JPH0458900A - Detection of expectedly existing nucleic acid and kit therefor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to nucleic acid extracts from blood (blood cells, serum, plasma), bile, pus, cerebrospinal fluid, feces, saliva, speech, animal and plant tissues, or cells. It relates to a method for detecting nucleic acids having a specific base sequence part that is expected to be present in biological samples such as . The present invention also relates to a reagent kit for carrying out such a detection method.

(Prior art and its problems) Conventionally, in fields such as clinical testing, for example, when a bacterial infection is suspected, the presence of the specimen is detected through a stained microscope, and then the presence of the disease is detected. A method is known in which the bacterial species is identified by culturing this specimen in various media.

Although this method has the characteristic of being able to identify the type of bacteria, etc., it requires a long period of time to perform as it requires culturing the specimen, and there is a risk of infection for the person performing the method. There are issues such as the need for culture equipment, etc.

On the other hand, DNA
A diagnosis is being made. For DNA diagnosis, a method is known that uses a nucleic acid probe having a complementary base sequence to a specific base sequence in a nucleic acid (usually a characteristic base sequence part in a nucleic acid). For more information on methods using nucleic acid probes, please refer to the dot plot method, Sand-Deutsch method,
There are column methods, etc. The generally widely used dot plot method immobilizes a sample nucleic acid converted into a single-stranded nucleic acid on a filter, and then labels it with a nucleic acid probe designed to be complementary to a specific base sequence in the predicted nucleic acid. This method consists of adding a sample labeled with a 2-labeled compound to the sample, washing and removing unhybridized nucleic acid probes, and finally detecting the labeled substance in the components that were not removed.

Detection methods using nucleic acid probes, such as the dot plot method, are frequently used because they have good detection sensitivity and can be carried out with simpler operations compared to the methods that involve culture operations described above. It's being used.

However, in the above methods using nucleic acid probes, either the probe or the nucleic acid to be detected is immobilized on an appropriate solid phase, such as immobilizing the sample nucleic acid on an appropriate filter in the dot plot method. There is a problem in that pretreatment operations such as immobilization operations are required. Furthermore, in the methods using these nucleic acid probes, one type of nucleic acid probe is usually used in one operation, and although it is suitable for detecting the presence or absence of one type of nucleic acid in a sample, However, in order to simultaneously detect the presence of multiple types of nucleic acids, there are problems such as the need to attach different labels to each probe and perform detection operations for each of these labels.

In view of the conventional techniques and their problems as described above, the present inventor has developed a method for determining whether or not multiple types of predicted nucleic acids exist in a sample without performing pretreatment such as fixation of nucleic acids. We have conducted extensive research with the aim of making it possible to detect specific nucleic acids by measuring their degree. the result,
Multiple types of nucleic acid primer sets consisting of two nucleic acid primers designed to produce a synthetic nucleic acid with a different number of bases than the base group when a nucleic acid elongation reaction is performed, and DNA polymerase necessary for the elongation reaction. The inventors have discovered that these problems can be solved by using a suitable substrate, etc., and have completed the present invention.

