DETECTION OF NUCLEIC ACIDS IN CELLS BY THE METHOD OF AMPLIFICATION OF THE HEBREWS BY DISPLACEMENT, IN A THERMOFILIC REACTIONFIELD OF THE INVENTIONThe present invention relates to the amplification of nucleic acids, and in particular, to the amplification of nucleic acids in morphologically intact cells.
BACKGROUND OF THE INVENTION Nucleic acid amplification techniques have proven to be powerful tools for the detection and analysis of small amounts of nucleic acids. The extreme sensitivity of these methods has given rise to attempts to develop them for the purposes of early diagnosis of infections and genetic diseases, isolation of genes for the analysis and detection of specific nucleic acids in forensic medicine. Nucleic acid amplification techniques can be grouped according to the temperature requirements of the procedure. For amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR) and transcriptional amplification require repeated cycles of reactions between elevated temperatures (85 ° C-100 ° C) and low temperatures (30 ° C-40 ° C) to regenerate single-stranded white molecules. In contrast, methods such as Displacement Strand Amplification (SDA), replication of the self-sustaining sequence (3SR) and the Qβ replicase system are isothermal reactions that can be carried out at a constant temperature. In the PCR, the temperature of the reaction rises after the extension of the primer or initiator to separate the freshly synthesized strand from the template. The temperature is then reduced to re-prime the primers and repeat the extension process. Therefore, the PCR steps are carried out in small phases or cycles as a result of the temperature limitations of the reaction. In contrast, in the Amplification of the Strand by Displacement (SDA), the extension of the primers, the displacement of the single-strand extension products, the annealing of the primers for the extension products (or the original white sequence) and the subsequent extension of the initiators occurs concurrently in the reaction mixture. Conventional SDA (performed at low temperatures, usually around 35-45 ° C) is described in G.T. Walker, et al. (1992a Proc. Na ti, Acad. Sci. USA 89, 392-396 and 1992b, Nac. Acids, Res. 20, 1691-1696). A version of the thermophilic SDA reaction (the SDAt, described below) has recently been developed and is carried out at a very high temperature, but still constant, using termostable poly erases and restriction endonucleases. The targets for amplification by SDA can be prepared by fragmenting the larger nucleic acids using the endonuclease that is used in the SDA reaction. However, when the target is not flanked by the restriction endonuclease recognition sites necessary for fragmentation, the target nucleic acids having the recognition sites for the restriction endonuclease suitable for notching the SDA reaction can be generated as it was described in Walker et al. (1992b, supra) and U.S. Patent No. 5,270,184. As in the SDA, the individual steps of the target generation reaction are presented concurrently and continuously generating target sequences with the terminal recognition sequences that are required for the formation of the notches by the restriction enzyme in the SDA. As all the components of the SDA reaction are present in the target generation reaction, the white sequences generated automatically and continuously enter the SDA cycle and are amplified. The in si t u methods of nucleic acid analysis allow the detection and localization of specific nucleic acid sequences within morphologically intact cells. These methods have traditionally been based on the direct hybridization of the labeled probes, for example, as described in U.S. Patent No. 4,888,278. However, these methods of direct hybridization, while specific for the nucleic acid of interest, may not be sensitive enough to detect very small numbers of copies of the nucleic acid in all cases. As a means to detect very small copy numbers, it has been of great interest the amplification in si tu of the white sequence before the detection ín si t? . The amplification methods m if your nucleic acid have the potential to be more sensitive than the amplification of conventional solutions, because the cell can concentrate the amplification product, by means of this the detection of the least amount can be made of molecules that are possible when the products of the amplification are free to diffuse or when they are diluted in the content of the cells that do not contain the sequence of interest. Since it is not necessary for the nucleic acid to be extracted from the cell prior to detection of the target sequence, the methods themselves provide information for which the cells in a population contain a specific nucleic acid and also allow acid analysis nucleic acid in the context of the biochemical and morphological characteristics of the cell. The amplification methods in itself have been developed mainly for PCR (0. Basgara and R. Pomerantz, 1993. AIDS Research and Human Retroviruses 9 (1), 69-76; G. Nuovo, et al. 1992. Diag. Mol ec. Pa thol. 1 (2), 98-102; M. J. Embleton, et al. 1992. Nuc. Acids Res. 20 (15), 3831-3837; J. Emmetson, et al. 1993. Proc. Nati Acad. Sci. USA 90, 357-361; P. Ko minoth, et al. 1992. Di ag. Molec. Pa thol. 1 (2), 58-97; K. P. Chile, et al. 1992. J. Hi stochem. Cytochem. 