JPH0484899A - Determination of nucleic acid - Google Patents

Abstract

PURPOSE:To ensure the title determination in high sensitivity and efficiency in a short time by ultrasonic treatment of nucleic acid. CONSTITUTION:Single-stranded nucleic acid obtained by thermal modification with e.g. NaOH of nucleic acid obtained by extracting e.g. leukocytes is put to a ultrasonic treatment such as thermal process ultrasonics into nucleic acid fragment of plural Kb (A). Second, (1) the component A containing the target nucleic acid and (2) a scavenger probe are mutually bound and immobilized on an immobilization filter into a two-body-bound hybrid (B). Third, the component B is washed and freed from the component A unimmobilized followed by contact of a labeled probe such as radioisotope into scavenger probe-target nucleic acid-labeled probe, a three-body-bound sandwich hybrid (C). Thence, the component C is washed and freed from free labeled probe for unformed hybrid followed by either directly or indirectly detecting the presence of label, thus determining the desired nucleic acid.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a method for more effectively carrying out nucleic acid hybridization, and is therefore suitable for, for example, nucleic acid measurement using sandwich hybridization. It concerns a method.

(Prior art and its section 8) By using nucleic acid hybridization and using a nucleic acid probe that has a specific base sequence, it is possible to measure nucleic acids that have a base sequence portion that is complementary to this specific base sequence. It is.

Conventional methods for measuring nucleic acids using nucleic acid probes include filter hybridization, liquid phase hybridization, sandwich hybridization, and the like (see DNA probes, CMC Publishing, etc.).

In the sandwich hybridization measurement method, at least two types of nucleic acid probes complementary to the target nucleic acid to be identified are used. One nucleic acid probe is bound to a solid phase and used as a capture or capturing reagent (hereinafter referred to as a capture probe), and the other nucleic acid probe is labeled and used as a detection reagent (hereinafter referred to as a labeled probe). Labeled probes can be labeled with radioisotopes, fluorescent dyes,
Enzymes and the like are used. Sand German hybridization can be carried out by adding a sample containing a denatured nucleic acid, a capture probe, and a labeled probe together or sequentially to a hybridization solution. If a target nucleic acid is present in a sample, a hybrid is formed in which the three are bound together, such as a capture probe, a target nucleic acid, and a labeled probe. Next, free nucleic acid fragments that did not form a hybrid and free labeled probes are separated. If the capture probe is immobilized on the filter, the filter can be washed in a wash solution.

If the capture probe is immobilized on a gel, the gel can be filtered on a filter or collected by centrifugation and separated from free labeled probe. The amount of target nucleic acid can be determined by measuring the amount of label contained in the hybrid after separation.

There are other methods, such as binding a sample nucleic acid to a suitable solid phase, adding a labeled probe thereto, washing it, and measuring whether or not there is a target nucleic acid that forms a hybrid with the labeled probe. The sandwich hybridization measurement method is considered to be more suitable for achieving higher sensitivity because of the ease of pretreatment such as binding the sample nucleic acid to a solid phase and the ability to reduce background.

In the above measurement method, the samples include animal cells such as white blood cells, kidney cells, liver cells, etc., microorganisms such as bacteria and viruses, and even nucleic acids (DNA, RNA) such as plant cells.
use. Nucleic acids (DNA, RNA) can be extracted from these samples using the phenol method, guanidine thiocyanate method, etc. (Biochemistry Experiment Course - Chemistry of Nucleic Acids 2)
, Tokyo Kagaku Doujin).

However, if a sample extracted by this method is used as is, the target nucleic acid is long, ranging from several kilobases to several hundred kilobases, so hybrids (capture probe...target nucleic acid, capture probe...target nucleic acid...・It takes several hours to more than 10 hours to form labeled probes, and it takes a long time to obtain results, and there are problems in that the sensitivity does not improve.

(Means for Solving the Problems) As a result of intensive research to solve the problems seen in the prior art, the present inventor has discovered that by applying ultrasonication to nucleic acids contained in a sample, a hybrid (capture probe) ...
It has been found that formation of target nucleic acid, capture probe, target nucleic acid, label probe) takes place within several minutes to one hour.