(Means for Solving the Problems) The present invention, which has been completed with the above-mentioned objectives, has the following features:
A first nucleic acid primer capable of converting these nucleic acids into single strands when they are expected to be double stranded and hybridizing to a specific base sequence portion in the single stranded nucleic acids that are expected to exist. A plurality of sets of nucleic acid primers consisting of a second nucleic acid primer capable of hybridizing to a specific base sequence portion in a nucleic acid complementary to this single-stranded nucleic acid, and for a nucleic acid elongation reaction using these nucleic acid primers as a starting point. DNA polymerase and its substrate, etc. are added to realize a nucleic acid elongation reaction, and a double-stranded nucleic acid containing a synthetic nucleic acid in which the first nucleic acid primer is located at least at its 5-terminus appears, and then these two The double-stranded nucleic acid is converted into a single-stranded nucleic acid, and a nucleic acid elongation reaction is further realized in the presence of the multiple types of nucleic acid primer sets, and the second nucleic acid primer is located at least at the 5' end of the nucleic acid primer, A synthetic double-stranded nucleic acid containing a synthetic nucleic acid having a complementary base sequence to the first nucleic acid primer appears at the 3-end, and further converting the double-stranded nucleic acid into a single-stranded nucleic acid as necessary. After repeating the elongation reaction of the nucleic acid, the synthetic nucleic acids appearing in the sample that have one of the nucleic acid primers at the 5' end and a base sequence complementary to the other nucleic acid primer at the 3' end are separated. A method for detecting a nucleic acid predicted to exist, which consists of adding the second nucleic acid primer to the 5' end and the first nucleic acid primer to the 3' end, which appear in relation to each nucleic acid primer set. The synthetic nucleic acid having a complementary base sequence and/or the first
A synthetic nucleic acid having a base sequence complementary to the second nucleic acid primer at its 3-end has a different number of bases from similar synthetic nucleic acids appearing in relation to other nucleic acid primer sets. and a second nucleic acid primer constituting each nucleic acid primer set are designed, and the method is characterized in that the separation of the synthetic nucleic acids is achieved by utilizing the difference in the number of bases of the synthetic nucleic acids. be. The present invention also provides at least a first nucleic acid primer capable of hybridizing to a specific base sequence portion in a single-stranded nucleic acid that is expected to exist, and a specific base sequence in a nucleic acid complementary to this single-stranded nucleic acid. A plurality of sets of nucleic acid primers consisting of a second nucleic acid primer capable of hybridizing to a portion of DNA and a DNA for a nucleic acid extension reaction using these nucleic acid primers as a starting point
This is a reagent kit for detecting a predicted nucleic acid containing polymerase and its substrate. The present invention will be explained in detail below.

The present invention utilizes a nucleic acid elongation reaction using DNA polymerase and a nucleic acid primer. This nucleic acid elongation reaction is generally called Polymerase Chain Reaction.
method (PCR method) (SCIENCE, 23 [1, 1350-1354゜1
985). Briefly, the PCR method consists of: - a nucleic acid primer that can hybridize to a specific base sequence part in a double-stranded nucleic acid; Nucleic acid primers made of nucleic acid primers that can hybridize to other specific base sequence portions present on the side were prepared, these nucleic acid primers were brought into contact with the single-stranded nucleic acid, and then DNA polymerase was applied to hybridize. A nucleic acid elongation reaction is performed in one direction (the 5' direction of the double-stranded nucleic acid serving as a template) of the primer. After that, the double-stranded nucleic acid consisting of the original nucleic acid and the newly synthesized synthetic nucleic acid in which the nucleic acid primer is located at the 5' end is converted into a single strand, and the same operation is repeated to convert both 5' One of the aforementioned nucleic acid primers is located at the end, and a synthetic double-stranded nucleic acid having a base sequence complementary to the other nucleic acid primer can appear at the 3' end. By carrying out 20 cycles of such a reaction, it is possible to obtain a synthetic nucleic acid that has been amplified approximately one million times.

The present invention is carried out on samples containing multiple types of nucleic acids. The nucleic acid may be DNA or RNA, but when implementing the present invention with RNA, reverse transcription is performed prior to the nucleic acid elongation reaction.
It is recommended to perform ptase (reverse transcriptase) treatment. Further, the nucleic acid only needs to exist in a single-stranded nucleic acid state in the sample. When carrying out the present invention on a double-stranded nucleic acid, it is sufficient to convert it into a -heavy chain. The nucleic acid referred to in the present invention means that either the first or second nucleic acid primer is located at the 5-end of the nucleic acid primer that finally appears in relation to a set of nucleic acid primers that will be explained later. 3'
It is a nucleic acid that includes a base sequence part consisting of the same base sequence as a synthetic nucleic acid that has a base sequence complementary to the other primer at its end. Note that, strictly speaking, the sample is a nucleic acid-containing solution that is expected to contain multiple types of nucleic acids. Specifically, these include biological samples such as blood (blood cells, serum, plasma), bile, pus, spinal fluid, feces, saliva, ruminants, and nucleic acid extracts from animal and plant tissues or cells. An example is a biological sample obtained from a certain patient.