40 (3), 333-341; Haase, et al. 1990. Proc. Na ti. Acad Sci. USA 87, 4971-4975; O. Basgara, et al. 1992. New Engl. J. Med. 326 (21), 1385-1391; Patterson, et al. 1993. Science 260,976-979). However, the multiple cycles of heating and cooling and the severe hybridization conditions that are required for PCR, to achieve their sensitivity, are not well tolerated by tissues and cells. The diffusion of the amplified sequences out of the cells can be increased by repeating the heating, resulting in a crescent diffuse signal throughout the sample. To try to reduce the loss of PCR products from the cell, prolonged fixation (15 hours to days) with fixative cross-links is often used for PCR amplification. In this treatment it is usually necessary to treat the protease of the fixed cells before the amplification (G. Nuovo, et al., 1992 Diag Molec, Pathol, 1 (2), 98-102). It has been found that the conventional low temperature of SDA, performed in itself, has advantages over PCR in itself, which include: 1) better conservation of the cellular structure that allows the determination of the immunological phenotype for identification cellular, and, 2) much greater retention of the plicons within the cell. However, the effect of the increased temperature of the SDAt in si tu on these characteristics was uncertain. While the increase in temperature (usually around 15-20 ° C compared to conventional SDA) can provide the advantages of increased specificity and speed of the reaction, it can also significantly increase cell destruction, possibly to a level that would interfere with or prevent the determination of the precise immunological phenotype and cell identification. The marked increase in the reaction temperature can also increase the diffusion of the ampicons out of the cell where they can be taken by negative cells and produce a false positive signal. However, it was unexpectedly found that the cellular structure after the SDAt in si tu p-was substantially intact as demonstrated by the normal lateral and frontal light scattering properties in the flow cytometry test. In this way, the determination of the immunological phenotype is compatible with the temperatures and protocols of the SDAt in si tu. The applicants' hypothesis is that maintaining the cells at elevated temperatures can be less harmful than subjecting them to repeated cycles of heating and cooling as in PCR. It was also unexpectedly discovered that the spread of ampicons usually did not increase significantly, possibly also because the cells may suffer less damage when kept at a constant high temperature than when subjected to thermocycling. The following terms are defined herein as follows: An amplification primer or primer is a primer for the amplification of a target sequence by means of hybridization and extension of the primer. For the SDA, the 3 'end of the amplification primer is a white binding sequence that hybridizes at the 3' end of the target sequence. The amplification primer further comprises a recognition site for a 5 'restriction endonuclease for the target binding sequence, usually near its 5' end. The restriction endonuclease recognition site is a nucleotide sequence recognizing the restriction endonuclease which will nick a site of double-stranded recognition by the restriction endonuclease when the recognition site has been semi-modified, as described in Walker, et al. (1992a), supra. A semi-modified recognition site is a double-stranded recognition site for a restriction endonuclease in which a strand contains at least one nucieotide derivative that prevents cutting one of the strands of the duplex by the restriction endonuclease. "Muting" refers to this modified activity, in which the restriction endonuclease only cuts one strand of the duplex, in contrast to the splitting of two strands, conventional. Any semimodified restriction endonuclease recognition site that is suitable for notching by a restriction endonuclease is suitable for use in SDA. The amplification primers for SDA are called Si and S¿ in Walker et al. (1992b), supra. Deoxyribonucleoside triphosphates modified by an alpha-thio are abbreviated NTPaS "," dATPaS "," dCTPaS ", etc. A" buffer "or external primer is a primer that is annealed to a white sequence upstream of an amplification primer, Thus, the extension of the external primer displaces the downstream primer and its extension product, ie, a copy of the target sequence containing the restriction endonuclease recognition site in which the amplification primer contributed is displaced. Both, the buffering primers consist only of white binding sequences and are designed so that they are tuned upstream of the amplification primers and displace them when they are extended.The external primers were designated Bi and B2 by Walker et al. (1992b). , supra The extension of the external primers is a method for displacing the extension products of the amplification primers, but the This may also be adequate in certain cases. The terms "white" or "white sequence" refers to the sequences of the nucleic acid (DNA and / or RNA) to be amplified. These include the sequence of the original nucleic acid to be amplified and its complementary secondary strand as well as any strand of a copy of the original white sequence produced by the amplification of the target sequence. Amplification products, extension products or amplicons are oligo and polynucleotides that contain copies of the white sequence produced during the amplification of the target sequence.
