JPH08500725A - Loop structure - Google Patents

Abstract

(57) [Summary] The present invention provides a method for introducing a 3'-end loop structure onto a target sequence of one strand of double-stranded DNA, wherein the target sequence has a region A at the 3'-end side thereof. In some cases, there is a DNA region B extending further from the region A to the 3'side, and it is the first primer that hybridizes to the 3'end side of the sequence complementary to the target sequence and is a solid A first primer, immobilized on a carrier or provided with means for binding to a solid carrier, and a 3'terminal sequence which hybridizes to at least part of A and / or B of the target sequence The double-stranded DNA is subjected to a polymerase chain reaction (PCR) amplification treatment using a second primer having a sequence substantially the same as A at its 5'end, and the target is obtained by the amplification treatment. Distribution A double-stranded target DNA having a region A, a region capable of forming a loop, and a region A'complementary to the sequence A at the 3'end of the above is produced, and then this amplified double-stranded DNA is added. Subjected to double-strand dissociation treatment in immobilized form, thereby releasing the non-immobilized target strand and allowing region A'to spontaneously or artificially hybridize to region A to form a loop. A method characterized by the above is provided.

Description

Detailed Description of the InventionLoop structure   The present invention relates to a single-stranded 3'-end loop structure of DNA.   The single-stranded nucleic acid rewinds itself to form a loop structure, which is paired by hydrogen bonding. It is well known in molecular biology that short double-stranded segments are formed.   Such secondary structures are associated with various enzymes (eg polymerase, hydrolase, etc.) It may affect the susceptibility to nucleic acid interactions with. In some cases, The formation of a loop structure by a single-stranded nucleic acid results in a probe or a target sequence of the nucleic acid. May be in competition with the primer hybridization reaction. An example For example, when using a primer for DNA sequencing, etc. When a loop is formed at or near the hybridizing sequence, the hybridase This may reduce the efficiency of the sequencing and affect the clarity of the sequencing results. It   The present invention uses PCR to prepare a primer on the 3'end side of a DNA strand of interest. It is based on the idea of introducing such a loop structure.   DNA molecules are often present only in trace amounts in a sample. Polymerase chain reaction (PCR) methods have been developed for amplification. This way Then, a pair of polymerization primers specific to the known sequence of the target DNA are selected. Immediately Then, one primer is at the 5'end of one strand of the DNA double strand or at the vicinity thereof. And the other primer hybridizes to the 5'end of the complementary strand or its Allow each primer to hybridize to its neighbors in the presence of polymerase. The extended DNA sequence from the mer to the end of the target DNA template. It The DNA thus synthesized is then subjected to a double-strand dissociation treatment (usually at a temperature of about 90 ° C). Melted) and the newly synthesized single-stranded DNA sequence is Hybridize with excess primer (usually temperature up to a range suitable for annealing) (After lowering), further DNA strands will be synthesized in the presence of polymerase. In the case of, only the part from both ends of both primers is extended. Polymerase Is preferably one that can withstand the high temperatures used in the above double strand dissociation step, but recently A thermophilic polymerase suitable for this, Taq, is now available. The above-mentioned two types of primers and various nucleotides required for DNA synthesis are overloaded in a medium. If the excess amount is maintained, each strand will be synthesized, dissociated, and the primer annealed. , Repeating the cyclic process of synthesizing a new chain, simply It can be performed only by raising or lowering the temperature to the optimum temperature. In this way The first target DNA can be amplified exponentially, and in a relatively short time The concentration can be increased by a million times. The DNA thus amplified has the sequence It can be used for decisions and other studies.   