The operation of converting a double-stranded nucleic acid into a single-stranded nucleic acid in the present invention can also be achieved by adding an appropriate chemical substance such as formaldehyde that reduces the stability of the double-stranded nucleic acid, but in this case, the When hybridizing a nucleic acid primer to a nucleic acid converted into a single strand, operations such as lowering the concentration of the chemical substance are required. Therefore, a conversion operation by thermal denaturation, which has been conventionally employed in PCR methods, is preferable.

In the present invention, - a first nucleic acid primer capable of hybridizing to a specific base sequence portion in a heavy chain nucleic acid; A plurality of nucleic acid primer sets consisting of the following nucleic acid primers are used. The multi-species set includes two types: a nucleic acid primer set designed as described above for at least one type of nucleic acid, and a nucleic acid primer set designed as described above for another type of nucleic acid different from this set. This means using more than one set. Here, it is preferable to select, as the specific base sequence portion, a portion that is characteristic enough to distinguish the nucleic acid expected to exist from other nucleic acids. In other words, a nucleic acid primer designed to hybridize with a specific base sequence part in a certain nucleic acid cannot (is difficult to) hybridize with other parts in the nucleic acid or other types of nucleic acids. Preferably, it is a specific part.

At present, as a result of advances in the analysis of nucleic acids of viruses, bacteria, etc. that cause various diseases, the specific base sequences of many viruses and nucleic acid species are now known. Furthermore, for example, a combinator search may be performed to compare the base sequence of the target nucleic acid with that of other nucleic acids, and characteristic portions may be selected. In order to give the nucleic acid primer specificity for a specific base sequence in the heavy chain nucleic acid, the number of bases may be 15 bases, preferably 20 bases or more.

Here, the specific base sequence part in the nucleic acid is usually
Nucleic acid probes are selected differently for each nucleic acid predicted to exist, and a nucleic acid probe is designed; however, in the present invention, the same base sequence portion for different nucleic acids can be selected as a specific base sequence portion as long as the conditions explained later are satisfied. You can.

That is, it is also possible to use a common primer for two or more primer sets. For example, even if there are two sets of nucleic acid primers in which both the first and second nucleic acid primers are the same, a synthetic nucleic acid with a different number of bases than the two types of nucleic acids will appear in relation to these nucleic acid primer sets. This is the case. In addition, only one primer may be common.

In the nucleic acid primer set used in the present invention, either the first or second nucleic acid primer is located at the 5-terminus, which appears after the nucleic acid elongation reaction is performed at least twice by PCR method, and the 3-terminus is located at the 5-terminus. A synthetic nucleic acid having a complementary base sequence to the other primer is designed to appear in a synthetic nucleic acid having a different number of bases from the synthetic nucleic acid that appears in association with another set of nucleic acid primers. That is, one set of nucleic acid primers and another set of nucleic acid primers are designed such that when the primers constituting each set hybridize to a predicted nucleic acid, the spacing between the primers is different. . In addition, as will be explained later, in the present invention, the difference in the number of bases is used when separating the synthetic nucleic acid from synthetic nucleic acids derived from other types of primers, so any primers used are The resulting synthetic nucleic acids are designed to have different numbers of bases.

