JPH10500313A - Detection of nucleic acids in cells by thermophilic strand displacement amplification - Google Patents

Abstract

(57) SUMMARY Thermophilic strand displacement amplification (tSDA) for in situ amplification of nucleic acid target sequences in cells in suspension, on slides or in tissue is described. Superior specimen formats can be preserved and either DNA targets, RNA targets, or both can be selectively amplified. Since in situ amplification by tSDA is compatible with immunochemical techniques, both amplification of the target sequence and immunological staining can be performed on the same specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION                 Detection of nucleic acids in cells by thermophilic strand displacement amplification                                 Field of the invention   The present invention relates to the amplification of nucleic acids, and in particular to the amplification of nucleic acids in morphologically complete cells. .                                 Background of the Invention   Nucleic acid amplification technology provides a powerful tool for the detection and analysis of small amounts of nucleic acids Came. The extreme sensitivity of such methods is important for the early diagnosis and analysis of infectious and genetic diseases. Genes for isolation and development of specific nucleic acids in forensic medicine Have brought you an attempt. Nucleic acid amplification technology is subject to the temperature requirements of the procedure. Can be classified. Polymerase chain reaction (PCR), ligase chain reaction (LC R) and transcription-based amplification require high temperatures (R) to regenerate single-stranded target molecules for amplification. It requires repeated circulation of the reaction from 85 ° C to 100 ° C) to low temperatures (30 ° C to 40 ° C). In contrast, strand displacement amplification (SDA), self-sustaining sequence replication (3SR) and Qβ rep Methods such as the lipase system are isothermal reactions that can be performed at a constant temperature.   In PCR, the reaction temperature rises after primer extension and newly synthesized The separated strands are separated from the template. Next, lower the temperature and reanneal the primer And the elongation process is repeated. Therefore, the PCR reaction step is constrained by the temperature constraints of the reaction. As a result, it occurs in discrete phases or cycles. In contrast, strand displacement amplification (SDA ), Primer extension, displacement of the single-stranded extension product, extension product (or the original Annealing of the primer to the target sequence) and subsequent primer Elongation occurs simultaneously in the reaction formulation. Conventional SDA (at lower temperatures, usually about 3 Performed at 5-45 ° C.) is described in G. T. Walker et al. (1992a. Proc. Natl. Ac. ad.Sci.USA89, 392-396 and 1992b.Nuc.Acids.Res.20, 1691-1696) Are listed. A thermophilic variant of the SDA reaction (tSDA, described below) has recently been developed. It consists of a thermostable polymerase and a restriction endonuclease. It is carried out at a higher, but still constant, temperature.   The target for amplification by SDA is the endonuclease used in the SDA reaction. And can be produced by fragmenting larger nucleic acids. . However, if the target is a restriction endonuclease recognition required for fragmentation If not adjacent to the site, a restriction engine suitable for nicking in SDA Target nucleic acids having a nuclease recognition site are described in Walker et al. (1992b, supra). And as described in US Pat. No. 5,270,184. S As in the case of DA, the individual steps of the target generation reaction occur simultaneously and sequentially, Contains the terminal recognition sequences required for nicking by restriction enzymes in SDA Generates the target sequence. Since all components of the SDA reaction are present during the target generation reaction, The generated target sequence automatically and continuously enters the SDA cycle and is amplified .   In situ nucleic acid analysis detects and detects specific nucleic acid sequences in morphologically complete cells. And localization. These methods are described, for example, in U.S. Pat.No. 4,888,278. Based on direct hybridization of labeled probes as described in It has been used conventionally. However, such direct hybridizations The method is specific for the nucleic acid of interest, but in each case has a very low copy number May not be sensitive enough to detect the nucleic acid. Detect extremely low copy numbers As an alternative, in situ amplification of the target sequence before in situ detection is extremely interesting Was. In situ nucleic acid amplification uses traditional solution amplification because cells concentrate the amplification products. Where the amplification product can diffuse freely. Or when they are diluted by cell contents that do not contain the sequence of interest. Allows for the detection of fewer molecules than is possible. Nucleic acids are required prior to target sequence detection Because there is no need to extract from cells, in situ methods are used to identify which cells in a population Provides information about the presence of nucleic acids and provides biochemical and morphological characteristics of cells. It further allows the analysis of nucleic acids for gender. In situ amplification is mainly performed by PCR Have been developed (O. Basgara and R. Pomeranz) erantz), 1993, AIDS Research and Human Retroviruses 9 (1), 69-76; Nuovo et al., 1992, Diag. Molec. Pathol. 1 (2), 98-102; M.J. Embleton et al., 1992, Nuc. Acids Res. 20 (15), 3831-3837; Emmetson (Emmetson) et al., 1993, Proc. Natl. Acad. Sci. USA 90, 357-361; Cominos (Komminoth) et al., 1992, Diag. Molec. Phatol. 1 (2), 85-97; K.P. Chile. Et al., 1992, J. Histochem. Cytochem. 40 (3), 333-341; Haase et al., 1990. Year, Proc. Natl. Acad. Sci. USA 87, 4971-4975; Basgara et al., 1992, New Engl .J. Med. 326 (21), 1385-1391; Patterson et al., 1993, Science 260. , 976-979). However, due to the multiple cycles of heating and cooling, and PCR, The stringent hybridization conditions necessary to achieve that sensitivity are And not well tolerated by cells. The extracellular diffusion of the amplified sequence is repeated. Increased heating can increase the diffusion signal in the sample. Attempts to reduce the loss of PCR products from cells often involve cross-linking fixatives. Long-term fixation (15 days to several hours) by PCR, in situ amplification by PCR Used for This treatment often requires protease treatment of fixed cells before amplification. (G. Nuovo et al., 1992, Diag. Molec. Pathol. 1 (2), 98-102).   Conventional low-temperature SDA performed in situ (1) immunophenotype for cell identification Improved maintenance of cell structure allowing determination and (2) amplicon in cells Having many advantages over in situ PCR, including significantly improved retention understood. However, the increased temperature of in situ tSDA is due to these features It was unknown what effect it had. Increased temperature (typically with conventional SDA (Approximately 15-20 ° C. in comparison) may provide the advantage of increased reaction specificity and speed. But did not prevent accurate immunophenotyping and cell discrimination. It could also significantly increase cell destruction to levels that would otherwise prevent it. reaction A significant increase in temperature may also increase the diffusion of amplicons outside the cell. Where they are taken up by negative cells and give rise to false positive signals was there. However, surprisingly, cell structures after in situ tSDA As demonstrated by normal forward and side light scattering in cytometry, It turned out to be in perfect condition. Therefore, immunophenotyping is Compatible with itu tSDA temperature and protocol. Applicant has to Cell maintenance involves subjecting cells to repeated cycles of heating and cooling, as in PCR. It is assumed that the damage may be less than the damage. Surprisingly, amplicon It was also found that the spread of the cells hardly increased, Little damage when maintained at a higher constant temperature than It is thought that this is possible.   The following terms are defined herein as follows:   Amplification primers are labeled by primer hybridization and extension. Primer for amplification of the target sequence. For SDA, 3 of amplification primer The 'end is the target binding sequence that hybridizes at the 3' end of the target sequence. Increase The width primer is also 5 'to its target binding sequence, generally at its 5' end. It contains a recognition site for restriction endonuclease near the end. Restriction endonuclease recognition The recognition site is defined as described above by Walker et al. (1992a), supra. Nicks at the double-stranded recognition site of the restriction endonuclease when semi-modified Sequence recognized by a restriction endonuclease that will contain It is. The semi-modified recognition site is a double-stranded recognition site for a restriction endonuclease. Yes, where one strand has a restriction endonuclease on one of the strands of the double helix At least one derivatized nucleotide that prevents cleavage by Including “Nicking” refers to the formation of double helixes, as opposed to typical double-strand breaks. This modified activity in which only one strand is cleaved by a restriction endonuclease Means Semi-modified restriction nickable by restriction endonuclease Any endonuclease recognition site is suitable for use in SDA . Amplification primers for SDA are described by Walker et al. (1992b),₁ And S_TwoIt is called. The α-thio modified deoxyribonucleoside triphosphate is Abbreviations such as "dNTPαS", "dATPαS", and "dCTPαS".   [Bumper] or outer primer binds to the target sequence upstream of the amplification primer. Since the primer is annealed, the extension of the outer primer is Displaces the imer and its extension products, i.e., A copy of the target sequence containing the resulting restriction endonuclease recognition site is replaced . The bumper primer therefore consists only of the target binding sequence and Anneal upstream of the amplification primers and displace them when extended Designed to be. Outer primers are described by Walker et al. (1992b), above. B₁And B_TwoIt is called. The extension of the outer primer is equivalent to the extension of the amplification primer. Although one method of replacing the material, in some cases, heating may also be appropriate.   