That is, the present invention is a method characterized by using a nucleic acid subjected to ultrasound treatment as a sample for nucleic acid hybridization. The present invention will be explained in detail below.

Since nucleic acids are fragmented into fragments ranging from hundreds of bases to several kilobases by sonication, it is necessary to select the base sequence so that the capture probe and/or labeled probe hybridizes to the fragmented nucleic acid fragments. . When a sample is a nucleic acid with a length of several hundred bases, the efficiency of hybrid formation is naturally high, so even if you apply ultrasound, it will be more effective than applying ultrasound to a nucleic acid with a length of a few kilometer to a few hundred kilobases. Since the effects are small, the invention is preferably directed to nucleic acids of a few kilobases.

In sandwich hybridization, if the capture probe and labeled probe are far apart, they may be cut by ultrasonication and a hybrid (capture probe, target nucleic acid, labeled probe) may not be formed. Therefore, it is best to keep the recognition sites of the capture probe and labeled probe close to each other. The distance between the portion of the target nucleic acid complementary to the capture probe and the portion complementary to the labeled probe is preferably within several kilobases, more preferably within several hundred bases. In addition, to increase sensitivity in measurements, one type of capture probe and two
It is good to combine more than one type of labeled probe, or to combine several types of capture probes and several types of labeled probes.

The present invention is applicable not only to sandwich hybridization measurement methods, but also to hybridization measurement methods that use only labeled probes. Further, there are no restrictions on other measurement methods as long as they involve hybridization of nucleic acids.

Ultrasonic treatment is a heat system called Ultrasonics.
(Heat Systems Ultrasonics
), Towy, Rapidi
s), Raytheon, Muraldo (
Mullard and BRANSON
This can be carried out using commercially available equipment such as. In addition,
Available commercial sonication devices use one of two modes: That is, (1) immersing the vibrating probe directly into the sample, and (2) immersing the container containing the sample into the bath in contact with the vibrating transducer.

The first mode is disadvantageous in that it introduces sample carryover from sample to sample, and is therefore problematic when implementing the invention for clinical trials. It is preferable to use the second mode because the liquid in the bath serves to transmit the ultrasonic vibrations to the sample in the container and also serves to cool and control the sample temperature. .

Sample nucleic acids of the present invention include animal cells such as white blood cells, kidney cells, liver cells, etc., microorganisms such as bacteria and viruses,
Furthermore, nucleic acids (DNA, RNA, etc.) extracted from plant cells, etc.
), but it is also possible to directly apply ultrasound treatment to cells, etc. from which nucleic acids have not been extracted.

The capture probe and/or labeled probe used in the nucleic acid hybridization of the present invention is synthesized by chemical synthesis, so in consideration of synthesis yield and specificity to the target nucleic acid, a synthetic NA with a length of up to 100 bases may be used. . A filter or gel can be used as the solid phase for immobilizing the capture probe or target nucleic acid. Radioisotopes, fluorescent dyes, enzymes, etc. can be used to label the labeled probe.

Below, a sandwich hybridization measurement method incorporating a step of fragmenting a sample nucleic acid by subjecting it to ultrasonication in a solution will be described in detail.

(1) During the step of fragmenting the sample nucleic acid into nucleic acid fragments by subjecting the sample nucleic acid to ultrasound treatment in a solution (hereinafter referred to as a), the sample nucleic acid is converted into single-stranded nucleic acid by heat or denaturing agent treatment. . Modification may be performed before step (a), simultaneously with step (a), or after step (a). Sodium hydroxide, formamide, etc. can be used as a denaturing agent.

Next, the step of contacting the nucleic acid fragment with a capture probe (
Perform the following). If the target nucleic acid is present in the sample, a hybrid is formed in which the three components bind together like the capture probe and the target nucleic acid.

Next, a first washing step (c) is performed to remove nucleic acid fragments not captured by the capture probe. If the capture probe is immobilized on the filter, the filter can be washed in a wash solution.

If the capture probe is immobilized on a gel, the gel can be filtered on a filter or collected by centrifugation to remove free nucleic acid fragments not captured by the capture probe.

