Patent Application
20090053820
The present invention relates to a method of testing whether a sequence of interest is contained in a sample nucleic acid. The nucleotide sequences of DNA possessed by an organism differ from each other by individual, species, habitat, and so forth. Therefore, by using the method of the present invention, it is possible to discriminate the individual, species, habitat, etc. of an organism. Recently, putting false labels on foods or mixing of genetically modified products have become issues. The method of the present invention is also applicable to judgment of such false labels or mixing.
Generally, tuna may be classified into bluefin tuna (Thunnus thynnus) grown in the Pacific Ocean, bluefin tuna grown in the Atlantic Ocean, Southern bluefin tuna (Thunnus maccoyii), big-eyed tuna (Thunnus obesus), Humphead thryssa (Thunnus albacares), albacore (Thunnus alalunga) and so forth. Conventionally, a technique called polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP; see, for example, Non-Patent Document No. 1) has been used for discriminating types of tuna (see, for example, Non-Patent Document No. 2). This technique comprises amplifying a sample DNA by PCR (see, for example, Non-Patent Document No. 3); digesting the DNA at sites of the nucleotide sequence occurring specifically in a species, lineage or individual; and making judgment. This method discriminate the species, lineage or individual of interest from the region of DNA amplified, the type of the restriction enzyme for digesting the DNA, the number of DNA fragments resulting from the digestion and the length of each DNA fragment. For example, when the tuna of interest is bluefin tuna grown in the Pacific Ocean, the ATCO region of mitochondrial DNA is selected as a sample DNA. When this sample DNA is digested with a restriction enzyme AluI after PCR amplification, five DNA fragments are generated of which the base lengths are 432, 295, 81, 66 and 41. When this sample DNA is digested with a restriction enzyme MseI eight DNA fragments are generated of which the base lengths are 255, 254, 194, 115, 41, 32, 15 and 9 are generated. When this sample DNA is digested with a restriction enzyme Tsp509I, six DNA fragments are generated of which the base lengths are 417, 217, 186, 72, 15 and 8 (see, for example, Non-Patent Document No. 2).
Similar techniques are also used in testing and judging genetically modified products.
[Non-Patent Document No. 1] G. R. Deng, “A sensitive non-radioactive PCR-RFLP analysis for detecting point mutations at 12th codon of oncogene c-Ha-ras in DNAs of gastric cancer”, Nucleic Acids Res. Jul. 11, 1988; 16(13): 6231
[Non-Patent Document No. 2] IAA Center for Food Quality, Labeling and Consumer Services/Fisheries Research Agency, “Technical Information: Manual for Typing Fishes of Thunnus”
[Non-Patent Document No. 3] Mullis, K. and five others, “Specific enzymatic amplification of DNA in vitro: The polymerase chain reaction”, Cold Spring Harb Symp Quant Biol. 1986; 51 Pt 1:263-73
The above-described technique of PCR-RFLP is a discrimination method using differences in the number of DNA fragments resulting from the digestion with a restriction enzyme and in the lengths of the individual DNA fragments. Specifically, when all the digested DNA fragments have been separated by electrophoresis, the separated individual DNA fragments appear as bands. At this time, the number of bands appearing is the number of DNA fragments; and the distance of migration of each band is the length of the relevant DNA fragment. Electrophoresis is a method of separation utilizing the effect that shorter DNA fragment migrate rapidly while longer DNA fragment migrate slowly. The lengths of DNA fragments can be determined from their distances of migration.
However, electrophoresis is not capable of sufficiently separating DNA fragments when a plurality of digested DNA fragments have similar lengths. Even when two DNA fragments are present, they appear as one band. (For example, in a fluorescently stained image of electrophoresis gel as shown in Non-Patent Document No. 2, two DNA fragments of 255 base length and 254 base length are only detected as one broad band when they were electrophoresed a distance of about 4 cm on 3% (w/v) agarose gel.) Therefore, when PCR-RFLP is performed under conditions which generate DNA fragments of similar lengths, the number of DNA fragments may be judged erroneously. Thus, the technique of PCR-RFLP is not highly reliable and unsuitable for important tests.