The term target or target sequence refers to the nucleic acid sequence (DNA and / or Means RNA). These include the original nucleic acid sequence to be amplified and its complementary Both the second strand as well as a copy of the original target sequence generated by amplification of the target sequence The chain is included.   Amplification products, extension products or amplicons are generated during amplification of the target sequence Oligo or polynucleotide containing a copy of the target sequence.                                 Summary of the Invention   Thermophilic strand displacement amplification (tSDA) is performed on cells in suspension, on slides or in tissue. For in situ amplification of nucleic acid target sequences over conventional in situ SDA Have been adapted by their speed, sensitivity and specificity. Excellent sample morphology As demonstrated by normal light scattering parameters in cytometry, Is preserved despite exposure to temperatures significantly higher than in situ SDA . In situ amplification with tSDA also results in increased immunity despite increased reaction temperature. Both compatible with chemical technology, so both target sequence amplification and immunological staining Can be performed on the same sample. This means that repeated temperature cycling frees cellular antigens of interest. In contrast to in situ PCR, which may be undetectable by epidemiological techniques is there.   The method of the present invention for in situ tSDA generally involves the simple preparation of cells or tissues. Following immobilization, permeabilization and addition of reagents required for tSDA are included. Target sequence If the DNA is DNA, the cells or tissues can be heated briefly before amplification to alter the target sequence. Sexuality. Due to the thermostability of the enzymes involved, heating may optionally involve enzymes. It can occur in a mixture of drugs. Alternatively, after denaturation, cool the sample to the desired reaction temperature. The enzyme can be added at the time. tSDA reactions are typically 55-65 ° C. For 1 minute to 2 hours, but if compatible with the selected enzyme Higher temperatures are also possible. Preheating is not required to denature the target sequence If not, all of the SDA reaction components can be transferred directly to the fixed, permeabilized cells. Typically, simply add at the desired reaction temperature to start the amplification. Unused ply After washing away the primers and enzymes, release the amplification products in situ or from the cells. Detect after leaving.                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the HIV target sequence and HLA-DQα exo by in situ tSDA. 3 shows the results of flow cytometry for amplification of the target sequence.                             Detailed description of the invention   The method of the present invention for in situ nucleic acid amplification by tSDA uses in situ tSD A shows in vitro (solution) tSD with little loss of cell structure and morphology To the discovery that it offers significantly improved sensitivity, speed and specificity of the A protocol Based. Generally, a sample of cells suspected of containing the nucleic acid target sequence (eg, a suspension) Cells or tissue sections) maintain the morphological integrity of the cells but not the cellular proteins. Is fixed by a fixative that does not crosslink or precipitate the material, so that Penetration of primers and other reagents is prevented. Therefore, in fixed cells By immobilized protease to achieve penetration of primers and reagents into the Processing is generally not required.   Crosslinkable or precipitating fixatives can be used in the practice of the present invention. Examples are paraformaldehyde, 4% glutaraldehyde, ethanol / vinegar Acid fixative, Carnowa fixative (acetic acid, ethanol, chloroform), 1% tetraoxide Smium, Bouin's fixative (1.21% picric acid, 11% formaldehyde, 5 . 6% acetic acid), Zenker fixative (5.0% mercury chloride, 2.5% potassium dichlorate) Acetic acid, 5.0% acetic acid, 1.0% sodium sulfate), and acetic acid / methanol fixative There is. FACS^TMThe use of lysing solution (Lysing Solution) is for lysis, fixation and permeability Application is enabled using one reagent. Fixation preferred for use in the present invention The solution is 1 to 4% paraformaldehyde, preferably using it for cells or Treats the tissue for about 1 minute to 1 hour. In general, fixed cells should be For example, making it permeable with detergents such as NP40, TRITON or saponin Useful. Under some circumstances, fixation may be optional. That is, tS DA is particularly important in unfixed cells when RNA targets are selectively amplified. can be performed in situ and does not require a preliminary heat denaturation step (see below) Want).   An important feature of the present invention is that the RNA or DNA target sequence, or both, That is, it can be directly amplified using the above-described method. RNA Reverse transcriptase to the tSDA reaction to amplify R (rtPCR-G.J. Nuovo et al., 1992, Diag. Molec. Pathol. 1, 98-102; G.J. Obo et al., 1991, Am. J. Pathol. 58, 518-523; G.J. Nuovo et al., 1991, Am. J. Patho. 1.139,1239-1244). However, tSDA Some of the DNA polymerases used in I know that. They use dNT or RNA or DNA as templates. Polymerize DNA copy of target sequence with inclusion of PαS and displacement from nick Can be done. RNA target sequence therefore adds another reverse transcriptase The same polymer that performs the DNA amplification portion of the tSDA reaction without the need for Can be reverse transcribed. RNA can be obtained by omitting the heat denaturation step or Is treated with DNase before initiating the tSDA reaction, (Ie, with little amplification of the DNA target). next Double-stranded DNA in cells remains double-stranded and unavailable as a template However, the primer hybridizes to the available single-stranded RNA. And increase the specificity of the RNA target sequence by generating cDNA. You can start the width. The cDNA is used in turn as a template for further amplification. Useful. Specific amplification of the RNA target sequence also requires that RN This can be achieved by treating the cells with a gauze-free DNase. Fixing is In the absence of a preliminary heat denaturation step, it helps maintain the integrity of the cells in heat. No fixation is required. However, it makes non-fixed cells or tissue permeable. It can still be useful.   Treatment of cells or tissues with RNase before heat denaturation of double-stranded DNA Degrades the target RNA target sequence and enables specific amplification of the corresponding DNA target sequence You. NaOH (approximately 0.1 M) is also selectively RNA for DNA-specific amplification. Can be used to degrade DNA and denature DNA.   A heat denaturation step (with RNase treatment) prior to annealing of the SDA primer If included, both DNA and RNA target sequences will be amplified There will be. In situ reverse of RNA by DNA polymerase used in tSDA Transcription is generally less effective than DNA synthesis, but surprisingly, in some cases. Therefore, it was found to be more effective than the conventional reverse transcriptase. However, RNA markers The target is usually present in cells in greater numbers than the corresponding DNA target, and quickly The high performance of the resulting amplification of the cDNA will result in any reduced efficiency in the reverse transcription step of the reaction. Overcome and supplement rates. Amplification of both RNA and DNA targets is Gives the maximum number of target sequences that can be amplified, and consequently the potential For most diagnostic applications of the present invention because it provides the greatest sensitivity and maximum number of positive cells to It is particularly preferred.   If the target is heat denatured prior to amplification, the fixed cells or Object (eg, dNTP, KiPO_Four, MgCl_Two, BSA, DMSO, outer primer Heating, amplification primers, and enzymes that are sufficiently thermostable). Can be. Polymerase and restriction endonucleases are sufficiently hot at denaturing temperature If not, cool the samples to the desired reaction temperature before adding them Can be. If the target is not heat denatured, restriction endonucleases and one or more Or amplification of an SDA reaction mixture containing multiple polymerases on a cell sample. It can simply be added at the reaction temperature chosen to start.   As with conventional SDA, the target for amplification by tSDA is the target The restriction of endonucleases that do not cut the sequence And can be manufactured by making the same into a pigment. However, in situ tS For both DA and traditional in situ SDA, the amplification reaction generally Nuclei with selected restriction endonuclease recognition / cleavage sites for tagging Acids were obtained from Walker et al. (1992, Nuc. Acids Res., Supra) and US Pat. No. 5,270,18. It is preferably produced as described in US Pat.   Prevent one SDA reaction from being cross-contaminated by the amplification products of another reaction To replace dUTP with dTTP without significantly inhibiting the amplification reaction. It can be included in an SDA amplicon. Next, the uracil-containing nucleic acid is: Specific recognition by treatment with UDG (uracil DNA glycosylase) and Can be deactivated. Therefore, dUTP is required to When included in a con, any subsequent SDA reaction will amplify the double-stranded target. Can be processed by UDG before and from previously amplified reactions Any of the dU-containing DNAs will not be amplifiable. Amplified in subsequent reactions Target DNA does not contain dU and is unaffected by UDG treatment Will. Next, UDG is ligated with Ugi (uracil DNA glycosylation) prior to target amplification. (Lase inhibitor). Or UDG is hot Can be deactivated. At tSDA, the higher temperature of the reaction itself (>50 ° C.) At the same time, UDG can be inactivated and the target can be amplified.   