The present invention introduces a 3'end loop structure on the target sequence of one strand of double-stranded DNA. In some cases, the target sequence has a region A on the 3'-end side thereof. In this case, there is a DNA region B extending 3 ′ from the region A. With the first primer that hybridizes to the 3'end of the sequence complementary to the target sequence Means for being immobilized on or attached to a solid support A given first primer and a small number of A and / or B of the above target sequence. Having a 3'terminal sequence that hybridizes to at least a portion and its 5'end Using a second primer having a sequence substantially identical to A for By adding the above double-stranded DNA to a chain reaction (PCR) amplification treatment, , At the 3'end of the target sequence, complementary to the region / sequence A capable of forming a region A / loop A double stranded target DNA having regions A'in the above order is generated, after which this By subjecting the amplified double-stranded DNA to a double-stranded dissociation treatment in immobilized form, The non-immobilized target strand is released, and the region A ′ is changed to the region A naturally or artificially. To form a loop.   The area that can form a loop is the entire area B (if the area B exists). Or a part and / or an optional element existing between the regions A ′ and A of the second primer It will be understood that it contains a sequence complementary to the loop-forming linker sequence of . If the second primer hybridizes to region A of the target sequence, the In order to form A'substantially completely hybridize to A, It is highly desirable that a loop-forming linker sequence be present in the primer . If the second primer is part of the region B existing away from the region A on the target sequence Between the region A and the hybridization region when hybridizing with The portion of B located in the Loop-forming linker sequence is present in Form a loop.   The second primer is A'-B (if B is present) and / or loop-forming The second primer has the sequence (A It may form a loop by its own rewind (without acting), but The annealing temperature should be higher than the temperature at which the primer preferentially anneals to itself. Avoiding this possibility of "self-priming" by choosing high Can be   Understand that both immobilized and non-immobilized strands can form loops Let's do it. However, the sequence A'in the correct orientation to serve as a primer for the chain extension reaction. Only the immobilized strand has. More importantly, is the array 5 '? The rule is that they are read in the 3'direction. Thus, with A The sequences of the primers identical to 5'to 3'are read substantially identically. .   One advantage of the present invention is that a primer is incorporated at the 3'end of one strand of DNA. Use that primer to perform, for example, sequencing of the strand or other manipulations. That is what you can do. Each immobilized template carries a built-in primer The ratio of the region to which the primer hybridizes due to the intervention of the linker sequence. It is clear that it remains relatively close. Therefore, even if the conditions change Strand dissociation occurs, the primer is linked to the template and It will hybridize easily.   If desired, create a restriction enzyme (RE) site near the loop to remove the loop. It may be possible to allow strand dissociation, for example during sequencing. Become so. By preparing such a procedure, for example, the Sanger method ( Sanger, F.et al(1977) PNAS (USA)74: 5463-5467 etc.) From the target DNA used as a new deoxynucleotide-terminated chain of various lengths It may be desirable when the synthetic DNA strands must be separated. Suitable RE section Positions and methods of incorporating them into primers are well known to those skilled in the art, For example, in the literature such as T. Maniatis et al.'S Molecular Cloning: a laboratory manual. Is taught. For example, the linker sequence in the second primer is attached to each of A and A '. It may contain palindromic sequences that are adjacent and complementary to each other, When these flanking regions are hybridized, the RE site (this Allows the removal of loops). Otherwise , The region AA ′ may be selected so that the region AA ′ includes the RE site.   Suitably, the invention relates to the same filing date of the Applicant entitled "Chemical Process" Combined with the invention taught in the pending application (Attorney Reference No .: 75.57465) be able to.   This applicant's pending application has amplified and then immobilized the DNA of interest. After that, four aliquots of the immobilized DNA in single-stranded form (split samples) It is based on the idea of carrying out the polymerase chain reaction. For each aliquot Using the same specific extension primer and a different dideoxynucleotide, No deoxynucleotides were used, thus ensuring that the Ensure that only xynucleotides are incorporated. At this time, the target position is Immediately adjacent to the 3'end of the specific extension primer that hybridizes to DNA. In other words, the target position on the immobilized strand is the extension primer specific to the DNA of interest. Is on the 5'side of the hybridizing site. Next, normal deoxynucleo A chain extension reaction using tide was carried out using the above-mentioned specific primer to give dideoxy. While nucleotide-blocked DNA remains unreacted, it is blocked Not allowed DNA to form double-stranded DNA. Then use various methods To distinguish double-stranded DNA from non-stretched strands (ie, substantially single-stranded DNA) Allows identification of the base at the position. Preferably, the DNA in question is PCR Amplify by.   