COMPENDIUM OF THE INVENTION The amplification of the strand by displacement, in a thermophilic reaction (SDAt) has been adapted for the amplification of the nucleic acid target sequences in itself in cells in suspension, in slides or in tissues, with speed, sensitivity and specificity, which is superior to the SDA in if your conventional. The excellent morphology of the sample is preserved despite exposure to significantly higher temperatures than in the SDA in if your conventional, as demonstrated in the normal light scattering parameters in flow cytometry. The in si t u amplification by the SDAt also remains compatible with the immunochemical techniques despite the increased reaction temperature, so that both the amplification of the target sequences and the immunological staining can be performed on the same sample. This is contrary to PCR in si t u, in which repeated thermocycling can make the cellular antigens of interest imperceptible to immunochemical techniques. The inventive methods for the SDAt in si tu usually consist of a brief fixation of the cells or tissue, followed by the permeabilization and addition of the reagents necessary for the SDAt. When the target sequence is DNA the cells or tissues are heated for a very short time before amplification to denature the target sequence. Due to the thermostability of the enzyme involved, the heating can optionally be carried out in a mixture of reagents that includes the enzymes. As an alternative, the enzymes can be added after denaturation, with heating of the sample to the desired reaction temperature. Typically, the SDAt reaction is incubated at 50-65 ° C for one minute or up to two hours, although higher temperatures are possible if compatible with the selected enzymes. If preheating is not required to denature the target sequence, all components of the SDA reaction are only added directly to the fixed cells, sensitized at the desired reaction temperature to initiate amplification. After washing to remove the primers and unused enzymes, the products of the amplification are detected in you or after they are released from the cells.
DESCRIPTION OF THE DRAWINGS Figure 1 shows the results of flow cytometry for the amplification of a white HIV sequence and a white HLA-DQ exon 3 sequence by means of the SDAt in si tu reaction.
DETAILED DESCRIPTION OF THE INVENTION The inventive methods for nucleic acid amplification in si tu by the SDAt reaction are based on the discovery that the SDAt in si tu provides the significantly improved sensitivity, velocity and specificity of the protocols for the SDAt in vi tro (solution) without significant loss of structure and cell morphology. In general, a sample of cells (e.g., suspension cells or tissue sections) suspected of containing nucleic acid target sequences are fixed with a fixative that maintains the morphological integrity of the cell, but not a reticule or precipitate cellular proteins, so that the penetration of the primers and other reagents is largely prevented. Therefore, protease treatment is usually not required after fixation to obtain the penetration of the primers and reagents into the fixed cells. In the practice of the invention, crosslinking or fixatives for precipitation can be used. Examples of fixatives include paraformaldehyde, 4% glutaraldehyde, ethanol / acetic acid, Carnoy's fixative (acetic acid, ethanol, chloroform), 1% osmium tetraoxide, Bouin's fixative (Picric acid 1.21%, folic acid). aldehyde 11%, acetic acid 5.6%), Zenker's fixative (5.0% mercuric chloride, 2.5% potassium diclorate, 5.0% acetic acid), sodium sulfate 1.0%) and acetic acid / methanol fixatives. The use of the FACS® Lysing Solution allows lysis, fixation and permeabilization using a single reagent. The preferred fixative for use in the invention is 1-4% paraformaldehyde which is preferably used to treat cells or tissues from about one minute to one hour. It is usually used to permeabilize fixed cells before amplification, for example, using detergents such as NP40, TRITON or saponin. Under certain conditions, fixation may be optional. That is, SDAt can be performed in itself in non-fixed cells, especially when the RNA targets are to be selectively amplified and no preliminary denaturation step is required by heating (see, in the following sections). An important feature of the present invention is that the target DNA or RNA sequences or both can be amplified directly using the methods of the invention. To amplify only the RNA, reverse transcriptase can be added to the SDAt reaction when it is found in the reverse transcription PCR (rtPCR-GJ Nuovo, et al., 1992. Diag. Molac. Pa thol. 1.98-102, GJ Nuovo, et. al., 1991, Am. J. Pathol, 58,518-523, GJ Nuovo, et al., 1991 Am. J. Pa thol., 139, 1239-1244). However, it has been found that various DNA polymerases that are used in SDAt exhibit reverse transcriptase activity. These can polymerize DNA copies of a target sequence using RNA or DNA as a template, with the incorporation of dNTPaS and displacement from a notch. Therefore, the RNA target sequences can be reverse transcribed by the same polymerase that performs part of the DNA amplification of the SDAt reaction, without the need to add a separate reverse transcriptase. The RNA can be amplified in the cells (ie, without substantial amplification of the ADÑ targets) by eliminating the denaturation step by heating or by treating with the DNase before initiating the SDAt reaction. The double-stranded DNA in the cells then remains double-stranded and is not available as a template, while the primers can hybridize to available single-stranded RNA and be specific for the amplification of the target sequences of the RNA generating the cDNA. The cDNA in turn serves as a template for further amplification. The specific amplification of the RNA target sequences can also be carried out by treating the cells with RNase-free DNase to initiate SDA. When the fixation helps to maintain the integrity of the cells during heating, fixation may not be necessary when there is no preliminary denaturation step by heating. However, it may still be useful to percept the non-fixed cells or tissues.