SDA lacks 5 'to 3' exonuclease activity and is single-stranded in double-stranded nucleic acids. Initiate polymerization at the nick and displace the strand downstream of the nick while simultaneously It requires a polymerase that uses the strand as a template to generate a new complementary strand. Po Rimerase displacement activity makes the target available for the synthesis of yet another copy and Single-stranded extension products capable of hybridizing two amplification primers in an exponential amplification reaction Which is essential for the amplification reaction. More progressive polymerase Can maximize the length of the target sequence that can be amplified, so they are Good.   Activity of thermophilic polymerases at temperatures conventionally considered appropriate for tSDA Little is known about. Furthermore, the activity of thermophilic restriction endonuclease The activity of the thermophilic polymerase at a temperature compatible with sex was not known. did Thus, screening assays may be performed using candidate restriction endonucleases, if any, And polymerases. Polymerase screening system The stem is a polymer that displaces the downstream strand starting at the single-stranded nick in the double-stranded template. This is an extension assay that tests the ability of the enzyme. It is 5 '→ 3' exonuclease Also check for the presence or absence of activity. 5 '→ 3' exonuclease activity Are known in the art when present in other suitable thermophilic polymerases. It can be inactivated by conventional methods (WO 92/06200). Polymer One of the most common methods to selectively inactivate exonuclease activity in One clones the polymerase gene and is involved in exonuclease activity Identify the portion of the gene sequence that encodes the protein domain, and Inactivation by in vitro mutagenesis. Or, Exonuclea The activity of the polymerase is determined by treating the polymerase with a protease to achieve the desired polymerization and Can be inactivated by isolating a fragment that exhibits only the substitution activity. You. Therefore, the thermophilic polymerase identified in the extension assay is activated at the appropriate temperature. Sex, initiates extension at the nick, and includes a modified dNTP but 5 ' → Having 3 'exonuclease activity, to remove exonuclease activity Therefore, it can be made suitable for tSDA.   Displacement of single-stranded from double-stranded nucleic acids and Starting with a nick, pair the two primers immediately next to each other with both primers. And annealing on the complementary complete sequence. Primer Are usually at their 5 'end,³²Labeled by P. Polymerase chain Having displacement activity, formed by adjacent hybridized primers The polymerization can be initiated with “nick” and 5 ′ → 3 ′ exonuclease If it lacks activity, both primers will be extended and the two extension products will be Will be detected. Polymerase lacks 5 '→ 3' exonuclease activity But cannot start extension at nick (eg it needs a gap) And / or if it lacks displacement activity, the extension of downstream primers Only adult products will be detected. Start with Nick but 5 '→ 3' Exonuclea Polymerases with protease activity produce only extension products of the upstream primer. There will be. The extension assay also determines the α-thio dNTP (dNTPαS) contained in the reaction. Needs to be able to be included by the polymerase. Upstream and downstream plastic Imers and their respective extension products are generally Are identified by dimension on the gel.   11 thermophilic DNA polymerases initially screened in the extension assay Exo ~ Vent (New England Biolabs ( New England Biolabs)), exo ~ DeepVent (New England Bath) Io Labs), Bst (BioRad), exo ~ Pfu (Stratagene) (Stratagene)), Bca (Panvera) and sequencing grade Taq (Promega) has all the properties necessary for use in the present invention. It has been certified. Others are routinely performed using the extension assay described above without using the technique of the present invention. Can be identified by means, and any such polymerase Will be suitable for use in tSDA. Polymerase Tth (Boehringer (Boeh ringer)), Tfl (Epicentre), REPLINASE (DuPont Pont)) and REPLITHERM (Epicentre) displace the strand from the nick, but It also has' '3' exonuclease activity. These polymerases, for example, After removing exonuclease activity by genetic engineering, the method of the present invention Useful. Most of the thermophilic polymerases identified so far are at about 50 ° C. Active at 75 ° C., optimally active at about 65 ° C. to 75 ° C., and about 50 ° C. to 6 ° C. Low activity at 5 ° C. However, thermostability of thermophilic restriction endonuclease Is generally limited to less than 65 ° C., so it is thermophilic with optimal activity at lower temperatures Polymerases (eg, Bst and Bca) are thermophilic restriction enzymes in the reaction. It is more compatible with nucleases and is therefore preferred. However, Polymer Restriction endonuclease that is active at higher temperatures compatible with the usual optimum temperature for Creases can be identified and are also useful in the present invention.   Restriction endonucleases suitable for SDA are two of the restriction endonucleases. Only one strand of the semi-modified recognition / cleavage site of the main strand needs to be cleaved ("d "). This nicking activity indicates that nicking makes the reaction permanent and increases the target. Very important because it starts the next cycle of width. Restriction enzymes are generally One strand break at the double helix break site is It must be selectively inhibited. This usually means that the modified or unmodified strand Nucleoside in one strand of the DNA during synthesis so that it is no longer sensitive to Introduction of a tide analog (eg, deoxynucleoside phosphorothioate) And is achieved by: Nucleotide similarity if unmodified strand is protected from cleavage The body can be included in the primer during its synthesis, thus The addition of nucleotide analogs to the reaction does not require the polymerase The requirement that a nucleotide analog can be included is also eliminated.   Nucleotide analog substitutions are nicked by any restriction endonuclease. Tests the nicking properties of restriction endonucleases as it does not cause kinging Is there a large number of thermophilic restriction endonucleases available? Needed to identify any suitable enzymes that may be present. Therefore, the desired properties Screening system for identifying thermophilic restriction endonucleases with System was included in one strand of the double-stranded restriction endonuclease recognition / cleavage site. Modified deoxynucleotides cause two strands from cleavage by the endonuclease. It was devised based on its ability to protect one. This is called the analog-induced nicking assay Or called a chain protection assay.   Single strand casting containing restriction endonuclease recognition / cleavage sites in strand protection assays A primer complementary to a part of the template other than the template and its recognition / cleavage site Combine. Next, the template and primer are combined, typically by radiolabeling. Label. The primer and the template are hybridized and the modified d A semi-modified restriction endonuclease including NTP by extension of the primer This results in a completely double-stranded molecule that contains a site for recognition / cleavage. This product is It is treated with a restriction endonuclease under conditions suitable for cleavage of the main strand. Using electrophoretic analysis of the reaction products under denaturing conditions, the size of the resulting fragments Depending on whether the recognition / cleavage site is nicked, cleaved or cleaved Check if it was not found. The size of the fragment in electrophoresis is Which of the two strands of the position (ie, modified or unmodified) was protected from cleavage It is also used to determine Strand protection assays are performed using conventional techniques. Without using any additional restriction endonucleases for use in the present invention. Can be adapted for screening.   Twenty-eight thermophilic restriction endonucleases were tested using a strand protection assay ( AccI, AspI, BsaI, BsaBI, BsiYI, BslI, BsmI BsmAI, BsmFI, BsmHI, BspWI, Bso Four degenerate sites of BI, two degenerate sites of BsoFI, BsrI, BsrBRI , Two degenerate sites of BsrDI, two degenerate sites of Bst71I, BstNI, BstOI, BstXI, DpnI, HaeII, Maml, MboII, Mv aI, MwoI, SfiI and TthllI). 11 out of 28 types That is, AccI, BslI, BsmI, BsmAI, BsoBI, BsrI, BsrDI, BstNI, BstOI, BstXI and MwoI are at least At least one control nicked during the introduction of one α-thio dNTP. It had a restriction endonuclease recognition / cleavage site. One of the recognition sites for BsmI is Unprotected strand protection was shown when dCTPαS was included. Heat at 50-65 ° C When tested for stability, all but one of the eleven (AccI) were adequate. Of common stabilizers such as double-stranded DNA or BSA The addition could be sufficiently stabilized. Therefore, these endonu The creatase was compatible with the thermophilic polymerase in the tSDA reaction.   In addition, partial or low nicking activity under initial screening conditions for strand protection assays Were identified (eg, Tthll II, BsiYI and BsoFI). Reduced nicking activity hinders SDA Nicking with restriction endonucleases By adjusting (eg, optimizing buffer or adjusting reaction temperature) Optimized) to make those conditions more effective in tSDA. Can be Additionally, sequences flanking the restriction endonuclease recognition / cleavage site May affect the extent of endonuclease activity. Changes in the template flanking sequence are only partially nicked In addition, the nicking activity of the endonuclease can be further improved.   