Further, the phenomenon that DNA fragments of similar lengths can not be separated is not a problem peculiar to PCR-RFLP, but it is a common problem encountered by every testing technique using electrophoresis. Therefore, not only PCR-RFLP but other testing techniques which determine the number of DNA fragments by electrophoresis such as qualitative PCR [for example, Japanese Unexamined Patent Publication No. 2003-009866 titled “Animal Species Discrimination Method with SINE Technique”; Notice regarding Testing Method for Foods Produced by Recombinant DNA Technique (SHOKU-HATSU No. 110 issued on Mar. 27, 2001) (attachment: “Testing Method for Foods Produced by Recombinant DNA Technique”, this document is available for download from the website of the Ministry of Health, Labor and Welfare http://www.mhlw.gojp/topics/idenshi/kensa/050517a.html)] are not highly reliable and thus unsuitable for important tests.
On the other hand, the most reliable testing technique is testing by reading the nucleotide sequence of sample DNA (for example, see Sambrook, J. & Russell, D. W., Chapter 12 DNA Sequencing in Molecular Cloning: A laboratory manual, 3rd Ed. 2001, pp. 12.1-12.120). However, this method requires high cost and can be performed only in special laboratories provided with special equipment and skilled technicians. Thus, this method is not suitable for ordinary, frequent tests.
The present invention has been made in view of these problems conventional tourniquets have. It is an object of the present invention to provide a more efficient testing method using DNA.
As a result of extensive and intensive researches toward the solution of the above-described problem, the present inventors have found that it is possible to perform a highly reliable test with low cost by dividing the test into two states of a simple test and a close examination when testing whether or not a sequence of interest is contained in a sample nucleic acid. The present invention has been achieved based on this finding.
The present invention provides the following (1) to (12).
The present invention produces the effects as described below.
By performing a two-stage test as shown in the method of (1) or (2) above, efficient and highly reliable testing becomes possible. That is, usually, a simple test of the first stage which does not require special equipment or skilled technicians and yields results with low cost and in a short period of time is performed; and when a problem has been found, then, a close examination of the second stage is performed.
Further, according to a method in which an examination by reading the nucleotide sequence of sample nucleic acid is employed as a close examination of the second stage as described in the method of (3) above, highly reliable testing can be secured regardless of what method has been used as a simple test in the first stage, because reading of the nucleotide sequence of nucleic acid is the most reliable analysis/test method for the primary structure of nucleic acid.
Further, according to a method in which a sample nucleic acid used in a simple test is recovered and recycled to a close examination as described in the method of (4) above, it is possible to provide a highly reliable test means with fewer mistakes during the test process (such as confusion of test samples). It is also possible to reduce the amount of nucleic acid used in the test by recycling.
Further, according to a method in which a sample nucleic acid is tagged in advance and the sample nucleic acid is recovered after a simple test using the tag as described in the method of (5) above, it is possible to efficiently recover the sample nucleic acid from non-tagged substances.
Further, according to a method in which a sample nucleic acid is recovered after a simple test using a physicochemical difference between the sample nucleic acid and other substance as described in the method of (6) above, it is possible to recover the sample nucleic acid without addition of a tag thereto.
Further, according to a method in which a simple test that does not disrupt a sample nucleic acid is employed as described in the method of (7) above, it is possible to recycle the sample nucleic acid used in a simple test of the first stage to a close examination of the second stage. Therefore, it is possible to provide a highly reliable test means with fewer mistakes during the test process (such as confusion of test samples). It is also possible to reduce the amount of nucleic acid used in the test by recycling.
Further, according to a method in which a simple test is capable of retaining a sample nucleic acid in a state that allows reading of the nucleotide sequence thereof as described in the method of (8) above, it is possible to recycle the sample nucleic acid used in a simple test of the first stage to a close examination of the second stage which reads the nucleotide sequence of the sample nucleic acid. Therefore, it is possible to secure eventually a highly reliable test and, at the same time, it is possible to provide a highly reliable test means with fewer mistakes during the test process (such as confusion of test samples). It is also possible to reduce the amount of nucleic acid used in the test by recycling.