The treatment of the cells or tissues with RNase before the denaturation by heating the double-stranded DNA degrades the target RNA of the potential RNA and allows the specific amplification of the corresponding DNA target sequences. NaOH can also be used(approximately 0.1M) to selectively degrade the RNA and denature the DNA for the specific amplification for the DNA. If the denaturation step is included by heating (without the RNase treatment) before priming the SDA primers, the DNA and RNA target sequences will be amplified. The reverse transcription of the RNA by means of the DNA polymerases used in SDAt is generally less efficient than DNA synthesis, but it has unexpectedly been found that in some cases it is more efficient than conventional reverse transcriptases. . However, in general, the RNA targets are present in the cell in larger quantities than the corresponding DNA blank, and the high efficiency of the amplification of the rapidly generated cDNAs solves and compensates for any reduced efficiency in the passage of the reverse transcription of the reaction. Amplification-of RNA and DNA targets are preferred for most diagnostic applications of the invention because it provides the greatest number of target amplifiable sequences per cell, and as a result, greater sensitivity and greater number of potentially positive cells per cell. sample. If the target is to be denatured by heating prior to amplification, fixed cells or tissues can be heated in the SDA reaction mixture (eg, dNTPs, KiP04, MgCl, BSA, DMSO, external primers, amplification primers and enzymes, if they are sufficiently thermostable). If the polymerase and the restriction endonuclease are not sufficiently thermostable at the denaturation temperature, it is possible to add them later when the sample has been cooled to the desired reaction temperature. If the blank is not denatured by heating, the SDA reaction mixture, in which the endonuclease and restriction polymerase (s) are included, can simply be added to the cell sample at the selected reaction temperature to initiate the amplification. As for conventional SDA, targets for amplification by means of SDAt can be prepared by fragmenting larger nucleic acids by restriction with an endonuclease which does not cut off the target sequence. However, both for the SDAt itself and for the conventional SDA in itself, it is generally preferred that the target nucleic acids have the restriction endonuclease recognition / cleavage sites selected to make the notch in the reaction. amplification to be generated as described in Walker et al. (1992, Nuc AcidsRes, supra) and in U.S. Patent No. 5,270,184. To avoid cross-contamination of an SDA reaction by the amplification products of another, dUTP can be incorporated into the SDA aplicons in place of dTTP without significant inhibition of the amplification reaction. Nucleic acids containing uracil can then be recognized and inactivated specifically by treatment with UDG (uracil DNA glycosylases). Therefore, if dUTP is incorporated in the SDA aplicons in a previous reaction, any of the subsequent SDA reactions can be treated with UDG before the amplification of the double-stranded targets, and any DNA containing dU of the reactions previously amplified will become non-amplifiable. The target DNA to be amplified in the subsequent reaction does not contain dU and will not be affected by the UDG treatment. Then, UDG can be inhibited by treatment with Ugi (inhibitor of uracil DNA glycosylase) before target amplification. Alternatively, the UDG can be inactivated by heating. In the SDAt the highest reaction temperature can be used (>50 ° C) to inactivate the UDG at the same time and amplify the target. SDA requires a polymerase lacking 5'-3 'exonuclease activity, initiates polymerization in a single-stranded nick in double-stranded nucleic acids, and moves the strand downstream of the notch while generating a new complementary strand using as a template the thread in which it was not notched. The polymerase shift activity is essential for the amplification reaction, since it makes the target available for the synthesis of additional copies and generates the single-stranded extension product for which a second amplification primer can hybridize in reactions of exponential amplification. Processive polymerases are preferred since they can increase the length of the target sequence that can be amplified. Previously little was known about the activities of thermophilic polymerases at temperatures that could be suitable for SDAt. In addition, the activities of thermophilic polymerases at temperatures compatible with the activity of thermophilic restriction endonucleases were not known. Therefore, screening assays were developed to identify candidate endonucleases and restriction polymers, if any. The polymerase screening system is an extension test that tests the ability of the