Thermophilic SDA can be used to produce the desired thermostable polymerase and thermostable restriction endonuclease. The procedure is essentially the same as with conventional SDA, with the replacement of the protease. Of course, The reaction temperature will be adjusted to a higher temperature suitable for the selected thermophilic enzyme, And conventional restriction endonuclease recognition / cleavage sites are used to select selected thermostable elements. Place the appropriate restriction endonuclease recognition / cleavage site on the nuclease. Will be replaced. In addition, in contrast to conventional SDA, the practitioner is not Prior to the step, if the enzyme is sufficiently stable at that temperature, the enzyme is included in the reaction mixture. Can be Preferred restriction endonucleases for use in tSDA are BsrI, BstNI, BsmAI, BslI and BsoBI (New In Grand By Labs) and BstOI (Promega). Preferred thermophilic The polymerases are Bca and Bst.   High amplification factors (eg, 10⁸-10⁹) To develop the best SDA system To this end, evaluation and optimization of the buffer system is recommended. This is the thermophilic S Evaluate new restriction enzyme / polymerase pairs for use in DA . Such an optimization method can be performed with any restriction endonuclease / portion for tSDA. The present invention can be applied to determine an appropriate buffer for a remelase combination. Only routine testing is required without the use of the techniques described above. In most cases, traditional KPO typically used in SDA_Four/ MgCl_TwoBuffer is suitable for tSDA As described or with some customary changes in component concentrations.   In in situ tSDA, reagents for amplification are transferred to non-fixed cells or as described above. Apply to fixed and permeabilized cells. After the start of the reaction, Amplification is generally performed at about 50-65 ° C. for 1 minute to 2 hours, preferably 10 minutes to 1 hour. Let time progress. When needed for in situ amplification with tSDA or conventional SDA Is the time required to obtain a similar level of target in situ amplification by PCR. Has been found to be significantly less. In some cases, trials for in situ SDA Increased drug (especially primer) concentration compared to in vitro tSDA It may be advantageous to ensure that sufficient amounts enter the cells for efficient amplification . Leakage of amplicons from cells is problematic in some in situ nucleic acid amplification methods Met. Such leakage can be caused by various parameters, such as the size of the amplicon. The consequences of the complex interactions of the method, temperature, temperature cycling, and the degree to which the cells have been It is considered to be. To promote the retention of amplicons in cells, digoxigeni ("Dig"), biotin or fluorescein isothiocyanate (FITC) ), Etc. attached to a residue such as deoxyribonucleoside triphosphate (dNTP) The dNTP analogs, including, optionally, are packaged in the amplification product together with dNTPαS. Can be included. Still other dNTP analogs may optionally amplify the amplification product. It can also serve as a tag or label used to detect. dig The inclusion of any dNTP analog is such that each included label residue is an alkaline Binds to anti-dig antibody conjugated to phosphatase (AP-α-dig) And has the advantage of providing an enhanced signal since . Since the amplified target sequence is generally smaller than the PCR amplicon, The inclusion of such dNTP analogs is particularly advantageous for in situ SDA. But But However, even though the SDA amplicon is generally smaller than the PCR amplicon, Leakage associated with upcoming in situ SDA is less than with in situ PCR Was observed. Due to the higher temperature of in situ tSDA, amplicon leakage is Expected to increase to the level observed in in situ PCR. I was imagined. However, in practice, there is a slight increase in amplicon leakage at high temperatures. Although possible, amplicon leakage in in situ tSDA was seen in in situ PCR. Still significantly less than   After target amplification, the resulting amplicons are used for detection of specific nucleic acid sequences. Detection can be by any of the methods known in the art. For example, amplification products may be produced in situ or after release of amplicons from cells. Specific hybridization to oligonucleotide detection probe Can be detected. The detection probe is a detectable label, A short oligonucleotide containing residues that produce or can be made to produce an emissible signal It is a lignonucleotide. The label is nicked in the oligonucleotide probe. By translation, by end-labeling or during chemical synthesis of the probe May be included. Numerous direct for use with oligonucleotide probes And labels that are indirectly detectable are known in the art. directly Detectable labels include those that require no further reaction to be detectable, e.g., For example, radioisotopes, fluorescent residues and dyes. Fluorescein isothiocyanate Fluorescent label such as in (FITC) and³²Radioactive isotopes such as P Preferred for use when labeling probes for direct detection of broadened target sequences . Indirectly detectable labels include those that have been reacted with additional reagents to make them detectable. Labels that need to be labeled, such as enzymes, There are otines, avidins, digoxigenins, antigens, haptens or fluorescent dyes. Yeast The signal from the elementary label is generally the enzyme, its substrate and the colored enzyme reaction product. Generated by reacting with any additional reagents necessary to produce Biotin (or avidin) label can be labeled avidin (or labeled Otin) or labeled anti-biotin (or labeled anti-avidin) antibodies And can be detected by binding. Digoxigenin and Hapte N The label is usually labeled anti-digoxigenin (anti-dig) or anti-hapten antibody Is detected by binding specifically to The enzyme is used in the present invention. Preferred for use as an indirectly detectable label. Most preferred is alkali Phosphatase (AP), which is stable and in tissues and cells It has been widely used for labeling. The presence of AP depends on the reaction with the substrate Can be detected by A preferred substrate for the detection of AP is Vector Red / Ve ctor Blue (Vector Labs, CA), 5-bromo-4-chloro B-3-Indolyl phosphate (BCIP) / nitro blue tetrazolium (NBT ) (Sigma Chemical Company, St. Louis) , MO) or Nuclear Fast Red (Sigma Chemical Company) . Vector Red has the additional advantage of fluorescence, which can be achieved by traditional light microscopy or by Allows visualization of positive signals by fluorescence microscopy. AP and these groups Methods for producing colored reaction products with quality are known in the art.   The amplified target sequence is hybridized to a detection probe. Cells or tissues are probed for single-stranded amplification products for detection. Under the appropriate reaction conditions for specific hybridization, the probe And expose. In general, a detection probe is a combination of two amplification primers. Hybridize to nucleotide sequences in the amplicon between the joining sites Would be chosen to be. However, the detection probe is May have the same nucleotide sequence as either one of the two. For detection probe For a suitable method for detection by in situ hybridization, see J.B. Lawrence et al. (1989, Cell 57,493-502); J.B. Lawrence et al. (1990) Natl. Acad. Sci. USA 87, 5420-5424) and U.S. Pat. No. 78.   Alternatively, the amplification product can be obtained as described by Walker et al. (1992b), supra. , In situ or after release from cells by primer extension Wear. In the primer extension method, an oligonucleotide primer containing a detectable label is used. Hybridize to amplification product and add polymerase And thereby elongate. For detection, the primer is preferably³²P also Is The 5 'end can be labeled using a fluorescent label. Alternatively, hybridization Extension of the resulting primer is directly or indirectly performed with a dNT containing a detectable label. P analogs can be included. For example, primer extension can be performed with a dig derivative dNTPs, which can then be combined with AP-α-dig and the appropriate Detected after extension by reaction with AP substrate. The extended primer is It may be the same as the primer or it may be between the binding sites of the amplification primers. Different probes that hybridize to nucleotide sequences in different amplicons May be a limer.   The detectable label is incorporated directly into the amplicon during target sequence amplification. You can also. For example, one of the dNTPs in a conventional SDA reaction is a direct Analogous to dNTPs, including dNTPs attached to a detectable label, either directly or indirectly Can be completely or partially replaced by the body. For example, the desired sign DUTP bound to is substituted for dTTP in the SDA reaction Can be Next, the polymerase was generated by extension of the amplification primer. Include the label directly in the amplification product. The label may be directly or indirectly Can be detected. Preferably, the label bound to dNTP is a fluorescent microscope. Direct detection in amplicons by microscopy or flow cytometry Is a fluorescent label.   In another preferred embodiment, the label attached to dNTP is biotin or Or digoxigenin, which reacts with streptavidin / FITC and And fluorescence microscopy or flow cytometry.   The second amplification product is used for hybridization of the signal primer on the target sequence. And copies of the target sequence generated by the extension. The second amplification product is amplified Internal segments of the target sequence Includes signs that can be issued. At least the 3 'end of the signal primer is the target sequence And sequences that hybridize to It is also the capture of the second amplification product. Features that facilitate capture or immobilization can be included so that they can be detected, quantified Or it can be isolated for further manipulations. In situ tSDA reaction The co-production of the second amplification product is uniform and can be performed simultaneously with the amplification. Extremely long in situ probe hybridizations provide another detection method The hybridization step is eliminated, and the concentration of signal primer is Lower than for the probe. Lower concentrations themselves reduce background And also allows for higher stringency washes that further reduce background You. In order to generate a second amplification product, at least one signal primer is used for i Include in the n situ tSDA reaction mixture. The signal primer is amplified The primer hybridizes to the target sequence downstream of the hybridization site. And extended by polymerase in a manner similar to extension of amplification primers Is done. Extension of the amplification primer is performed by extending the signal primer downstream from the target sequence. Replace the product. Next, the opposite amplification primer was Can hybridize to the null primer, and itself Longer double helix extended by limerase, resulting in target amplification This results in the inclusion of the signal primer therein. Remaining unextended signal plug Since all immers are small, they can be washed out of the cell, The extended signal primer is retained in the cell. Therefore, target amplification characteristics Differential signals become associated with the cell in which the target resides, and the target Almost absent in non-existent cells.   