Further, according to a testing method in which a sample nucleic acid is divided into two aliquots in advance, and one is used in a simple test and the other in a close examination as described in the method of (9) above, it is possible to secure the sample for the close examination even when the simple test of the first stage disrupts the sample nucleic acid. Further, it is also possible to reduce mistakes such as confusion of test samples by retaining the divided aliquots on the same device.
Further, according to a method in which a plurality of sequences of interest can be tested in one simple test as described in the method of (12) above, it is possible to test a plurality of sequences of interest at one time.
Hereinbelow, the present invention will be described in detail.
The first testing method of the invention is a method of testing whether a sequence of interest is contained in a sample nucleic acid or not, comprising performing a simple test on the sample nucleic acid and performing a close examination on the same sample nucleic acid when it has been judged that the sequence of interest is contained therein or when it has been judged that the sequence of interest is not contained therein. The second testing method of the invention is a method of testing whether a sequence of interest is contained in a sample nucleic acid in more than a specific amount or not, comprising performing a simple quantitative test on the sample nucleic acid and performing a close examination on the same sample nucleic acid when it has been judged that the sequence of interest is contained in more than the specific amount. By thus dividing a test into two stages and performing a close examination only when a problem is found, it is possible to provide highly reliable testing with optimum cost. This technique is a testing method suitable for situations where problems occur only in a small number of cases.
Sequences of interest to be tested are not particularly limited. Since the testing methods of the present invention are used in judging the species of organisms or judging genetically modified organisms, sequences occurring specifically in the species, habitats or individuals of organisms or sequences occurring specifically in genetically modified organisms may be used as sequences of interest to be tested. Information about such specific sequences is widely known. For example, information about sequences specific to species or habitats of tuna fishes is disclosed in Non-Patent Document No. 3 (this document is available for download from the website of IAA Center for Food Quality, Labeling and Consumer Services (<URL:http://www.cfqlcs.go.jp/technical_information/hinpyou/pdf/maguromanual.pdf>). Information about species specific sequences of Alaskan king crab (Paralithodes camtschatica) and blue king crab (Paralithodes platypus) is disclosed in H. Uyama, M. Ikeda and N. Taniguchi, “Discrimination of Paralithodes camtschatica and Paralithodes platypus by PCR-RFLP of mtDNA”, Fish Genetics and Breeding Science, 2005, 34:111. Information about species specific sequences of Hexagrammidae fishes is disclosed in S. Yanagimoto and Y. Hamatsu, “Discrimination of 7 Species of Hexagrammidae Fishes Grown in Japan by PCR-RFLP Analysis of mtDNA”, Journal of Japan Society of Fisheries Science, 2003, 69 (5): 726. Information about species specific sequences of sea bream is disclosed in “Technical Information: Species Discrimination Manual for Pagrus major, Evynnis japonica and Dentex tumifrons” (this document is available for download from the website of IAA Center for Food Quality, Labeling and Consumer Services, <URL:http://www.cfqlcs.go.jp/technical_information/hinpyou/pdf/madai_manual.pdf>). Information about species specific sequences of Bass (Leteolabrax japonicus) and Nile perch (Lates niloticus) is disclosed in “Technical Information: Species Discrimination Manual for Leteolabrax japonicus, Leteolabrax sp. and Lates niloticus” (this document is available for download from the website of IAA Center for Food Quality, Labeling and Consumer Services, <URL:http://www.cfqlcs.go.jp/technical_information/hinpyou/pdf/suzuki_manual.pdf>). Information about species specific sequences of fishes belonging to the genus Scomber grown in Japan is disclosed in Japanese Unexamined Patent Publication No. 2002-345498 titled “Species Discrimination Method for Japanese Scomber Fishes”. Information about sequences specific to genetically modified organisms that were approved of 1st type use and 2nd type use under Cartagena Protocol is obtainable by consulting LMO related information “Search System for Genetically Modified Organisms Approved under Cartagena Protocol” (this document is available for download from the website of Japan Biosafety Clearing-House (J-BCH); <URL:https://ch.biodic.go.jp/bch/OpenSearch.do>).