polymerase to move a downstream strand starting in a single-stranded notch in a double-stranded template. It also tests the presence or absence of the exonuclease activity 5'- > 3'. The activity of 5'-3 'exonuclease, if present in a suitable thermophilic polymerase, can be inactivated using routine methods known in the art (WO 92/06200). One of the most common methods to selectively inactivate the exonuclease activity in a polymerase is the cloning of the gene for the polymerase, the identification of the part of the genetic sequence that encodes the protein domain responsible for the exonuclease activity and the inactivation by mutagenesis in vi tro. In an alternative mode, the exonuclease activity can be inactivated by treating the polymerase with protease to isolate the fragments having only the desired polymerization and displacement activities. Therefore, a thermophilic polymerase that is identified in the extension assay that is active at a suitable temperature initiates extension in a notch and incorporates the modified dNTPs but has 5'- exonuclease activity > 3 'may be suitable for SDAt by elimination of exonuclease activity.
In the extension test for the polymerases, the displacement of the individual strand of a double-stranded nucleic acid and the initiation in a notch is graduated by tuning two primers immediately adjacent to each other in an intact sequence, complementary to both primers. The primers are labeled at their 5 'ends usually with 3¿P. If a polymerase has strand displacement activity, it is capable of initiating polymerization in the "notch" formed by the adjacent hybrid primers and lacks 5'-3 'endonuclease activity, both primers are extended and two products of the extension. If the polymerase lacks the 5'-3 'exonuclease activity but can not initiate extension in the notch (e.g., requires a gap) and / or if it lacks displacement activity, only the product of the extension of the downstream primer. A polymerase that starts in a notch but has 5'- exonuclease activity > 3 'will only generate the product of the upstream primer extension. The extension test also requires that the polymerase be capable of incorporating an α-thio dNTP (dNTPaS) that is included in the reaction. The upstream and downstream primers and their respective extension products are usually identified by autoradiography by their size on the gel.
Of the eleven thermophilic DNA polymerases that were initially selected in the extension test, 6 were identified with all the characteristics necessary for their use in the invention: exo-Vent (New England Bíolabs), Exo-Deep Vent (New England Biolabs), Bst (BioRad), exo- Pfu (Stratagene), Bca (Panvera), and Sequencing Grade Taq (Promega). Others can be identified routinely using the aforementioned extension assay without the exercise of inventiveness and all these polymerases would be suitable for use in the SDAt. The polymerases Tth (Boehringer), Tfl (Epicenter), REPLINASE (DuPont) and REPLITHERM (Epicenter) displace the strand from a notch, but also have exonuclease activity 5'- > 3", These polymerases are useful in the methods of the invention after eliminating the exonuclease activity, for example, by genetic engineering.Most of the thermophilic polymerases that have been identified to date are active between about 50 ° C and 75, ° C, with optimal activity around 65-75 ° C and reduced activity around 50-65 ° C. However, as the thermostability of thermophilic restriction endonucleases is generally limited to less than 65 ° C, Thermophilic polymerases with optimal activity at lower temperatures (eg, Bst and Bca) are more compatible with thermophilic restriction endonucleases in the reaction and therefore are preferred, however, restriction endonucleases that are active at compatible higher temperatures with the optimum temperatures common for the activity of the polymerase can be identified and are also useful in the invention. Suitable restriction sites for the SDA must unfold only one strand of a double-stranded semi-modified recognition / cleavage site for the restriction endonuclease ("notch processing"). This notching activity is of great importance, since the "notch" which perpetuates the reaction and allows subsequent rounds of target amplification to begin. Because restriction enzymes generally produce double-strand breaks, the cleavage of one of the two strands at the duplex cleavage site must be selectively inhibited. This is usually carried out by introducing nucleotide analogs (eg, deoxynucleoside phosphorothioates) into a strand of the DNA during synthesis, so that either the modified strands or the unmodified strand are no longer susceptible to unfolding. In cases where the unmodified strand is protected from splitting, nucleotide analogs can be incorporated into the primer during its synthesis, thus eliminating the need to add nucleotide analogs to the amplification reaction and