Next, a step (hereinafter referred to as d) in which the nucleic acid fragment captured by the capture probe is brought into contact with a labeled probe is carried out, and a l form the body.

Next, a second washing step (hereinafter referred to as e) in which free labeled probes that have not formed a hybrid is washed is performed in the same manner as the first washing step (c).

Finally, a step (hereinafter referred to as f) of directly or indirectly detecting the presence of the label is performed.

(2) The step (a) and the modification at that time are the same as in (1) above. Next, the nucleic acid fragment is brought into contact with the capture probe for a certain period of time and then brought into contact with the labeled probe (b) to form a hybrid body (capture probe...target nucleic acid...labeled probe). let

Next, a first washing step (C') is performed to remove nucleic acid fragments that were not captured by the capture probe and labeled probes that were not captured. If the capture probe is immobilized on the filter, the filter can be washed in a wash solution. If the capture probe is immobilized on a gel, the gel can be filtered on a filter or collected by centrifugation to remove free nucleic acid fragments and labeled probes that are not captured by the capture probe.

Finally, a step (f) of directly or indirectly detecting the presence of the label is performed.

(3) The step of (a) and the modification at that time are the same as in the above-mentioned (1). Next, a step (b') of simultaneously contacting the nucleic acid fragment with a capture probe and a labeled probe is carried out to form a hybrid (capture probe...target nucleic acid...
...labeled probe) is formed.

Next, a first washing step (C') is performed to remove nucleic acid fragments that were not captured by the capture probe and labeled probes that were not captured. If the capture probe is immobilized on the filter, the filter can be washed in a wash solution. If the capture probe is immobilized on a gel, the gel can be filtered on a filter or collected by centrifugation to remove free nucleic acid fragments and labeled probes that are not captured by the capture probe.

Finally, a step (f) of directly or indirectly detecting the presence of the label is performed.

(4) Step (a) and its denaturation are the same as in (1) above, but these steps are performed in the presence of a complementary probe and a labeled probe. However, if a fluorescent dye or enzyme is used to label the labeled probe, conditions should be selected so that it will not be degraded or deactivated by the heat or denaturing agent applied during nucleic acid denaturation. Next, a first washing step (c) is performed to remove nucleic acid fragments that were not captured by the capture probe and labeled probes that were not captured. If the capture probe is immobilized on the filter, the filter can be washed in a washing solution.

If the capture probe is immobilized on a gel, the gel can be filtered on a filter or collected by centrifugation to remove free nucleic acid fragments and labeled probes that are not captured by the capture probe.

Finally, a step (f) of directly or indirectly detecting the presence of the label is performed.

(Effects of the Invention) According to the present invention, the time for forming a hybrid (capture probe...target nucleic acid, capture probe...target nucleic acid...labeled probe) can be shortened.

This makes it possible to more efficiently perform measurement methods that utilize nucleic acid hybridization, such as sandwich hybridization measurements. That is, if the time for hybrid formation in such measurements is shortened, it is possible to increase the number of specimens that can be processed in a given time.

Furthermore, according to the present invention, since the efficiency of hybrid formation increases, it is also possible to achieve an increase in sensitivity in a method for measuring nucleic acids using nucleic acid hybridization.

(Examples) The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 ■ Fragmentation of plasmid DNA by ultrasound Plasmid DNA incorporating hepatitis B virus as a sample
Cplasmjd Hepatjtis B vir
us.

Hereinafter abbreviated as pHBV; Proc, Natl, Sci
, LISA, Vol. 78, pp. 2602-2610, 1981) was used. pHBV is a 7.6 kilobase circular DN
It is A.

After dispensing a solution containing 800 ng of pHB V in 20 ul of pure water into a microcentrifuge tube, add aqueous sodium hydroxide solution (
Add 20u1 of 0.6M) to convert double-stranded DNA to single-stranded DNA.
degenerated into. The above microcentrifuge tube was cleaned using a Velveosonic ultrasonic cleaner (model VS-20, manufactured by Honda Electronics Co., Ltd.).
Ultrasonication was performed at room temperature for 1 minute at a rated power consumption of 32 watts and a rated frequency of 50/60 hertz. As a result of 1% agarose electrophoresis, it was confirmed that the DNA fragments were fragmented into 200 to 400 base DNA fragments.