Usually, a primer is used for each aliquot for the dideoxy and extension reactions. Add. However, using the present invention, the target DNA is a loop-ligated primer. The primer is stable to changes in conditions as described above. To substantially reduce or avoid loss when transferring from dideoxy reaction to chain extension reaction it can.   In particular, the invention taught in the above-mentioned pending application of the applicant is in accordance with the present invention, The sample DNA is equipped with a loop binding 3'primer, and this primer is fixed Can be modified to hybridize immediately adjacent to the target position to the target DNA. it can. Each of the four aliquots of this immobilized single-stranded DNA was then treated with one Subject to polymerase chain reaction in the presence of oxynucleotides, Uses different dideoxynucleotides to create a dideoxy complementary to the base at the target position. Ensure that only xynucleotides are incorporated. Next, these four aliquots Each aliquot was subjected to an extension reaction in the presence of all four nucleotides. DNA that has not reacted with dideoxynucleotides in the While forming A, the dideoxynucleotide blocked DNA is non-elongating Leave as strand DNA. Then double-stranded and / or non-elongated DNA Identify which dideoxynucleotide was introduced and therefore which salt Determine if the group is at the target position.   Sufficient DN, for example, with a relatively low background giving clear results It is desirable to assess the efficiency of PCR to determine if A was synthesized . Various test methods are known, but in the applicant's opinion, the applicant previously reported Solid-phase method called DIANA (PCT / EP9) for detecting immobilized amplified nucleic acid 0/00454) is preferably used. This DIANA method is, for example, In a specific embodiment, in vitro (in in vitro)Colorimetric detection of DNA amplified by Used for. This assay is based on PC biotinylated or otherwise functionalized. Based on the use of the R primer, which is an in vitro amplified product For example on streptavidin coated magnetic beads. Also The other PCR primerlacA "handle" part like an operator array Have,LacUsing I repressor-β-galactosidase fusion protein The captured DNA can be colorimetrically assayed. (Wahlberg, colorimetric method for DNA diagnostics allowing direct solid-phase geno mic sequencing of the positive samples ”Proc. Natl. Acad. Sci. U.S.A.87 , 6569-6573). This preferred form of qualitative DIANA assay is The advantages of the PCR method and the high specificity and safety of the biotin-streptavidin system It has both qualitative and easy colorimetric assay based on β-galactosidase. Strong interaction between biotin and streptavidin (K_d= 10^-15M^-1) Is the system efficiency Further increase. By using magnetic beads as a solid carrier, centrifugation,   Many proteins that bind to a specific DNA sequence are known, and the operation of the operon It is also often involved in various genetic processes such as repression and suppression. Tampa like this One of the qualitylacRepressorLacI, this islacoperator (lacOP) to suppress transcription. Therefore, if the recognition site is DNA ArraylacIf OP,LacIt is possible to attach a label via the I protein. it can.LacA fusion of a DNA-binding protein such as I with another protein We devised synthetic proteins so that the latter protein chamber could be based on, for example, color, fluorescence, or chemiluminescence. It is particularly preferable to make it available for subsequent detection using the method described below. This Specific examples of such proteins include β-galactosidase, alkaline phosphatase, and Such as oxidase.   As the label, one of the primer pairs (preferably immobilized primer) is used. 21 base pairs introduced at the end of the amplified DNAlacRecognize operator sequence likeLacOf I repressor-β-galactosidase fusion protein Is preferred. This fusion protein is DNAlacONPG linked to OP sequence (O-nitrophenyl-β-D-galactoside) is added, so the color develops. Can be measured spectrophotometrically. Use this fusion protein and ONPG Allows for a quick and easy colorimetric assay, which is safer to use with radiolabels. There is no issue of integrity. For example, IPTG (n-isopropyl-β-D-thiogalactopi Lanoside) allows the fusion protein to be dissociated from the DNA. It   The specificity of this method is significantly increased by providing a first-stage PCR amplification step. be able to. Due to such pre-amplification, there is a possibility that they may be mixed in the sample. Significantly increases the concentration of target DNA compared to other DNA, and As described in PCT / EP90 / 00454, in another sequence of the target DNA If second-stage amplification is performed using at least one specific primer, The signal of the target DNA is significantly increased as compared with the "ground noise".   