also eliminating the requirement that the polymerase be capable of incorporating these nucleotide analogs. Since substitutions of the nucleotide analogue do not induce nicking by all restriction endonucleases, a means was needed to test the nicking characteristics of the restriction endonucleases in order to identify suitable enzymes that could exist among the multiple endonucleases of thermophilic restriction available. Therefore, a selection system was devised to identify the thermophilic restriction endonucleases, with the desired properties, based on the capacity of a modified deoxynucleotide, incorporated in a strand of the recognition / unfolding site of the double-stranded restriction endonuclease. , to protect one of the two strands of the unfolding by the endonuclease. This is known as nicking test induced by the analog or thread protection assay. During the strand protection assay, a single-strand template containing the recognition / cleavage site of the restriction endonuclease and a primer complementary to a template portion different from the recognition / cleavage site is synthesized. Then, the template and the primer is usually labeled with a radiolabel. The primer and template are hybridized and the modified dNTPs are incorporated by primer extension producing a complete double-stranded molecule containing a semi-modified restriction endonuclease recognition / cleavage site. This product is treated with the restriction endonuclease under conditions suitable for double-strand cleavage. The electrophoretic analysis of the products of the reaction under denaturing conditions is used to determine, by means of the size of the fragments generated, whether or not the formation of the notch was carried out, the unfolding, or if there was no cut in the recognition / unfolding site. The size of the fragments in the electrophoresis is also used to determine which of the two strands of the recognition / unfolding site (modified or unmodified) was protected from splitting. The strand protection assay can be routinely adapted to select additional restriction endonucleases for use in the invention without exercising the skill of the inventive. Using the strand protection assay, we tested 28 thermophilic restriction endonucleases (Accl, Aspl, Bsal, BsaBI, BsiYI, Bsll, two degenerated sites of Bsml, BsmAI, BsmFI, BsmHI, BspWI, four degenerated BsoBI sites, BsoFI, two degenerate sites of Bsrl, BsrBRI, two degenerate sites of BsrDI, Bst71l, two degenerated sites of BstNI, BstOI, BstXI, Dpnl, HeII, MamII, Mval, Mwol, Sfil and Tthllll). Of the 28, 11 had at least one recognition / unfolding site of the restriction endonuclease in which the notch was made with the introduction of at least one dNTPa-thio. One of the recognition sites of the Bsll presented protection of the unmodified strand when the dCTPaS was incorporated. When thermostability was tested at 50-65 ° C, all but one of the eleven (Accl) were sufficiently stable or could be sufficiently stabilized with the addition of common stabilizers, such as double-stranded DNA or BSA. These endonucleases, therefore, were compatible with the thermophilic polymerases in the SDAt reaction. In addition, several thermophilic endonucleases having partial or low activity for nicking were identified under the initial screening conditions of the strand protection assay (e.g., Tthllll, BsiYI and BsoFI). Although the reduced activity for dying does not prevent the SDA, the notching of a restriction endonuclease can be optimized by adjusting the reaction conditions (for example, by optimizing the buffer or adjusting the reaction temperature), making them more efficient in SDAt. In addition, as it is well known that by blanking a recognition / cleavage site of the restriction endonuclease the degree of activity of the endonuclease can be affected, the alteration of the sequences of the flanking of the templates can also improve the nicking activity of the endonucleases. which notched only partially. The thermophilic SDA is performed essentially as the conventional SDA, with the substitution of the thermostable polymerase and the desired thermostable restriction endonuclease. Of course, the temperature of the reaction will be adjusted to the highest suitable temperature for the selected thermophilic enzymes, and the conventional restriction endonuclease recognition / unfolding site will be replaced by the appropriate restriction endonuclease recognition / cleavage site for the selected thermostable endonuclease. Also in contrast to conventional SDA, the practitioner can include the enzymes in the reaction mixture before the denaturation step by initial heating if they are sufficiently stable at that temperature. Preferred restriction endonucleases for use in SDAt are Bsrl, BstNI, BsmAI, Bsll and BsoBI (New Englan BioLabs), and BstOI (Promega). Preferred thermophilic polymerases are Bca and Bst.