In the present invention, multiple types of nucleic acid primers and DNA
Substrates necessary for the elongation reaction of volumerase and nucleic acids may be added to the sample after the above-mentioned conversion of double-stranded nucleic acid into single-stranded nucleic acid, or may be allowed to coexist during this conversion. . Klenov f'ra as DNA polymerase
gment (klenov fragment)
Escherichia coli DNA poly
When using an enzyme that is easily heat-inactivated such as merase I), and when converting double-stranded nucleic acid to single-stranded nucleic acid by heat denaturation, take into account the heat denaturation of the enzyme. This is added after the conversion operation, and is added every time the thermal denaturation operation is performed in the subsequent PCR reaction cycle. Conversion of a double-stranded nucleic acid into a single-stranded nucleic acid is preferably carried out by heat denaturation for the reasons mentioned above. T to do
It is preferable to use heat-resistant DNA polymerase (Taq polymerase) isolated from herIIlus aquatlcus YTI. By using this enzyme, there is no need to add a new enzyme each time double-stranded nucleic acid is converted to single-stranded nucleic acid, and it becomes possible to perform multiple cycles of PCR by simply repeating heat treatment. .

By causing a nucleic acid elongation reaction in the coexistence of multiple sets of nucleic acid primer sets, a double-stranded nucleic acid consisting of a synthetic nucleic acid in which the first nucleic acid primer is located at the 5' end and a single-stranded nucleic acid that is predicted to exist is produced. appears.

If a nucleic acid complementary to the predicted nucleic acid exists in the sample, a synthetic nucleic acid with a second nucleic acid primer located at the 5' end and a single strand predicted to exist. Double-stranded nucleic acids consisting of single-stranded nucleic acids complementary to nucleic acids also appear. If the nucleic acid predicted to exist in the sample does not actually exist, the nucleic acid primer will not hybridize with the nucleic acid in the sample, and as a result, the nucleic acid elongation reaction using the primer as the starting point will not occur, resulting in a synthetic nucleic acid. does not appear. Furthermore, even if a synthetic nucleic acid appears as a result of hybridization of a nucleic acid primer designed for a nucleic acid predicted to exist by chance with an unrelated nucleic acid, it is possible that a synthetic nucleic acid with the expected number of bases will ultimately appear. The gender is extremely low.

Next, after converting the double-stranded nucleic acid into a single-stranded nucleic acid, a second cycle of PCR is performed to position the second nucleic acid primer at the 5' end and the first nucleic acid primer at the 3' end. A synthetic nucleic acid with a complementary base sequence appears for the first time. If a nucleic acid complementary to the predicted nucleic acid is present in the sample, a first nucleic acid primer is further located at the 5' end, and a second nucleic acid primer complementary to the second nucleic acid primer is located at the 3' end. Synthetic nucleic acids with similar base sequences have also appeared.

In any case, in the present invention, a synthetic nucleic acid with a predetermined number of bases appears by performing two cycles of PCR. In the present invention, PCR is performed for 10 or more cycles to position the second nucleic acid primer at the 5' end, and the 3''
A synthetic nucleic acid having a base sequence complementary to a first nucleic acid primer at the end; and a synthetic nucleic acid having the first nucleic acid primer at the 5' end and a base sequence complementary to a second nucleic acid primer at the 3' end. It would be good if it appeared sufficiently.

In the present invention, multiple sets of nucleic acid primers for multiple types of nucleic acids predicted to exist are used. Even if two or more types of these predicted nucleic acids exist, the synthetic nucleic acids that appear have different numbers of bases, so they can be easily separated. Moreover, if a classification test is conducted using the same separation method for nucleic acids whose number of bases is known, it can be determined which of the predicted nucleic acids the separated synthetic nucleic acids are related to. can also be easily known. There are no restrictions on the separation of synthetic nucleic acids as long as the difference in the number of bases is used. For example, electrophoresis using SDS polyacrylamide gel, liquid chromatography using size fractionation gel, etc. Can you give examples of laws, etc.

Particularly when fractionation is performed by electrophoresis or the like, detection is facilitated if the nucleic acid is later stained with a fluorescent substance that intercalates with the nucleic acid, such as ethidium bromide. Furthermore, if a suitable label is attached to the primer, its detection can be performed more easily and accurately than, for example, a method of detecting by determining UV absorption by 11Fl.