To increase the signal-to-noise (S / N) ratio, the applicant's pending application PC Two-step PCR method (nested (nested) as described in T / EP 90/00454. ted) using a primer) and by doing so The sensitivity of the method can be improved.   Of amplified DNA such as when one or more primers are bound to a carrier Immobilization may occur as part of PCR amplification, or the primer may be Functional groups (eg biotin or It may have a thiol group). Immobilizing the 5'end of the primer The DNA chain extended from the primer can be bound to a solid support, Since the 3'end of the A chain becomes far away from the carrier, the polymerase in the latter stage Can be used for the chain extension reaction.   The solid support is preferably a microtiter well (conventional 8 × 12 format). Convenient) or dipstick shape. They can be made of polystyrene activated to bind to primer DNA (K. Almer, PhD dissertation, Royal Institute of Technology, Swe Stockholm, 1988). The carrier may also be, for example, agarose, cellulose. Particles, fibers or caps made of sutures, alginates, Teflon or polystyrene. It may be a pillary. The carrier is a magnetic particle, such as Dynal AS (Norr). Superparamagnetic beads manufactured by Oslo, Way, etc. may be used.   The solid support is used to bind a primer, such as a hydroxyl group, a carboxyl group, or an aldehyde. Functional groups such as hydrido and amino groups or moieties such as avidin and streptavidin You may own the minutes. These are generally groups bearing any of these functional groups. Given by subjecting the carrier to a treatment which gives a surface coating of the limer For example, polyurethane and polyglycol were used together to give a hydroxyl group. Give a hydroxyl group with a cellulose derivative, To give a carboxyl group with a polymer or copolymer, or to give an aminoalkylated polymer. An amino group can be provided with a limer. In US Pat. No. 4,654,267 Have described many methods of introducing such surface coatings.   Any suitable polymerase may be used, including Taq Polymer It is preferable to use a thermophilic enzyme such as For example, without adding a polymerase such as Klenow fragment, The temperature cycle of can be repeated.   The target DNA may be cDNA synthesized from mRNA in the sample, and The method of Ming is thus also applicable to diagnostics based on characteristic mRNAs. like this Pre-synthesis can be performed by pre-treatment with reverse transcriptase, but preferably , In the same system as the buffer and base system used in the subsequent PCR step. PCR Since the operation requires heat treatment to dissociate the double strands, the reverse transcriptase is the first P It should deactivate in the CR cycle. When mRNA is sample nucleic acid , Treating the first sample (eg serum sample) with immobilized polydT oligonucleotide So that all the mRNA can be recovered via its terminal poly A sequence. It is advantageous. Alternatively, to recover RNA via a specific RNA sequence, Specific oligonucleotide sequences can also be used. Such oligonucleoti Later, as described in International Patent Application PCT / EP89 / 00304, It can serve as a primer for cDNA.   While PCR has been described as the preferred method of first amplifying the target DNA, It will be apparent to those skilled in the art that alternative methods may be used instead of combining with PCR. Will. Developed in recent years without the need for temperature cycling or thermostable polymerases As an amplification technology, Self Sustained Sequence Replication 3SR method is abbreviated). The 3SR method is modeled on the replication of retroviruses. The prepared product can be used for amplification. (Gingeras, T.R.et al, PNAS (USA) 87: 1874-1878; and Gingeras, T.R.et al, PCR Methods and Applications Vol.1, pp 25-33 etc.).   The primer should be large enough to allow adequate hybridization. On the other hand, it is convenient to be short to some extent to avoid unnecessary chemical synthesis. . Since the number of hydrogen bonds involved in the binding is larger in the pairing between C and G, Big And the stability of hybridization to CG base pairs of AT base pairs. It will be apparent to those skilled in the art that there is some dependence on the ratio of In addition, Naturally, the homologous sequence between the extension primer and the rest of the amplified sequence The condition setting (stringency) should be considered accordingly. This Guidelines for such routine experiments can be found, for example, in Sambrook, J., Fritsch, E.F., and  Maniatis, T., Molecular Cloning: a laboratory manual (1989), etc. Can be found in the offering.   