To develop an optimal SDA system capable of higher amplification factors (eg, 108-109), evaluation and optimization of buffer systems is recommended. This is also the case when a new restriction enzyme / polymerase pairing is evaluated for use in thermophilic SDA. These optimization methods can be applied to determine a suitable buffer solution for any restriction endonuclease / polymerase combination for SDAt, in which only routine tests are required without the exercise of inventiveness. In most cases the buffer solution of KP04 / MgCl2, which is commonly used in conventional SDA, is suitable for SDAt, as described, or with some routine modification in the concentrations of the components. In the SDAt in si tu, the reagents for the amplification are applied to the non-fixed cells or to the cells that have been fixed and perceptible as described above. After initiation of the reaction, amplification of the target sequence is generally allowed to continue at about 50-65 ° C for about one minute to two hours, preferably about 10 minutes to one hour. It has been found that the time required for amplification in itself for SDAt or conventional SDA is significantly less than the time needed to obtain a comparable level of target amplification in si tu by PCR. In certain cases it may be advantageous to increase the concentration of the reagents (especially the primers) for the SDAt in itself in comparison with the SDAt in vi tro to ensure that sufficient amount is entered into the cells for efficient amplification. The leakage of the amplicons from the cells has been a problem in certain nucleic acid amplification methods in itself. It is considered that these leaks are the result of the complex interaction of a series of parameters, for example, the size of the amplicon, the temperature, the temperature cycles and the extent to which the cell has been permeabilized. To facilitate the retention of the amplicons within the cell, a deoxyribonucleoside triphosphate (dNTP) analog containing the dNTP conjugated to a moiety such as digoxigenin ("dig"), biotin or fluorescein isothiocyanate (FITC) can optionally be incorporated in the amplification products together with the dNTPaS. Likewise, the additional dNTP analog can optionally serve as a label or label that can be used to detect the products of the amplification. The incorporation of the dNTP analogues, such as the digit, also has the advantage of providing an improved signal since each half of the incorporated label can generate a signal through the binding to anti-dig antibodies conjugated to alkaline phosphatase. (AP-a-dig). The incorporation of these dNTP analogues is particularly advantageous for SDA in si tu because the amplified target sequence is generally smaller than the PCR amplicon. However, it has been observed that although the amplicons of the SDA that are usually smaller than the PCR amplicons, there is less leakage associated with the conventional SDA in si tu than with the PCR in si tu. Given the higher temperature of the SDAt in si tu, it was anticipated that amplicon leakage could increase, possibly to the level seen in the PCR in si tu. In practice, however, although there could be a slight increase in amplicon leakage at elevated temperatures, there was still significantly less amplicon leakage in the SDAt in si tu than in the PCR itself. After the target amplification, the produced amplicons can be detected using any of the methods known in the art for the detection of specific nucleic acid sequences. For example, the products of the amplification can be detected in or after the amplicons are released from the cells using specific hybridization to an oligonucleotide detector probe. The detector probe is a short oligonucleotide that includes a detectable label, that is, a half that generates or can be made to generate a detectable signal.by translating the notch, the labeled end or during the chemical synthesis of the probe. Many direct and indirectly detectable labels are known in the art for use with oligonucleotide probes. Directly detectable labels include those labels that do not require a greater reaction to make them detectable, for example, radioisotopes, fluorescent moieties and dyes. Fluorescent labels, such as fluorescein isothiosyanate (FITC), or radioisotopes such as 1: > P are preferred for use in marker probes for the direct detection of amplified white sequences in itself. Indirectly detectable labels include those labels that must be reacted with additional reagents to make them detectable, for example, enzymes capable of producing a colorful reaction product, biotin, avidin, digoxigenin, antigens, aphenes and / or fluorochromes. The signal enzymatic markings are usually developed by reacting the enzyme with its substrate and any additional reagents that are required to generate a colorful enzi ethical reaction product. Biotin (or avidin) tags can be detected by binding to labeled avidin antibodies (or labeled biotin) or labeled antibiotin (or labeled antiavidin). The digoxigenin and aphene tags are usually detected by specific binding to an anti-digoxigenin (anti-dig) or labeled anti-aphene antibody. Enzymes are preferred for use as indirectly detectable labels in the present invention. More preferred is alkaline phosphatase (AP) because it is suitable and has been widely used to label tissues and cells. The presence of AP can be detected by reaction with a substrate. The preferred substrates for the detection of AP are Vector Red / Vector Blue (Vector Labs. CA), 5-bromo-4-chloro-3-indolyl phosphate (BCIP) / tetrazolium