Information about specific sequences other than those described above is also readily available for those skilled in the art. It should be noted here that sequences to be tested include not only DNA sequences but also RNA sequences.
The method of simple test is not particularly limited. However, when a nucleic acid that has been judged to contain or not to contain a sequence of interest by a simple test is recovered and used in a close examination, a method which does not disrupt the sample nucleic acid is preferable. When a sample nucleic acid is divided into two aliquots and one is used in a simple test and the other in a close examination, either a method which disrupts the sample nucleic acid or a method which does not disrupt the sample nucleic acid may be used as a simple test. Specific examples of methods which disrupt a sample nucleic acid include, but are not limited to, RFLP method, PCR-RFLP method, AFLP method, Southern blotting, chemical cleavage of mismatch (CCM) and methods using nuclease. Specific examples of methods which do not disrupt a sample nucleic acid include, but are not limited to, methods using hybridization in solution, real time PCR, primer extension method, sequencing method, random amplified polymorphic DNA method, TaqMan assay and Invader assay.
The method of recovering nucleic acid is not particularly limited. Preferably, a method used in which a sample nucleic acid is tagged in advance and the sample nucleic acid is recovered using the tag after a simple test. Alternatively, a method may be used in which a sample nucleic acid is recovered after a simple test using a physicochemical difference between the sample nucleic acid and other substance. Specific examples of recovering methods using a physicochemical difference include use of columns, electrophoresis and extraction with organic solvents.
The device for a simple test of the first stage may be made into a microchip using the microfluidics technology or the like. This enables automatic testing with a small-sized instrument. Further, a sample nucleic acid to be recovered after the simple test can be retained sealed in the microchip and delivered to the place of a close examination of the second stage easily and safely. In the close examination, the sample nucleic acid can be recovered from this microchip and used.
The method of close examination is not particularly limited. Preferably, a testing method of reading the nucleotide sequence of a sample nucleic acid is used.
Hereinbelow, preferred embodiments of the testing method of the invention are described with reference to Figures.
As described above, according to the preferred embodiments of the present invention, it becomes possible to perform a two-step testing. As a first stage, a simple test which does not require special equipment or skilled technicians and can be done easily and at low cost is performed. Then, when a problem is found in the results, a highly reliable close examination of the second stage is performed using the same sample DNA recycled. Thus, it is possible to provide a testing much more reliable than conventional testing. As a close examination of the second stage, the present invention employs a method of reading the nucleotide sequence of DNA which is currently the most reliable method. By employing this method, it is possible to provide ultimately the most reliable results even when the results of the simple test at the first stage have some problem.
Subsequently, a best mode for carrying out a method where a simple test of the first stage does not disrupt a sample DNA and the sample DNA recovered therefrom is used in a close examination of the second stage will be described below.
First, as shown in (b) in
After preparation of the group of beadbound sample DNA molecules 7, DNA hybridization (simple test of the first stage) is performed. For this purpose, a fluorescently labeled probe 10 is prepared by attaching a fluorescent dye 8 to an end of a probe DNA 9 that has a complementary nucleotide sequence to nucleotide sequence of interest 4, as shown in (c) in
However, since the group of fluorescently labeled probe 11 is present in the reaction solution in excess to the group of beadbound sample DNA 7, a group of excessive, unreacted fluorescently labeled probe 12 remains and also emits fluorescence. Therefore, for accurate testing, it is necessary to remove this group of unreacted fluorescently labeled probe 12 and to detect only the fluorescence from the fluorescently labeled probe 10 which has undergone double strand formation. For this purpose, in the present invention, a magnetic pipette 14 is used to attract and fix the magnetic beads in the reaction solution. By this operation, it becomes possible to recover the beadbound sample DNA 6 and the fluorescently labeled probe 10 (forming a double strand with nucleotide sequence of interest 4 within sample DNA 6) alone, as shown in (e) in
At this stage, the reaction solution after simple test A of the first stage contains the beadbound sample DNA 6 and the fluorescently labeled probe 10 (forming a double strand with nucleotide sequence of interest 4 within sample DNA 6). Of these, what is necessary for close examination B of the second stage is the beadbound sample DNA 6 alone. Since the fluorescently labeled probe 10 becomes a contaminant that inhibits close examination B of the second stage, it is necessary to purify and recover the beadbound sample DNA 6 alone prior to the close examination. For this purpose, after the completion of test A of the first stage, as shown in (g), reaction vessel containing the reaction solution is heated to dissociate hydrogen bonds forming the double strand by heat denaturation, followed by recovery of the bead-bound sample DNA 6 alone from the reaction solution with a magnetic pipette 14 as shown in (h). Thus, it is possible to recover only the necessary molecules while leaving the dissociated fluorescently labeled probe 10 in the reaction vessel.