Polymerase reactions in the presence of dideoxynucleotides can be performed, for example, by T7 polymerase. , Klenow or Sequenase Ver. 2.0 (made by US USB company) Which is done using a polymerase that does the incorporation of dideoxynucleotides Be done. However, as is known, many polymerases proofread (calibrate) ) Activity, that is, 3'which has error-checking activity and is used for chain extension Sometimes the terminal one or more nucleotides are digested. When such digestion occurs in the method of the present invention, background noise increases. . To prevent this problem from occurring, fluorine that inhibits 3'digestion by polymerase It is desirable to add ionic or nucleotide monophosphates to each aliquot.   Identification of double-stranded DNA and / or non-extended DNA can be accomplished in a variety of ways. It Regarding double-stranded DNA, conventional techniques such as introduction of radiolabel during chain extension reaction Is possible withlacIt is preferable to use an operator sequence,lacOh Pelator sequences are preferably incorporated into the DNA during amplification as described above. Stretching the entire chain Results in a double-stranded DNA sequencelacI repressor-β-galacto The dase fusion protein binds. The bound fusion protein is colorimetric as described above. The three aliquots in which the extension reaction occurred Determine the dideoxy base added to the remaining aliquot.   Extension of loop-bound 3'primer was blocked with dideoxynucleotides Numerous identification methods are possible for non-extended DNA and are readily apparent to those of ordinary skill in the art. Will. Preferably, a hybridizer is provided downstream of the 3'end of the loop binding primer. Probe, that is, between the 5'end and 3'loop structure of the immobilization site of the immobilization strand Use a probe that hybridizes to. Probe may be labeled or not Is Suitably it has a label binding means. Such probes are single-stranded DNA , But not double-stranded DNA.   If desired, both double-stranded and single-stranded DNA may be identified, and And the accuracy of the results can be further checked. Usually, dideoxy nucleotide A control experiment containing no octides and all four dideoxynucleotidies It is preferred to perform a "zero control experiment" that includes a mixture of actives.   Another means of identification is a pending application for the same filing date of the applicant (see agent). No .: 75.5777)), and is released during the chain extension reaction. It is to detect pyrophosphate. When each nucleotide is incorporated, Pyrophosphate dissociates from cleotide triphosphate, leaving the remaining nucleotide monophosphate Incorporated at the end of the growing nucleic acid strand. Stopped dideoxynucleotides are incorporated into the chain In the non-aliquot, pyrophosphate is actively released during the chain extension reaction. this Release of such pyrophosphate should be measured using luciferin and luciferase Can be. The luciferin / luciferase system is substantially positive for the abundance of pyrophosphate. It emits light in proportion.   For diagnostic applications, such as genetic testing of carriers of genetic disease, the sample is Contains terrorism-bonding material. That is, half of the DNA has one nucleotide at the target position. It has an otide, but the other half has another nucleotide. Therefore , Of the four aliquots used in the method of the invention, two give a positive signal. Well, the two will give a semi-positive signal. Therefore, the detection label in each sample It is desirable to quantitatively determine the amount. For homozygous samples, 4 aliquots It will be clear that out of the three, three give a negative signal and one gives a positive signal.   Hereinafter, the present invention will be described by way of non-limiting examples with reference to the drawings. To explain   FIG. 1 shows a process for identifying bases at a single target position using the method of the present invention. Showing a tocol,   2 and 3 show the oligonucleotide primers used in Example 1, And   FIG. 4 is a graph showing the results obtained in the examples.Materials and methods Bacterial strains and enzymes. Acids Res.,Ten, 5765-5772) was used as the bacterial host. Used plasm M. (1988) “Approaches to solid phase DNA sequencing” Nucleosides and Nu cleotides7, 629-638). Various restriction enzymes, DNA polymerase I (cle Know fragment), T7 DNA polymerase, CIP and T4 polynucleotides. Otidokinase was obtained from Pharmacia (Sweden). use WasTaq  The DNA polymerase is Perkin-Elmer (California, USA). (AmpliTaq) purchased from Nia.Example 1 Synthesis of oligonucleotides.   