For labeling, for example, a method such as using mononucleotide triphosphate labeled with a radioactive substance as a substrate for DNA polymerase can be exemplified.

Note that the generated synthetic nucleic acids may be fractionated on double-stranded nucleic acids at the time of completion of PCR, or after this,
Synthetic double-stranded nucleic acids may be converted into single-stranded nucleic acids.

The present invention further provides a reagent kit for detecting the predicted presence of a nucleic acid as described above.

This kit includes at least a first base that can hybridize to a specific base sequence portion in a single-stranded nucleic acid that is predicted to exist.
A plurality of nucleic acid primer sets consisting of a nucleic acid primer and a second nucleic acid primer that can hybridize to a specific base sequence portion in a nucleic acid complementary to this single-stranded nucleic acid, and nucleic acid extension using these nucleic acid primers as a starting point It contains DNA polymerase for reaction, its substrate, etc. More specifically, this kit may be one in which the reagents and the like are sealed in a freeze-dried state in a reaction cup that holds a sample and provides a reaction space.

(Effects of the Invention) According to the present invention, it is possible to detect what kind of nucleic acids are present in an unknown sample.

Moreover, in the present invention, it is possible to simultaneously detect the presence of a plurality of nucleic acids, and the only operation involved is substantially converting a double-stranded nucleic acid into a single-stranded nucleic acid. Furthermore, the present invention can detect the presence of multiple types of nucleic acids by performing the test twice.

In other words, if multiple types of nucleic acid primers are designed and used for multiple types of nucleic acids that are expected to exist, then synthetic nucleic acids that appear later can be separated based on the difference in the number of bases, which is an extremely simple operation. This makes it clear which nucleic acids are actually present in the sample.

For example, in the field of clinical diagnosis, it is possible to identify, to some extent, the type of bacteria or virus infected by a patient's symptoms. Therefore, if a set of nucleic acid primers is designed based on conventional knowledge for a plurality of nucleic acids that are expected to cause infection, and the present invention is implemented, it is believed that specific infectious bacteria or viruses can be identified. Moreover, for closely related viruses, it is possible to simultaneously use a set of nucleic acid primers for nucleic acids that are common to them, and a set of nucleic acid primers designed for nucleic acid parts that code for proteins, etc. specific to each species. , it is possible to simultaneously know the presence or absence of virus infection and the type of virus.

The present invention can be carried out using widely used equipment and reagents, such as containers for holding samples and reagents for carrying out nucleic acid elongation reactions using the PCR method. Furthermore, regarding the nucleic acid primer of the present invention, 15 to 2
Since it is sufficient to have about 0 bases, it can be easily prepared using a DNA synthesizer or the like. The above shows that implementing the present invention does not require large-scale facilities and equipment as seen in the prior art.

Furthermore, the present invention does not require complicated pretreatment such as immobilization of sample nucleic acids or nucleic acid probes (nucleic acid primers), and can be easily carried out.

(Example) Examples will be described below to explain the present invention in more detail, but these examples are only examples of the present invention and do not limit the present invention.

Example BLV (Bovine Leukemia
Vjrus S VIROLOGY.

138, p. 82, 1984), plasmids plB+76Bl and plB1 according to the reference example.
7EiB2 was prepared (hereinafter referred to as BLVl and BLV2). Furthermore, D of urokinase precursor (Pro-UK)
NA (Agrlc, BiolCheI!, 52(
2), p329-336.1988) (hereinafter referred to as UK) and M13 phage DNA
(hereinafter referred to as M 13, manufactured by Takara Shuzo Co., Ltd., M 13 m
p18) were prepared, E. coli JM 109 was transformed, and 2XTY medium (composition of TY medium;
1.6% tryptone, 1% yeast extract, 0
．． 5% NaCl) overnight.