(2) Sandwich hybridization measurement The pHBV fragment prepared in (3) was brought into contact with a capture probe and a labeled probe to perform sandwich hybridization measurement.

Capture probe for pHBV (synthetic NA.

21mer, 5--TGTACAGACTTGGCC
1 mg of gel (non-porous carrier NPR, 2.5 um, manufactured by Tosoh Corporation) on which CCCAAT-3-) was immobilized,
Hybridization buffer (Na
CI 75mM, 5odiulcitrate d
ihydrate 75mM, Bovjne seru
IIlalubumin 0.5%, Polyvjny
pyroljdone 0.5%, sodium do
decyl 5ulfate 1%) and the pHBV fragment prepared in ① was added to 2M Atpmniug Ace.
After neutralization with tate, 1 oopg of pHBV fragment ~11
00n was added and allowed to react for 10 minutes. After 10 minutes, a labeled probe labeled with alkaline phosphatase (synthetic NA moiety, 21mer, 5--QGTAACAGC) was added to the mixture after 10 minutes.
GGTATAAAAGGGAC3', 9 bases apart from the capture probe) was added and further hybridized at 50°C for 10 minutes.

Next, in order to separate the free labeled probe, the mixture was centrifuged (centrifuge MR-150 (manufactured by Tomy Co., Ltd.)) to sediment the gel, and the supernatant was discarded. Washing solution (KSCN 0.15M, Sodium curat) heated to 50°C
e dIhydrate 155M, Tveen2D
1%), centrifuged again to sediment the gel, and discarded the supernatant. This washing operation was repeated five times. Furthermore, the gel was soaked in an enzyme reaction buffer (2-Amjno-2-netyl
-1-propanol 0°5M, pH 9,0),
The fluorescent substrate for alkaline phosphatase (4-methylumbelliferyl-
phosphate, 21M) solution was added to measure the decomposition rate of the fluorescent substrate at 37°C (AIA-1200 (Tosoh)
Co., Ltd.)).

The results are shown in Figure 1.

Comparative Example 1° To examine the effect of ultrasonication, an experiment similar to Example 1 was conducted using pHBV that was not subjected to ultrasonication as a control. The results are shown in Figure 1.

According to FIG. 1, it can be seen that the decomposition rate of the fluorescent substrate is more than 10 times faster when using ultrasonicated pHBV as a sample than when untreated, and the decomposition rate also has good dependence on the sample amount.

Example 2゜The pHBV fragment prepared in Example 1 was used as a capture probe and
Sandwich hybridization measurements were performed by contacting each type of labeled probe separately.

capture probe, gel, hybridization buffer,
The same cleaning solution as in Example 1 was used.

1 mg of gel immobilized with capture probe for pHBV
was suspended in hybridization buffer heated to 50°C, and the pHBV fragment prepared in Example 1 was added to 2M Am
After neutralization with mnium acetate, 1100 n of pHBV fragment was added and reacted for 10 minutes. After 10 minutes, labeled probe 1, labeled probe 2, and labeled probe 3 labeled with alkaline phosphatase were separately added to this mixture, and the mixture was reacted for an additional 10 minutes.

The labeled probes are as follows.

Labeled probe 1 (synthetic NA moiety, 21mer, 5--
TGGCACTAGTAAACTGAGCCA-3-1
65 bases apart from the capture probe) Labeled probe 2 (synthetic NA moiety, 21 mar.

5″-GACAAACGGGCAACATACCT
277 bases apart from the T-3-1 capture probe) Labeled probe 3 (synthetic NA moiety, 21mer.

5=-ATTGAGAGAAGTCCACCACGA-
3-1 capture probe and 479 bases apart). To separate the free labeled probe, the mixture was then centrifuged to sediment the gel and the supernatant was discarded. The gel was suspended in a washing solution heated to 50°C, centrifuged again to sediment the gel, and the supernatant was discarded. This washing operation was repeated five times. As in Example 1, the gel was further suspended in an enzyme reaction buffer, an equal volume of a fluorescent substrate solution was added, and the decomposition rate of the fluorescent substrate was measured at 37°C.