A region (base 6) encoding part of the active site of the HIV reverse transcriptase gene (RT) 25-1165; Myers, G., Korber, B., Berkovsky, J.A., Smith, R.F. and P. avlakis, G.N., Human Retroviruses and AIDS 1991 (Los Alamos National Lab oratory, New Mexico 1991)) 5 complementary oligonucleotide primers -: RIT321, RIT322, RIT331, RIT333 and RIT33 8 (see drawing) is an automatic DNA synthesizer (KABI-Pharm, Sweden) Manufacturer's description on Gene Assembler Plus from Acia Synthesized exactly by phosphoramidite chemistry. RIT322 is biotin phospho Biomass using luamidite (Clonetech, California, USA) Tinted. Purification was carried out using a pepRPC 5/5 reverse phase column (Swedish KABI- (Pharmacia). PCR cloning   Clinical samples taken from HIV-1 patients (Swedish, Stockholm, Sweden) From the Swedish Bacteriology Laboratory (SBL) , By amplification with 5 pmol each of RIT331 and RIT333 (FIG. 3). The IV RT fragment was cloned. RIT331 and RIT333 Are both upstreamBamHI and downstreamEco"Handle for introducing RI recognition site Included part. The PCR reaction mixture contains 200 μM of each dNTP and 20 mM of T ris-HCl (pH 8.7), 2 mM MgCl₂, 0.1% Tween 2 Included 0 and 0.5 units of AmpliTaq with a final volume of 50 μl did. The temperature profile is a denaturation step of 0.5 minutes at 95 ° C followed by 0 at 55 ° C. ． Set the primer annealing step for 5 minutes and the extension step for 2 minutes at 72 ° C. I got it. These steps were performed on the Gene Amp PCR System PE 9 30 using 600 (Perkin-Elmer, California, USA) Repeated times. HIV RT fragment and vector thus PCR amplified together with pRIT28BamWith HIEcoRestricted by RI, cut out from agarose After purification, the ligation reaction was performed at room temperature for 1 hour. Competent R with this construct RIΔM15 cells were transformed and blue / white selection (Langley, E.K.et al (1975) PNA S (USA)72, 1254-1257) so that IPTG (n-isopropyl-β-D-thio Galactopyranoside), X-gal (5-bromo-4-chloro-3-indolyl-β-D-ga Lactoside) and TBAB containing ampicillin (Sambrook, J. et al., Supra). ) A powder was inoculated on the plate. Solid-phase sequencing (Hultman, T.et al (1991) Biotec hniquesTen, 84-93), and 5 white colonies have the correct insert. It was confirmed to be a rasmid. One of these colonies was designated pRIT-R Named T and selected for further study. This clone is from Sweden It is stored in the Department of Biochemistry, Royal Institute of Technology, Kholm. Preparation of template for mini-sequencing for DIANA detection   Transfer pRIT28-RT colonies to vials and transfer 10 μl of 20 mM Tr Lysis treatment was carried out in is-HCl (pH 8.7) at 99 ° C for 5 minutes. Then 1 μl The lysate was treated with 5 pmol of RIT135 and RIT322 (biotinylated), 0.2 5 pmol RIT321, 200 μM each dNTP, 20 mM Tris-HC 1 (pH 8.7), 2 mM MgCl₂, 0.1% Tween 20 and 0.5 Transferred to a unit AmpliTaq PCR mix to a final volume of 50 μl . Primer RIT322 was later bound to streptavidin-coated solid support. With 5'biotin forlacIt consists of 21 bases that define the OP recognition sequence. Amplification was performed as described above and the resulting PCR product was then prewashed Vidin-coated paramagnetic beads (Lea, T.et al (1988) J. Mol. Recognit1, 9-18) , Ie, Dynabeads that had been pre-washed with the binding solution recommended by the manufacturer Fixed on M280-Streptavidin (Dynal AS, Norway) (Hultman, T.et al (1989) Nuc .Acids Res.17, 4937-4946). Immobilization After treatment, the beads are washed with 50 μl of binding / wash solution to assay for bound DNA. It was 50 μl of the DNA-immobilized beads was used as the fusion protein lacI-β-gala. Ink for 20 minutes after mixing with Ctosidase (Dynal AS, Norway) Was added. Beads in DIANA buffer (Dynal AS, Norway) ) Four times to remove excess fusion protein and back-wall with a wall cap. The tube was changed to a new tube at the final stage to eliminate the band. 100 μl of o-nit Rophenyl-β-D-galactoside (ONPG, 1.25mg / ml) is a chromogenic substrate And after 6 minutes 100 μl 1M Na₂CO₃To stop the reaction did. The absorbance at 405 nm of the supernatant was measured by EAR340AT ELISA plate Measurement (DLT) (SLT-Labinstruments, Austria) did. Incubate with 20 μl of 0.1 M NaOH for 5 minutes and lyse by thawing. The single strands are dissociated to generate a single-stranded immobilized DNA template, which is reconstituted with 50 μl The binding solution was washed with 50 μl of 1 × TE. Mini sequencing reaction   For the compatible dideoxynucleotide, six separate extension reactions (one for ddA TP only, one ddCTP only, one ddGTP only, one ddT TP only, one with all four types of ddNTP and one with no ddNTP 2 μl of annealing mix, 17 mM Tris-HCl (pH 7.5), 6 mM MgCl₂1 mM DTT, 1 μM compatible dideoxynuc Reotide and 0.13 units of Sequenase Ver. Total amount including 2 Performed in 10 μl. A schematic diagram of this experiment is shown in FIG. Anti-dideoxy uptake The reaction was carried out at room temperature for 5 minutes and stopped by the addition of 20 μl of 0.5 M EDTA. It was Thereafter, the beads were added to 30 μl of 10 mM Tris-HCl (pH 7.5). ) Washed twice. The next extension step used a dNTP concentration of 200 μM and 5 mM Tris-HCl (pH 7.5), 12.5 mM MgCl₂1 mM DTT and And 0.13 units of Sequenase to make the total volume 10 μl. Dideo For aliquots that do not incorporate xynucleotides, use Sequenase Strand elongation occurs, allowing full-length double-stranded DNA to bind to the beads. It After incubating at room temperature for 5 minutes, add 20 μl of 0.5 M EDTA. In addition, 40 μl of beads were added to DIANA buffer (Dynal AS, Norway). Manufactured) (0.1 M Tris-HCl (pH 7.5), 0.15 M NaCl, 0) ． 