Next, the hybridized detection probe, extended primer, Label the recon or second amplification product, preferably in situ, with the amplified label. As an indicator of the presence of a genetic sequence. This can be AP, biotin or dig, etc. Addition of reagents to the cells to produce an indirectly detectable label signal May be required. Microscopic analysis of cells indicates that the detectable label is an enzyme. Is preferred. Microscopic analysis involves visual observation of cells or tissue (fluorescence or light Microscopy or DISC0VERY (Becton Dickinson Image Cytometry (Becton Dickinson Image Cytometry), Leiden, The Netherlands Automated video analysis using such devices as Can be evaluated. If the label is radioactive, scintillate the cells Suspended in liquid and detecting the signal by scintillation counting Can be. The use of directly detectable fluorescent labels has been demonstrated by flow cytometry (eg, For example, FACSCAN, Becton Dickinson Immunocytometry Systems (B ecton Dickinson Immunocytometry Systems), San Jose, CA) Enables fluorescence analysis of cells in liquid. Peaks on plot of cell number vs. fluorescence intensity The shift of the fluorescence to the right indicates an increase in the number of cells containing the target sequence. Reverse In the plot, the shift of the peak fluorescence to the left indicates the number of cells containing the target sequence. It shows a decrease. Alternatively, the amplification products are obtained from the cells prior to detection, as described above. Can be released or hybridized, eg, with EtBr staining, detection probes After gel electrophoresis by lysis or primer extension, the amplification product Can be visualized as Radiolabel to primer or detection probe When used, amplification products are visualized by gel autoradiography. Can be.   Produce cells for analysis by in situ amplification and flow cytometry Conventional methods include peripheral blood mononuclear cells (P) from whole blood prior to antibody staining and / or expansion. BMC-eg, FICOLL gradient centrifugation). PBMC to T An additional step of isolating the cells may also be necessary. Such an idiomatic protocol Requires about 2 days to obtain flow cytometry results on whole blood samples . Currently, FACS is used to prepare and fix whole blood samples by fixing and in situ amplification.^TM Lysis solution (Becton Dickinson Immunocytometry Systems, Sun ・ Jose, California) for flow cytometric analysis in just one day I know I can do that. The lysis reagent is diethylene glycol, It contains phosphorus, citrate buffer and formaldehyde and has a pH of 7.2. Hol FACS due to the presence of aldehyde^TMLysates are biological obstacles from biological samples. Provide the advantage of reducing. The new sample preparation protocol uses FACS^TM After being easily dissolved by the lysis solution, fix as described above, make it permeable, and  Amplify in situ. Cells in suspension or in tissue can be in situ tSDA and When analyzing by both immunostaining, the antibody is Binding to antibodies or antigens, and indirectly detectable labeling of antibodies For example, it is preferable to bind to biotin. Next, the antibody conjugate is Stabilize on cells by fixation. After in situ SDA, the bound antibody was This Signal-generating reagents, such as streptavidin or fluorescent Detection is by reaction with anti-biotin conjugated to the photopigment. For detection of target amplification Fluorescent dyes and antibody fluorescent dyes can be detected separately by flow cytometry. Yes, the practitioner confirms the presence of the target in the cell and the type of cell in which the target is found Can be identified at the same time. A new sample preparation protocol, amplification Further shortening by the inclusion of fluorescently labeled signal primers in the reaction Can be. As described above, the signal primer is a target amplification characteristic during the amplification reaction. Additional post-amplification steps that are extended and double-stranded in an heterogeneous fashion to detect amplification products Eliminate the need.   The design of amplification primers for SDA is generally directed towards the target moiety of interest. Following the synthesis of a number of primers, the pair of primers in the SDA reaction Need to be tested to determine the amplification efficiency of each pair. This This is because parameters affecting SDA primer performance are well understood. It depends. The present inventors have developed a target binding portion of an amplification primer for SDA. Changes that appear to be insignificant have significant and unexpected effects on amplification efficiency And the melting point of its target binding moiety Has not always been involved. For in situ tSDA development Then, a first pair of amplification primers specific to the gag gene of HIV was designed. S A target portion of the gag gene which does not contain a recognition site for DA restriction endonuclease was selected. I chose. The targeting moieties are also approximately 100 bp apart and are relatively invariant. Selected based on having two parts of 50 bp. Next, the primer Using design software, determine the melting point of target binding for each primer candidate And the possibility of primer dimer formation was determined. Primers are Modified or abandoned according to preliminary results. 8 types of candidates "left" Amplification primers (directed to the 3 'end of the target on the first strand) and 9 Candidate "right" amplification primer (directed to the 3 'end of the target on the second strand The final list was compiled for experimentation. Bumper primer and detection primer The lobes are designed with only the proper placement with respect to melting point and amplification primers. Was.   The amplification efficiency of the "left" and "right" primer pair combinations was Plasmid of gag target, Bca polymerase and BsoBI in tSDA It was determined experimentally using a clone. Amplicon,³²Pro for P label detection Hybridization and extension followed by gel electrophoresis And quantified. The amplification primer pair with the best amplification efficiency in vitro is in situ  Selected for further development at tSDA. Buffer optimized as above In the system, this primer set is ga in vitro in tSDA. g Less than 10 copies of target sequence detected (BsoBI site in italics, The target binding sequence is underlined).   A pair of amplification primers specific for exon 3 of the HLA-DQα gene was Similar for human candidate placental DNA for evaluation of candidate amplification primer pairs in itro Designed to. Since the HLA gene is present in all cells, this target is u was to be used as a positive control for tSDA. Amplicon leakage is important It was found that this was not a problem. Selected for initial identification of the mer (about 75-100 bp). Three types of "left" amplification Primers and three candidate "right" amplification primers were first designed and then paired. Were tested experimentally. Buffer solution optimized as above The following primer set gives the best amplification results and the in vitro tSD In A, less than 5 copies of the HLA-DQα exon 3 target sequence were detected.   The target binding sequence confers target specificity on the amplification primer. Amplification of the invention The target binding sequence of the primer may therefore be a nucleic acid amplification protocol other than SDA, For example, it is also useful in PCR and 3SR. Specifically, for the target sequence Specific hybridization of primers using target sequence as template Using primer extension and displacement of the extension product from the target sequence Amplification protocols can also use the target binding sequence of the amplification primers of the invention. You. Restriction endonuclease recognition site of amplification primer shown in attached sequence listing And amplification methods that do not require any special non-target binding sequences (eg, PCR). Thus, the amplification primer can consist only of the target binding sequence. Shown in Sequence Listing Amplification methods that require special non-target binding sequences different from those performed (eg, SR) is the target binding sequence of the listed amplification primers and known in the art. Sequence or structure required by the selected amplification method as is known Included amplification primers can be used. In addition, another restriction factor appropriate for tSDA The nuclease recognition site may also be determined using methods known in the art. And replaces the restriction endonuclease recognition site shown in the sequence listing be able to.   The following experimental examples are provided to illustrate some embodiments of the present invention. , Should not be construed as limiting the invention as defined by the claims.                                 Example 1   HLA-DQα exon 3 targets were expressed in human acute myeloid leukemia (AML) cells (K G-1a) In-situ amplification using the primer set selected above. Width and detected. First, cells are fixed in 4% paraformaldehyde for 30 minutes. And washed three times in 1 × phosphate buffer solution (PBS). Then put them Permeabilized with 0.01% saponin for 20 minutes and with 1 × PBS Washed three times. Fixed and permeabilized cells (35 mM Kp_i  pH 7.6 Cells 10⁸5 ml of 35 mM Kp_i, PH 7.6, 3 mM MgCl_Two, 50 μM each of dGTP, TTP and dATP, 1 . 