50 μl of culture medium containing each plasmid.
5X SSC solution (0,75M NaCl1,0
゜075M sodium citrate) and further 30
A 0 μm guanidine thiocyanate solution was added and stirred.

Add another 1 ml of ethanol to the solution and dilute at OoC for 15
After centrifugation at 000 rpm and discarding the supernatant, ethanol rinsing was performed.

Add each precipitate to 50μm TE warm solution 1.0mM
Tris-IICI, lmM EDTA), and a mixture of four sets of nucleic acid primers for PCR reaction was added to each solution or a mixture of the solutions at a distance of 5 μm for each sample. These primer sets were designed so that synthetic nucleic acids of 300 bases for BLV 1 i, 400 bases for BLV2, 200 bases for UK, and 142 bases for M13 appeared. It consists of a first primer and a second primer of 21 bases.

Further, a PCR reaction buffer and TAQ polymerase were added, and a temperature cycle of 94° C.-37° C.-72° C. was performed 25 times to perform 25 cycles of PCR reaction.

After the reaction was completed, 5 μm of the solution was subjected to electrophoresis using 296 agarose gel. The gel after electrophoresis was later stained with ethidium bromide and photographed under υ■ light irradiation.

The results are shown in Figure 1. According to FIG. 1, the presence of each plasmid DNA is clearly shown.

Reference Example p1317BBl and pBI76B used in Example
2 was prepared as follows.

BLV (BovJne Leukemia Vir
us, VIROLOGY.

138, p. 82, 1984), synthesized into double strands, treated with Sac I to obtain a fragment, and treated with Pst I to create plasmid pBR3.
It was incorporated into 22. The base sequences of a total of four DNA fragments obtained by treating this plasmid with Ban old were confirmed, and the fragments containing the base sequence portions shown in Figure 2 or Figure 3 were named B1 and B2, respectively, and the following operations were performed. used for.

Plasmid al1317B (manufactured by IB1) contains B
1 and B2 to prepare plasmids pI3178B1 and pB176B2 containing the respective fragments.

[Brief explanation of drawings]

FIG. 1 shows the results of Example 1. In the diagram, the first lane is M13 and the second lane is UK. 3rd lane is 13LV 1, 4th lane is IDLY
2. The fifth lane is a mixture of M13 and UK,
The 6th lane is a mixture of LIK and BLV 2,
The seventh len is a mixture of BLV 1 and BLV 2,
The eighth lane shows the results for a mixture of M 13, UK and BLV 2. Further, the numbers on the side indicate the positions when separately prepared nucleic acids each having a number of bases of 400, 300, 200, and 100 were subjected to electrophoresis under the same conditions. FIG. 2 shows a part of the base sequence of BLV I DNA fragment B1 used in Examples and the nucleic acid primer set used. The symbols in the diagram have the same meanings as those used in conventional genetics. It should be noted that there are four types of BLv gene, RNA, sonobase + 1A, G, C, and U, but in this example, since complementary NA was used, U is written as T. The numbers assigned to the base sequences are the same as those assigned in the above-mentioned report. In the figure, the primers marked with ■ are:
This is the first nucleic acid primer as used in the present invention, and is complementary to the specific underlined base sequence portion in the base sequence. The primer indicated by (2) is a second nucleic acid primer and is complementary to a specific base sequence portion that is complementary to the double underlined portion. These primer sets are shown in the figure.
The 400 base nucleic acid indicated by the broken line or its complementary 4
00 base nucleic acid appears. FIG. 3 shows a part of the base sequence of BLV 2 DNA fragment B2 used in Examples and the nucleic acid primer set used. The meanings of symbols in the figure, numbers attached to base sequences, nucleic acid primers indicated by ■ or ■, underlines attached to base sequences, etc. are the same as those in FIG. 1. These primer sets are 3 indicated by broken lines in the figure.
A 00 base nucleic acid or a 300 base nucleic acid complementary thereto appears. FIG. 4 shows a part of the base sequence of Pro UK DNA used in Examples and a set of nucleic acid primers used. The meanings of symbols in the figure, numbers attached to base sequences, nucleic acid primers indicated by ■ or ■, underlines attached to base sequences, etc. are the same as those in FIG. 1. These primer sets are designed to generate a 200 base nucleic acid indicated by a broken line in the figure or a 200 base nucleic acid complementary thereto. FIG. 5 shows a part of the base sequence of M 13 DNA used in Examples and a set of nucleic acid primers used. The meanings of symbols in the figure, numbers attached to base sequences, nucleic acid primers indicated by ■ or ■, underlines attached to base sequences, etc. are the same as those in FIG. 1. These primer sets produce a 142 base nucleic acid shown by a broken line in the figure or a 142 base nucleic acid complementary thereto.

