correction.

16. A detector that measures the synthesized biological sample via a label attached to the biological sample, and has a function of analyzing one or more biological samples based on the spectrum measured by the detector. In a biological sample analyzer comprising a computing device,

When analyzing two or more types of mixed biological samples, the arithmetic unit determines the rate of addition of a label or a label-binding medium added to at least one type of biological sample for synthesis. When the rate of addition of a label or a label binding medium added to a substance for synthesis of a biological sample of a different type is made smaller than the rate of addition, data relating to the rate of addition can be input, and each biological sample can be input based on the rate of addition data and measurement data. A biological sample analyzer that calculates the amount of a sample.