4 mM dCTPαS, 500 nM amplification primer, 50 nM bumper ply And 40 μL of 15% glycerol. After gently mixing, The sample was incubated at 95 ° C for 2 minutes and maintained at the reaction temperature (52 ° C). THERMAL-LOK^TMTransferred to temperature block. Next, 5 microliters of the enzyme mixture (10 NEB2 0.5 μL, 22 units / ml Bca 0.36 μL, 160 units / ml BsoBI 1.0 μL and water 3.14 μL) and mix To start the amplification reaction. Final reaction volume was 50 μL. After 30 minutes, the reaction was cooled to ice Stopped by placing on top and detecting amplification products.   Radiolabeled detection probe (2 × 10⁶cpm) to the amplification reaction. And 30 minutes at 95 ° C. followed by 60 minutes at 37 ° C. hybridized with tu. Wash twice in 200 μL of 1X SSC for 25 minutes After cleaning, cells were counted in scintillation fluid. The conclusion of one such experiment The results were as follows.   In test tube 1, the amplified KG1a cells are labeled with HLA-DQα exon 3 Probed by a heterogeneous detection probe. Test tube 2 contains restriction endonuclease Is a negative control omitted from the amplification reaction and prevented tSDA. Test tubes 3 and 4 Corresponded to tubes 1 and 2, but were independent of the target sequence, a gag-specific detection program. Explored by Loeb. Amplified in the presence of all necessary enzymes and HLA-D All samples detected by the Qα exon 3 specific probe were in situ Showed specific amplification of the target.   Incubation of unamplified cells with amplicons generated in situ The experiment was repeated to include the option. This can lead to amplicon metastasis to negative cells. Amp, by attaching amplicon to cell surface or by negative cells It was to evaluate by taking in the recon. After in situ amplification, centrifuge the reaction. The cells were sedimented apart. The supernatant is incubated at 70 ° C. for 15 minutes and Bso BI activity was removed and fixed and pre-heated to 95 ° C for 2 minutes. Was added to the obtained KG1a cells. Incubate the mixture at 52 ° C for 30 minutes After that, cells are probed for specific and non-specific (irrelevant) detection as described above. Both were hybridized, washed and counted. The results are shown below You.   Restriction endonuclease was omitted from tube 1 negative control reaction. Test tube 2 And 3 showed a complete amplification reaction by specific detection. Test tube 3 is a test tube In situ under the same conditions as in Example 2, but with a three-fold increase in polymerase concentration   The signal obtained from tSDA is shown. In test tube 4, tS generated in situ After incubating the DA amplicon with non-amplified cells, This is hybridized, washed and tested as for the amplified sample. Issued. The test tube 4 shows how much the positive signal of the test tube 2 is not amplified. Checking for non-specific attachment of amplicons to cells was included. Trial Test tubes 5-8 were hybridized using an irrelevant gag detection probe as a negative control. Except that the reaction was performed, the reaction conditions corresponded to the test tubes 1 to 4. As in the previous experiment In addition, specific in situ amplification of the target is clearly Occurs in the sample and is detected by a specific HLA-DQα exon 3 probe Was. Amplicons that may be found in the supernatant False positive signal generation due to the mechanism of However, the substantially higher signals in tubes 2 and 3 are clearly in situ Indicates that SDA is occurring. While most signals are target-specific, Not for the amplicon transition.   In another detection system, a signal probe labeled with fluorescein The experiment was repeated with the immer. Signal primer was added to the amplification reaction at 100 Added at a concentration of nM. As a result, the fluorescent probe is extended by the polymerase. And was displaced from the target by extension of the upstream amplification primer. Increase on ice After stopping the width reaction, the samples were each diluted with 150-200 μL of phosphate buffer solution. And washed by washing for 5 minutes. Smaller unstretched signal ply Was washed out of the cells but longer target amplification-specific fluorescent signal primers The mer was retained by the cells in which the amplification occurred. In these experiments, washing Cells were visualized by fluorescence microscopy, where a strong fluorescent signal Found in positive cells and negative cells were non-fluorescent. However, the signal Primer extension involves detecting fluorescent cells by flow cytometry and / or Suitable for counting.   In a third of a similar experimental series, the amplification products were detected by a colorimetric assay. 4 cells Fix in 20% paraformaldehyde for 20 minutes, wash 3 times in 1X PBS, For 10 minutes in 0.01% saponin. SDA buffer (35 mM KPO_Four  Wash in pH 7.5, 15% glycerol, 4 mM MgOAc) After the cell suspension (cells 5 × 10^Five5 μL of the primers and And 40 μL of SDA buffer containing dNTP. HLA- Using the DQα exon 3 primer set, add 5 μL of enzyme mixture after target denaturation. The target was then amplified to a final reaction volume of 50 μL. Human HLA and mouse MH Despite homology to C, the mouse MHC target was amplified with this amplification system Mouse cells served as a negative cell line. Gene sequence database Analysis of the mouse and human at the binding site of the primers used here. Indicates that the homology between them is insufficient, and the purified mouse DNA is The inability to amplify using these primers confirmed the negative. After denaturation at 94 ° C. for 2 minutes, a detection probe labeled with digoxigenin (1-1 to 1) 0 nM) was hybridized at 33 ° C. for 2 hours. For hybridization Subsequently, after hybridization three times at room temperature in 1X SSC, washing and washing were performed. And buffer exchange to 100 mM Tris pH 7.5 / 150 mM NaCl. Was. Next, an anti-digoxigenin antibody conjugated to alkaline phosphatase (AP) The body (Fc fragment) was incubated with the cells for 2-4 hours at room temperature . Tris / NaCl wash and alkaline eluate to remove unbound antibody from negative cells Sphatase buffer (100 mM Tris pH 9.5 / 100 mM NaCl / 50 mM MgCl_Two), Amplification occurs using NBT / BCIP. Color was developed in those cells. Cells are spread on slides (cytospun) and And visualized by microscopy. Human cells were detected for HLA-DQα exon 3 Strong colorimetric signal when hybridization probe is amplified in amplified cells Occurred. Probe for detecting HLA-DQα exon 3 in non-amplified cells Hybridization to negative or very weak but still obvious A negative colorimetric response resulted. Other negative controls include absence of amplification (ie, SDA enzyme Element), the absence of the detection probe, and the non-specific detection probe (ie, The gag or chicken β-actin detection probe in the amplified cells is Included, all of which were negative in the test. In mouse cells alone, HL Both A-DQα exon 3 and the gag probe produced little color, but did not And Colorimetric signal in mouse cells increases when mouse cells are mixed during amplification reaction did. However, the result is that this is an artifact of the colorimetric detection system itself , And negative cells suggest non-specific absorption of the dye.                                 Example 2   Venous blood, EDTA VACUTAINER^TMBlood collection tube (Becton Dickinson Bactina) (Becton Dickinson Vacutainer Systems). DNP Bound anti-CD4 (L120) and biotinylated anti-CD3 (Leu4) antibodies were Added to 50 μL of blood and stained for 20 minutes at room temperature. Lyse red blood cells 1X FACS^TMLysis solution (Becton Dickinson immunocytometry • Systems) 1.0 mL was added to each tube for 10 minutes. 4% paraphor for cells Fix in room temperature for 20 minutes at room temperature and 1X FACS^TMLysate and And made permeable by adding 0.5 mL of 0.025% Tween 20 . Transfer cells to 35 mM KPO_FourWashed twice with 35 mM KPO_FourBuffer 5μ L and transferred to a 0.5 mL microcentrifuge tube.   35 mM KPO for in situ tSDA_Four, 1.4 mM dCTPαS , 200 μM each of dATP, dGTP and dTTP, 4 mM Mg acetate , 15% glycerol, 0.05 μM of each bumper primer and 0.05 μM 40 μL of each of the M amplification primers was added to the prepared blood sample. sample Was heated at 95 ° C. for 3 minutes followed by 52 ° C. to 55 ° C. for 3 minutes to allow the primer to be annealed. Was ringing. After primer annealing, the enzyme formulation (10X NEB2 buffer) 0.5 μL of the solution, 160 units of BsoBI, 8 units of Bca polymerase) In addition, the amplification reaction was started. The tubes were incubated at 55 ° C. for 30 minutes. Increase The width product was hybridized to a fluorescein-labeled detection probe. Detected in situ by quantification. Hybridization is specific SDA reaction buffer containing 100-150 ng of target or irrelevant detection probe 5 minutes at 25 ° C. followed by 60 minutes at 33 ° C. to 37 ° C. in 25 μL of solution Was.   Alternatively, the amplification product is used as a primer for the fluorescein-labeled signal primer during amplification. Detected by inclusion. In this case, the amplification reaction buffer is specific or irrelevant 5 'fluorescein-labeled signal primer at 100 nM was also included. initial The heating step, addition of the enzyme mixture and amplification were performed as described above.   After in situ hybridization of the detection probe or signal After completion of tSDA in the presence of the immersed cells were washed with 1 × SSC at room temperature for 30 minutes. Washed for minutes. Antibody staining for cell surface markers was performed using anti-DNP-phycoerythrin ( PE) and Cy5 / PE labeled streptavidin. Cells Wash once with 1 × PBS and add 1 × for flow cytometry analysis. Resuspended in X PBS. Side scatter vs forward scatter (SSC vs FSC) dots On the plot, it was shown that leukocytes were not affected by the experimental treatment. Sand HLA of the CD4 / CD3 immunophenotype (using the above primer set) -Cells subjected to in situ tSDA for DQα exon 3 target include lymphocytes, condyles A typical population of granulocytes and monocytes was shown. The lymphocyte population also contains CD4 versus CD3 Looked normal on the fluorescent dot plot. These experiments were performed by FACS^TMLysate and Immunophenotyping demonstrated that both were compatible with in situ tSDA.   