Subsequently, as shown in (i), the bead-bound sample DNA is suspended in a solution for reading the nucleotide sequence thereof and recycled to close examination B of the second stage, in which the nucleotide sequence thereof is read by known methods (for example, see Sambrook J. et al. mentioned above). Then, the presence of nucleotide sequence 15 corresponding to nucleotide sequence of interest 4 in sample DNA 3 is detected (j).
As described above, according to the embodiment of the present invention, it is possible to perform test A of the first stage simply and with low cost; and when there is any problem in the results, then it is possible to perform highly reliable close examination B of the second stage recovering and recycling the sample DNA used in test A of the first stage.
Subsequently, a preferred embodiment for carrying out a method where simple test A of the first stage disrupts a sample DNA will be described below. In this method, the sample DNA are divided into aliquots for test A of the first stage and close examination B of the second stage in advance and stored on the same device.
First, a necessary amount of sample DNA 3 is obtained in the same manner as in the above-described case where sample DNA is recycled, according to
Since sample DNA 3 used in the PCR-RFLP test of the first stage was digested into a long DNA fragment 16, a medium-length DNA fragment 17, and a short DNA fragment 18 in the test, it is impossible to read the nucleotide sequence of the DNA in close examination B of the second stage even when those DNA fragments are recovered. Then, the aliquot of the sample DNA stored at place (B) for close examination B of the second stage as shown in (c) is used for reading the nucleotide sequence by known methods (for example, see Sambrook J. et al. mentioned above). Thus, the presence of nucleotide sequence 15 corresponding to nucleotide sequence of interest 4 in sample DNA 3 is detected (g).
As described above, according to the embodiment of the present invention, it is possible to perform test A of the first stage simply and with low cost; and when there is any problem in the results, then it is possible to perform highly reliable close examination B of the second stage using the sample DNA stored in aliquots on the same device.
Either when sample DNA is recovered and recycled or when sample DNA is stored in aliquots for individual tests, the simple test of the first stage is not limited to those described in the embodiments of the present invention. Any technique capable of identifying one or a plurality of specific nucleotide sequences of DNA may be used.
Needless to say, the present invention is not limited to the embodiments described above and may take various other constitutions without departing from the gist of the present invention.
Hereinbelow, the first example pertaining to the present invention will be described specifically. However, the present invention is not limited to these Examples.
Whether or not nucleotide sequence of interest (i) 27 as shown in
According to the flow as shown in
Subsequently, 100 pmol of DNA having probe nucleotide sequence 28 and labeled at its 5′ end with fluorescent dye Cy5 in aqueous solution (2 μl) was added to 50 μl of the suspension of beadbound DNA (
After the hybridization, magnetic beads in the reaction solution were recovered with a magnetic micropipette PickPen (e), and released and agitated in 50 μl of TE buffer placed in a separate tube to thereby wash the bead-bound DNA and the probe hybridizing thereto at room temperature. This washing was repeated three times to remove unreacted DNA. Then, the contents of the tube were suspended in 50 μl of TE buffer placed in a separate tube (
Subsequently, the suspension was heated at 94° C. for 2 minutes and then ice-cooled for 5 minutes to thereby dissociate the double strand (
A sequencing reaction was performed with the recovered bead-bound sample DNA 6 and a sequence primer having sequence primer nucleotide sequence 29 using BigDye Terminator v1.1 Cycle Sequencing Kit (ABI) according to the Kit's protocol. Then, sample DNA 6 was sequenced using ABI PRISMR 310 Genetic Analyzer.