1% Tween 20, 1 mM MgCl 2₂And 10 mM β-mercaptoetha Washed). Detection by DIANA   The result is the DIANA method (Wahlberg, J., Lundeberg, J., Hultman, T. and allowing direct solid-phase genomic sequencing of the positive samples ” Proc. Natl. Acad. Sci. U.S.A.87, 6569-6573). Immobilization of DNA 50 μl of the fused protein lacI-β-galactosidase (nor Way Dynal AS) and mixed for 20 minutes. Bee The cells were washed 4 times with DIANA buffer (Dynal AS, Norway). To remove excess fusion protein and eliminate wall coat background I changed to a new tube at the final stage. 100 μl of o-nitrophenyl-β-D-moth Lactoside (ONPG, 1.25 mg / ml) was added as a chromogenic substrate, and After 6 minutes 100 μl of 1M Na₂CO₃Was added to stop the reaction. 40 of supernatant EAR340AT ELISA plate reader (SLT-L) abinstruments, Austria). The result As shown in FIG. In this assay, all four dideoxynucleotides (dd Lower signals were obtained with NTP) and with ddATP alone. It was The next complementary base at the 3'end of the sequencing primer was dideoxythymidine. Therefore, the above results are based on the nucleotide sequence at a specific location using this assay. Indicates that can be detected.

─────────────────────────────────────────────────── ─── [Continued summary] A method characterized by allowing soybeans to form loops I will provide a.

Claims (1)

[Claims] 1. Method for introducing 3'terminal loop structure on target sequence of one strand of double-stranded DNA Thus, the target sequence has a region A at the 3'-end side thereof and, in some cases, has a region A. DNA region B extending further 3'from region A of The first primer that hybridizes to the 3'end of the sequence complementary to Providing means for immobilizing on a solid carrier or for binding to a solid carrier The first primer and at least one of A and / or B of the above target sequence Has a 3'terminal sequence which hybridizes to the part and has an A at its 5'end Polymerase chain reaction using a second primer having a substantially identical sequence (PCR) The above double-stranded DNA is attached to the amplification treatment, and the target treatment is performed by the amplification treatment. A region A and a region capable of forming a loop and a region complementary to the sequence A at the 3'end of the row A double-stranded target DNA having A'in the above order is generated, after which this amplification is carried out. Double-strand DNA is subjected to double-strand dissociation treatment in an immobilized form, and thus fixed The unstranded target strand is released, and the region A'is changed to the region A naturally or artificially. A method comprising the step of bridging to form a loop. 2. The method according to claim 1, wherein after the final strand dissociation treatment of the amplified double-stranded DNA, By annealing at a temperature higher than the self-annealing temperature of the second primer The region A'on the immobilized single-stranded DNA strand is hybridized to the region A. How to collect. 3. Immobilized single-stranded DN after loop formation according to the method of claim 1 or 2. A contains a restriction site within the double-stranded region adjacent to the loop. Method. 4. The method according to any one of claims 1 to 3, wherein the first primer is A method characterized in that biotin is retained as a means for immobilization. 5. The method according to any one of claims 1 to 4, wherein the first primer is Characterized by having a recognition site for a label-bearing DNA binding protein How to do. 6. The method according to any one of claims 1 to 5, after loop formation The immobilized single-stranded DNA was used as a sequencing primer at the 3'end of the A'sequence. A method characterized by being attached to the used sequencing. 7. 7. The method according to claim 6, wherein said region A'is the one to be determined in the target sequence. Immobilized single-stranded DNA selected to hybridize immediately adjacent to the target base Chain extension reaction in the presence of deoxynucleoside or dideoxynucleoside The presence or absence of the above target base indicates whether or not the chain has been extended. A method characterized by using as an index of.