Cells containing HIV genome for HIV detection by in situ tSDA The system (H9 +) is mixed with normal whole blood and processed as described above, and selected as described above. The gag target sequence was amplified using the primers thus obtained. In addition, a probe for detection Or yet another oligonucleotide that can be used as a signal primer Was designed to be used as an alternative to SEQ ID NO: 5.   On a dot plot of SSC vs. FSC, H9 + cells were in any of the leukocyte populations. Apparently separate from lymphocytes, monocytes and granulocytes with higher forward scatter Crab was distinguished. HLA-DQα exon 3 and HIV gag target were Amplified by in situ tSDA using primer set and Detection by hybridization or inclusion of probe for detection of primers did. A histogram plot of FL1 versus cell count is shown in FIG. HLA-DQα Exon 3 positive controls were either no enzyme added or irrelevant probe or When compared to a negative control reaction where the signal primer was not used for detection, A substantial shift to the right of the peak fluorescence (approximately 100 channels) was shown. HIV increase The width response is similar to the fluorescence peak shift on a histogram plot of FL1 versus cell count. Movement. These experiments confirmed that amplification occurred in situ, but only HIV target is also amplified by flow cytometry in lysed whole blood. And demonstrated that it could be detected. Detection probe and signal ply The magnitude of the peak shift for the mer detection method was similar.   Alternatively, use venous blood as EDTA VACUTAINER^TMCollect in blood collection tube, and FICOLL-PAQUE^TM PBMCs were isolated by centrifugation according to Collect the cells with 1X PBS. And washed using a human T-cell enhanced column (R & D Systems). And B cells were removed. The T cell enriched fraction was analyzed for DNP-conjugated anti-CD4 and biotini. Stained for 20 minutes at room temperature with a conjugated anti-CD3 antibody. 4% paraforma in PBS After 20 minutes of fixation in aldehyde, cells were washed with PBS and counted. Fine The vesicles are permeabilized with 10 μg / mL saponin and 35 mM KPO_Four Was washed twice. Cell 5X10^FiveKPO_FourResuspend in 5 μL of buffer , And transferred to a 0.5 mL microcentrifuge tube. in situ tSDA, amplification product Detection and immunophenotyping by FACS^TMThe lysate sample preparation method described above Went like so. The experimental results were almost identical for the two sample preparation methods.

[Procedure amendment] [Submission date] June 2, 1997 [Correction contents] (1) The claims are corrected as follows. [1. A method of in situ amplification of a target sequence,   (A) In a cell sample, the first amplification primer is The 3 'is hybridized in situ and the first amplification primer is Contains a restriction endonuclease recognition site 5 'to the binding sequence and Hybridize the primer to the first strand of the target sequence upstream of the amplification primer Forming;   (B) a first amplification primer and a first outer primer,     (I) a thermophilic polymerase, which is active at about 50 ° C. to 75 ° C .; The thermophilic polypeptide having an exchange activity and lacking 5 'to 3' exonuclease activity. Remelase,     (Ii) α-thiodeoxynucleoside triphosphate, and     (Iii) a thermophilic restriction endonuclease, wherein the restriction endonuclease is Is partially modified by the inclusion of the α-thiodeoxynucleoside triphosphate Nick the restriction endonuclease recognition site if The thermophilic restriction endonuclease is active at 0 ° C to 75 ° C. In the presence of a restriction endonuclease recognition site. To generate the first amplification primer extension product containing Thus displacing the first amplification primer extension product from the first strand of the target sequence;   (C) synthesizing a first complementary strand to obtain a first amplification primer extension product and And the restriction endonuclease recognition site is double-stranded, thereby Nicking the nuclease recognition site with a restriction endonuclease;   (D) extending from the nick using a polymerase, thereby allowing the Displacing the pea from the double-stranded first amplification primer extension product;   (E) nicking, extending and displacing such that the target sequence is amplified in situ The above method comprising the step of repeating the step of:   2. At the double-stranded restriction endonuclease recognition site, AccI, BslI, Bs ml, BsmAI, BsoBI, BsrI, BsrDI, BstNI, BstO 2. The method of claim 1 wherein the nick is nicked using I, BstXI or MwoI.   3. Thermophilic polymerase is exo-Vent, exo-DeepVent, Bst Exo-Pfu, Bca and sequencing grade Taq. Item 1. The method according to Item 1.   4. The amplified target sequence is compared with the signal primer on the target sequence being amplified. Detection by second amplification product generated by hybridization and extension The method of claim 1.   5. A method for amplifying a double-stranded HIV gag target sequence, comprising:   (A) a first amplification primer comprising the target binding sequence of SEQ ID NO: 1 Hybridized 3 'to the target sequence on the first strand, and SEQ ID NO: 2 A second amplification primer comprising a target binding sequence of Hybridized 3 'to the   (B) extending the first and second amplification primers with a polymerase to Producing first and second amplification primer extension products;   (C) displacing the first and second amplification primer extension products from the target sequence; hand   (D) the steps of hybridization, extension and displacement so that the target sequence is amplified. The above method comprising the step of repeating the steps.   6. The first and second amplification primers are each α-thiodeoxynucleotide Restriction endonuclease recognition site is semi-modified by inclusion of dotriphosphate Restriction endonucleus nicked by its restriction endonuclease in case A restriction endonuclease that is a recognition site for a protease and is 5 'to the target binding sequence. Contains a lease recognition site, and   (A) the outer primer pairs with the target sequence upstream of the first and second amplification primers; And hybridize; and   (B) the first amplification primer, the second amplification primer and the outer primer are restricted In the presence of endonuclease and α-thiodeoxynucleoside triphosphate And the first and second primers are displaced from the target sequence by extension of the outer primer. 6. The method of claim 5, wherein a second amplification primer extension product is produced.   7. A method for amplifying a double-stranded HLA-DQα exon 3 target sequence, comprising:   (A) a first amplification primer comprising the target binding sequence of SEQ ID NO: 6 Hybridized 3 'to the target sequence on the first strand, and SEQ ID NO: 7 A second amplification primer comprising a target binding sequence of Hybridized 3 'to the   (B) extending the first and second amplification primers with a polymerase to Producing first and second amplification primer extension products;   (C) displacing the first and second amplification primer extension products from the target sequence; hand   (D) the steps of hybridization, extension and displacement so that the target sequence is amplified. The above method comprising the step of repeating the steps.   8. The first and second amplification primers are each α-thiodeoxynucleotide Restriction endonuclease recognition site is semi-modified by inclusion of dotriphosphate Restriction endonucleus nicked by its restriction endonuclease in case A restriction endonuclease that is a recognition site for a protease and is 5 'to the target binding sequence. Contains a lease recognition site, and   (A) the outer primer pairs with the target sequence upstream of the first and second amplification primers; And hybridize; and   (B) the first amplification primer, the second amplification primer and the outer primer are restricted In the presence of endonuclease and α-thiodeoxynucleoside triphosphate And the first and second primers are displaced from the target sequence by extension of the outer primer. The method of claim 7, wherein a second amplification primer extension product is produced.   9. The method according to claim 7, wherein the target sequence is amplified by a polymerase chain reaction.   10. Target binding sequence of SEQ ID NO: 1, target binding sequence of SEQ ID NO: 2, SEQ ID NO: : Oligonucleotide comprising the target binding sequence of 6 or the target binding sequence of SEQ ID NO: 7 De. 』

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), AU, BR, CA, J P, KR, MX, SG (72) Inventor Van Cleave, Mark             North Carolina, United States             27707, Durham, Caliber Park de             Live 7301, number 202 (72) Inventor Reed, Robert Allan             North Carolina, United States             27713, Durham, Limerick Lane             923

Claims (1)