As a result, nucleotide sequence of interest (i) 27 was detected in a part of the resultant nucleotide sequence as shown in
As stated above, according to the subject Example, it is possible to perform a simple and highly reliable testing by carrying out a simple test by hybridization to detect a specific nucleotide sequence, recovering the sample DNA used in the test, and reading the nucleotide sequence of the recovered sample DNA as a close examination.
Hereinbelow, the second example pertaining to the present invention will be described specifically. However, needless to say, the present invention is not limited to these Examples.
Whether or not it is possible to quantitatively detect the relative amount of nucleotide sequence of interest (ii) 31 present in nucleotide sequence 26 of sample DNA was examined by the testing method of the invention. Real time PCR was used as a simple test of the first stage, and the reading of the nucleotide sequence of DNA was used as a close examination of the second stage. Although real time PCR is a testing method that does not disrupt sample DNA, the sample DNA was not recovered after testing because an extremely small amount of sample DNA is sufficient for this method. The sample DNA was divided into two aliquots in advance, one of which was used in the real time PCR of the first stage and the other was used in the reading of nucleotide sequence in the second stage.
For the real time PCR, a pair of primers were used as shown in
First, according to
From tube (A), a 2 μl aliquot was taken three times and diluted with distilled water to prepare 2 μM solution, 20 fM solution and 0.2 fM solution. Then, 2 μl each of these solutions (containing 4 amol, 40 zmol and 400 ymol of sample DNA, respectively) was mixed with 5 μl of 10×EX Taq buffer (attachment to Takara EX Taq, cat #RR001A; Takara Bio), 4 μl of 2.5 mM dNTP mix (attachment to Takara EX Taq, cat #RR001A; Takara Bio), 1 μl each of 10 μM RT primer [1] and 10 μM RT primer [2] (10 pmol each), 1.25 units of Taq polymerase (Takara ExTaq, cat #RR001A; Takara Bio) and 5 μl of 3,500 dilution of SYBR Green I (cat #F0512; Takara Bio) to prepare 50 μl of aqueous solution. Each of these aqueous solutions was added to a 96-well microtiter plate (Micro Amp Optical 96-well Reaction Plate, cat #N801-0560; ABI) and sealed tightly with caps (ABI PRISM OpticalCap, 8 caps/strips (Flat), cat #4323032; ABI). These samples were retained at 95° C. for 2 minutes and then subjected to 40 cycles of the two steps of 95° C. for 15 seconds and 60° C. for 1 minute using ABI PRISM 7000 Sequence Detection System (ABI). Thus, PCR reaction was performed. During the PCR reaction, amplification of the DNA was monitored by measuring the fluorescence intensity of SYBR Green I. The results of measurement were summarized in graphs. As shown in
Subsequently, a sequencing reaction was performed with 2 pmol of sample DNA having nucleotide sequence 26 dispensed into tube (B) in advance and a sequence primer having sequence primer nucleotide sequence 29 using BigDye Terminator v1.1 Cycle Sequencing Kit (cat #4337449; ABI) according to the Kit's protocol. Then, sample DNA was sequenced using ABI PRISMR 310 Genetic Analyzer.
As a result, as shown in
When there are a plurality of nucleotide sequences of interest, the presence or absence of the nucleotide sequences of interest is judged by performing real time PCR reactions on these sequences simultaneously in one reaction vessel and examining whether or not amplification of DNA is detected. When amplification of any of the nucleotide sequences of interest has been detected, a close examination of the second stage is performed and the type of the nucleotide sequence of interest present in the sample is identified. In this case, the simple test only detects the presence or absence of nucleotide sequences of interest. Therefore, the fluorescence dye used in a plurality of real time PCR reactions may be either single or plural.
The present specification encompasses the contents disclosed in the specification and/or the drawings of Japanese Patent Application No. 2006-072581 based on which the present application claims priority. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-072581 | Mar 2006 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2006/325982 | 12/26/2006 | WO | 00 | 3/13/2007 |