The results are shown in Figure 2. It can be seen that the decomposition rate of the fluorescent substrate decreases as the distance between the capture probe and the labeled probe increases.

Example 3゜■Plasmid D incorporating human urokinase gene as a fragmentation sample of plasmid DNA by ultrasound
NA (hereinafter abbreviated as pSV2UK; Agric, Bi
ol, Chem, volume 52(2), pages 329-338,
(1988) was used. pSV2UK is a circular DNA of approximately 6 kilobases.

After dispensing a solution containing 400 ng of pSV 2 U K in 20 μl of pure water into a microcentrifuge tube, add 20 μl of aqueous sodium hydroxide solution (0.6 M). In addition, double-stranded DNA was denatured into single-stranded DNA. The above microcentrifuge tube was subjected to ultrasonication for 1 minute at room temperature in the same manner as in Example 1.

In addition, as a result of 1% agarose electrophoresis, 200
It was confirmed that the DNA was fragmented into 400 base DNA fragments.

(2) Sandwich hybridization measurement The pSV2UK fragment prepared in (3) was brought into contact with the capture probe and the labeled probe, and sandwich hybridization measurement was performed in the same manner as in Example 1.

Capture probe for pSV2UK (synthetic NA,
21mar, 5--ACCAGCCCTGGTT-
111 g of gel immobilized with TGCGGCCA-3-) was suspended in a hybridization buffer heated to 50°C, and the pSV2UK fragment prepared in ① was added to 2M^mmmn.
After neutralizing with 1u Acetate, the pSV2UK fragment was
00pg-1100p were added and reacted for 10 minutes.

After 10 minutes, a labeled probe labeled with alkaline phosphatase (synthetic NA moiety, 21mer, 5-
-TTTCAGTGTGGCCAAAGACT-3-
1 capture probe (49 bases apart) was added and further hybridized at 50°C for 10 minutes. To separate free labeled probe, the mixture was then centrifuged to sediment the gel and the supernatant was discarded. The gel was suspended in a washing solution heated to 50°C, centrifuged again to sediment the gel, and the supernatant was discarded.

This washing operation was repeated five times. As in Example 1, the gel was further suspended in an enzyme reaction buffer, and the fluorescent substrate solution was added in equal amounts to measure the decomposition rate of the fluorescent substrate at 37°C.

The results are shown in Figure 3.

Comparative Example 2 In order to examine the effect of ultrasonication, an experiment similar to Example 3 was conducted using pSV2UK which was not subjected to ultrasonication as a control.

Figure 3 shows the experimental results. According to FIG. 3, it can be seen that the decomposition rate of the fluorescent substrate is more than 10 times faster when using ultrasonicated pSV2UK as a sample than when untreated, and the decomposition rate also has good dependence on the sample amount.

FIG. 2 shows the measurement results of Example 2.

As in FIG. 1, the vertical axis shows the decomposition rate of the fluorescent substrate, and the horizontal axis shows the distance between the capture probe and the labeled probe.

FIG. 3 shows the measurement results of Example 3 and Comparative Example 2. As in Figure 1, the vertical axis shows the decomposition rate of the fluorescent substrate, and the horizontal axis shows the decomposition rate of the fluorescent substrate added as a sample ff1 (pSV2UKj; t
) is shown. The solid line shows the calibration curve when pSV2UK subjected to ultrasonic treatment is used as a sample, and the broken line shows the calibration curve when pSV2UK is not treated.

[Brief explanation of the drawing]

FIG. 1 shows the measurement results of Example 1 and Comparative Example 1. The vertical axis shows the decomposition rate of the fluorescent substrate (4-methylumbelliferyl-phosphate) by the labeled probe alkaline phosphatase, and the horizontal axis shows the amount of sample added as a sample ('pHBvjl). The solid line shows the calibration curve when pHBV subjected to ultrasonic treatment is used as the sample, and the broken line shows the calibration curve when the sample is untreated.

Claims (1)

[Claims] (1) A method characterized in that a nucleic acid subjected to ultrasound treatment is used as a sample for nucleic acid hybridization.