[Claims]   1. A method of in situ amplification of a target sequence,   (A) In a cell sample, the first amplification primer is The 3 'is hybridized in situ and the first amplification primer is Contains a restriction endonuclease recognition site 5 'to the binding sequence and Hybridize the primer to the first strand of the target sequence upstream of the amplification primer Forming;   (B) a first amplification primer and a first outer primer,     (I) a thermophilic polymerase, which is active at about 50 ° C. to 75 ° C .; The thermophilic polypeptide having an exchange activity and lacking 5 'to 3' exonuclease activity. Remelase,     (Ii) α-thiodeoxynucleoside triphosphate, and     (Iii) a thermophilic restriction endonuclease, wherein the restriction endonuclease is Is partially modified by the inclusion of the α-thiodeoxynucleoside triphosphate Nick the restriction endonuclease recognition site if The thermophilic restriction endonuclease is active at 0 ° C to 75 ° C. In the presence of a restriction endonuclease recognition site. To generate the first amplification primer extension product containing Thus displacing the first amplification primer extension product from the first strand of the target sequence;   (C) synthesizing a first complementary strand to obtain a first amplification primer extension product and And the restriction endonuclease recognition site is double-stranded, thereby Nicking the nuclease recognition site with a restriction endonuclease;   (D) extending from the nick using a polymerase, thereby allowing the Displacing the pea from the double-stranded first amplification primer extension product;   (E) nicking, extending and displacing such that the target sequence is amplified in situ The above method comprising the step of repeating the step of:   2. At the double-stranded restriction endonuclease recognition site, AccI, BslI, Bs ml, BsmAI, BsoBI, BsrI, BsrDI, BstNI, BstO 2. The method of claim 1 wherein the nick is nicked using I, BstXI or MwoI.   3. Thermophilic polymerase is exo-Vent, exo-DeepVent, Bst Exo-Pfu, Bca and sequencing grade Taq. Item 1. The method according to Item 1.   4. 2. The method of claim 1, further comprising detecting the amplified target sequence.   5. The method according to claim 4, wherein the amplified target sequence is detected in situ.   6. The method according to claim 5, wherein the amplified target sequence is detected by flow cytometry. The described method.   7. 6. The method of claim 5, wherein the amplified target sequence is detected by microscopy. Law.   8. The amplified target sequence is compared with the signal primer on the target sequence being amplified. Detection by second amplification product generated by hybridization and extension The method of claim 5.   9. The target sequence is double-stranded,   (A) The second amplification primer is placed in situ 3 ′ to the second strand of the target sequence. And the second amplification primer is 5 'to the second target binding sequence. A restriction endonuclease recognition site and a second outer primer Hybridizing to a target sequence upstream of the primer;   (C) extending the second amplification primer and the second outer primer, thereby Generates Second Amplification Primer Extension Product Containing Restriction Endonuclease Recognition Site And the second amplification primer is extended from the second strand of the target sequence by extension of the second outer primer. Displacing the immersion extension product;   (D) synthesizing a second complementary strand to obtain a second amplification primer extension product and And the restriction endonuclease recognition site is double-stranded, thereby Nicking the nuclease recognition site with a restriction endonuclease;   (E) extending from the nick using a polymerase, thereby allowing the Displacing the DNA from the double-stranded second amplification primer extension product; and   (F) nicking, extending and substituting such that the target sequence is amplified in situ The method of claim 1, further comprising the step of:   10. A method for amplifying a double-stranded HIV gag target sequence, comprising:   (A) a first amplification primer comprising the target binding sequence of SEQ ID NO: 1 Hybridized 3 'to the target sequence on the first strand, and SEQ ID NO: 2 A second amplification primer comprising a target binding sequence of Hybridized 3 'to the   (B) extending the first and second amplification primers with a polymerase to Producing first and second amplification primer extension products;   (C) displacing the first and second amplification primer extension products from the target sequence; hand   (D) the steps of hybridization, extension and displacement so that the target sequence is amplified. The above method comprising the step of repeating the steps.   11. The first and second amplification primers are each α-thiodeoxynucleotide. The restriction endonuclease recognition site is semi-modified by the inclusion of sidotriphosphate. Restriction endonuclease nicked by the restriction endonuclease when A restriction endonuclease that is a recognition site for a creatase and is 5 'to the target binding sequence. Contains a nuclease recognition site, and   (A) the outer primer pairs with the target sequence upstream of the first and second amplification primers; And hybridize; and   (B) the first amplification primer, the second amplification primer and the outer primer are restricted In the presence of endonuclease and α-thiodeoxynucleoside triphosphate And the first and second primers are displaced from the target sequence by extension of the outer primer. 11. The method of claim 10, wherein the method produces a second amplification primer extension product.   12. The method according to claim 10, wherein the double-stranded target sequence is amplified by a polymerase chain reaction. the method of.   13. A method for amplifying a double-stranded HIV gag target sequence, comprising:   (A) A first amplification primer consisting of SEQ ID NO: 1 is labeled on the first strand of the target sequence. Amplification 3 'to the target sequence and consisting of SEQ ID NO: 2 Primer hybridizes 3 'to the target sequence on the second strand of the target sequence And hybridize the outer primer upstream of the first and second amplification primers. To form;   (B) The first and second amplification primers and the outer primer have strand displacement activity. 5 ′ → 3 ′ exonuclease deficient polymerase, Polymerase active at C, α-thiodeoxynucleoside triphosphate and B extension in the presence of soBI, thereby extending the extension of the outer primer. And a BsoBI recognition site that is displaced from the first and second strands of the target sequence. And generating a second amplification primer extension product;   (C) First and second amplification primer extension generation by synthesizing complementary strands And the BsoBI recognition site are double stranded, thereby allowing double stranded BsoBI recognition. Nick the site with BsoBI;   (D) extending from the nick using a polymerase, thereby allowing the Displacing the primers from the double-stranded first and second amplification primer extension products; and   (E) Repeat nicking, extension and displacement steps so that the target sequence is amplified. The above method comprising the step of returning.   14． SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 12, 2. The method according to claim 1, further comprising detecting using SEQ ID NO: 13 or SEQ ID NO: 14. 3. The method according to 3.   15. A method for amplifying a double-stranded HLA-DQα exon 3 target sequence, comprising:   (A) a first amplification primer comprising the target binding sequence of SEQ ID NO: 6 Hybridized 3 'to the target sequence on the first strand, and SEQ ID NO: 7 A second amplification primer comprising a target binding sequence of Hybridized 3 'to the   (B) extending the first and second amplification primers with a polymerase to Producing first and second amplification primer extension products;   (C) displacing the first and second amplification primer extension products from the target sequence; hand   (D) the steps of hybridization, extension and displacement so that the target sequence is amplified. The above method comprising the step of repeating the steps.   16. The first and second amplification primers are each α-thiodeoxynucleotide. The restriction endonuclease recognition site is semi-modified by the inclusion of sidotriphosphate. Restriction endonuclease nicked by the restriction endonuclease when A restriction endonuclease that is a recognition site for a creatase and is 5 'to the target binding sequence. Contains a nuclease recognition site, and   (A) the outer primer pairs with the target sequence upstream of the first and second amplification primers; And hybridize; and   (B) the first amplification primer, the second amplification primer and the outer primer are restricted In the presence of endonuclease and α-thiodeoxynucleoside triphosphate And the first and second primers are displaced from the target sequence by extension of the outer primer. 16. The method of claim 15, wherein the method produces a second amplification primer extension product.   17． The method according to claim 15, wherein the target sequence is amplified by a polymerase chain reaction. .   18. A method for amplifying a double-stranded HLA-DQα exon 3 target sequence, comprising:   (A) A first amplification primer consisting of SEQ ID NO: 6 is labeled on the first strand of the target sequence. Amplification 3 'hybridized to the target sequence and consisting of SEQ ID NO: 7 Primer hybridizes 3 'to the target sequence on the second strand of the target sequence And hybridize the outer primer upstream of the first and second amplification primers. To form;   (B) The first and second amplification primers and the outer primer have strand displacement activity. 5 ′ → 3 ′ exonuclease-deficient polymerase, Polymerase, α-thiodeoxynucleoside triphosphate and Bs elongation in the presence of oBI, whereby by extension of the outer primer A first and second sequence containing a BsoBI recognition site that is displaced from the first and second strands of the target sequence. And generating a second amplification primer extension product;   (C) First and second amplification primer extension generation by synthesizing complementary strands And the BsoBI recognition site are double stranded, thereby allowing double stranded BsoBI recognition. Nick the site with BsoBI;   (D) extending from the nick using a polymerase, thereby allowing the Displacing the primers from the double-stranded first and second amplification primer extension products; and   (E) Repeat nicking, extension and displacement steps so that the target sequence is amplified. The above method comprising the step of returning.   19. Target binding sequence of SEQ ID NO: 1, target binding sequence of SEQ ID NO: 2, SEQ ID NO: : Oligonucleotide comprising the target binding sequence of 6 or the target binding sequence of SEQ ID NO: 7 De.   20. SEQ ID NO: 5, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, An oligonucleotide consisting of SEQ ID NO: 13 or SEQ ID NO: 14.