Claims (2)

[Claims] (1) For samples that are expected to contain multiple types of nucleic acids, if these nucleic acids are expected to be double-stranded, convert them to single-stranded, and convert them to single-stranded nucleic acids that are expected to exist. A nucleic acid primer set consisting of a first nucleic acid primer capable of hybridizing to a specific base sequence in the single-stranded nucleic acid and a second nucleic acid primer capable of hybridizing to a specific base sequence in a nucleic acid complementary to this single-stranded nucleic acid. and DNA for nucleic acid elongation reactions using these nucleic acid primers as starting points.
A polymerase, its substrate, etc. are added to realize a nucleic acid elongation reaction, and a double-stranded nucleic acid containing a synthetic nucleic acid in which the first nucleic acid primer is located at least at its 5' end appears, and then these double-stranded nucleic acids are The nucleic acid is converted into a single-stranded nucleic acid and further a nucleic acid elongation reaction is realized in the presence of the plurality of sets of nucleic acid primers, and the second nucleic acid primer is located at least at the 5' end of the nucleic acid primer, and the second nucleic acid primer is located at the 3' end of the nucleic acid primer set. A synthetic double-stranded nucleic acid containing a synthetic nucleic acid having a complementary base sequence to the first nucleic acid primer appears at the end, and further converting the double-stranded nucleic acid into a single-stranded nucleic acid as necessary. After repeating the extension reaction, a synthetic compound having a base sequence similar to one of the nucleic acid primers at the 5' end that appears in the sample and a base sequence complementary to either of the other nucleic acid primers at the 3' end A method for detecting nucleic acids predicted to exist, which consists of fractionating nucleic acids, and in which the second nucleic acid primer is added to the 5' end that appears in relation to each nucleic acid primer set, and 3
A synthetic nucleic acid having a base sequence complementary to the first nucleic acid primer at the 5' end and/or a synthetic nucleic acid having a base sequence complementary to the first nucleic acid primer at the 5' end and/or a synthetic nucleic acid having a base sequence complementary to the first nucleic acid primer at the 5' end that appears in association with this nucleic acid primer set by a nucleic acid extension reaction performed as necessary. A synthetic nucleic acid having a base sequence complementary to the second nucleic acid primer at its 3' end has a different number of bases from similar synthetic nucleic acids appearing in relation to other nucleic acid primer sets. A method characterized in that first and second nucleic acid primers constituting each nucleic acid primer set are designed, and the separation of the synthetic nucleic acids is realized by utilizing the difference in the number of bases of the synthetic nucleic acids. (2) At least a first nucleic acid primer that can hybridize to a specific base sequence part in a single-stranded nucleic acid that is expected to exist, and a specific base sequence part in a nucleic acid that is complementary to this single-stranded nucleic acid. Detection of multiple types of nucleic acid primer sets consisting of hybridizable second nucleic acid primers, and nucleic acids predicted to exist containing DNA polymerase and its substrate for nucleic acid elongation reactions using these nucleic acid primers as starting points. Reagent kit for.