blue nitro(NBT) (Sigma Chemical Company, St. Louis, MO) or Nuclear Fast Red (Sigma Chemical Company). The Vector Red substrate has the additional advantage of fluorescence, which allows the visualization of a positive signal either by means of the conventional light microscope or by fluorescence microscopy. Methods for developing the colorful AP reaction product with these substrates are well known in the art. To detect white amplified sequences by hybridization to a detector probe, the cells or tissues are exposed to the labeled probe under reaction conditions suitable for specific hybridization of the probe to the single-stranded amplification products. In general, the detector probe will be selected so as to hybridize to a nucleotide sequence in the amplicon, which is between the binding sites of the two amplification primers. However, a detector probe may also have the same nucleotide sequence as any of the amplification primers. Suitable methods for detection by in-situ hybridization for a detector probe are described in J. B. Lawrence, et al. (1989 Cell 57,493-502), J. B. Lawrence, et al. (1990. Proc. Nati, Acad. Sci. USA 87.5420-5424) and in U.S. Patent No. 4,888,278. Alternatively, the amplification products can be detected in-house, or after being released from the cells, by means of the extension of the primer as described in Walker, et al. (1992b), supra. In the primer extension method, an oligonucleotide primer containing a detectable label hybridizes to the products of the amplification and is extended by the addition of polymerase. For the detection of the primer, it can be labeled at the 5 'end, preferably using 32P or a fluorescent label. Alternatively, the extension of the hybrid primer may incorporate a dNTP analog containing a direct or indirectly detectable label. For example, the extension of the primer can incorporate a dNTP derived from the dig, which is subsequently detected after extension by means of the reaction with the AP-a-dig and a suitable AP substrate. The primer to be extended may be the same as an amplification primer or may be a different primer that hybridizes to a nucleotide sequence in the amplicon that lies between the binding sites of the amplification primers. The detectable label can also be incorporated directly into the amplicons during the amplification of the target sequence. For example, one of the dNTPs in the conventional SDA reaction can be completely or partially substituted with a dNTP analog containing a conjugated dNTP for a direct or indirectly detectable label. For example, the dUTP conjugated to the desired label can be replaced by dTTP in the SDA reaction. Then, the polymerase is incorporated into the label directly in the amplification products generated by the extension of the amplification primer. The brand can be detected directly or indirectly. Preferably, the label conjugated to dNTP should be a fluorescent label that can be detected directly in the amplicones by means of fluorescence mocroscopy or flow cytometry. In an alternative preferred embodiment, the label conjugated to the dNTP is biotin or digoxigenin, which can be detected by the reaction with streptavidin / FITC and fluorescent microscopy or flow cytometry. The secondary amplification products are copies of the white sequence generated by hybridization and the extension of a signal primer on the target sequence. The products of the amplification contain an internal segment of the amplified target sequence and a detectable label that is associated with the signal primer. At least the 3 'end of the signal primer contains a sequence that hybridizes to the target sequence. It may also include features that facilitate the capture or immobilization of secondary amplification products, so that they can be isolated for detection, quantification or other manipulation. The concurrent generation of the secondary amplification products in the SDAt reaction in itself provides another detection method which is homogeneous and can be performed concurrently with the amplification. The prolonged step of hybridization of the probe in itself and the concentrations of the signal primer are lower than the hybridization probes are eliminated. The low concentration reduces the bottom and also allows the superior rigorous washing which also reduces the bottom. To generate the secondary amplification products, at least one signal primer is included in the reaction mixture of the SDAt in si tu. The hybrid signal primer (s) (n) to the target sequence downstream of the hybridization site of an amplification primer and extended by the polymerase in a manner similar to the extension of the amplification primer. . The extension of the amplification primer displaces the extension product downstream of the signal primer of the target sequence. The opposite amplification primer can then hybridize to the extended and shifted signal primer and itself can be extended by means of the polymerase, resulting in the incorporation of the signal primer in a longer duplex indicating the target amplification. Since any of the non-extended signal primers are small, they can be washed from the cell while the extended signal primers are kept inside it. By this means, the specific signal of the target amplification is associated with the cells where the target is present and is substantially absent from the cells where there is no target. The mark of the hybrid detector probe, the extended primer, the amplicon or the secondary amplification product can then be detected, preferably in itself, as an indication of the presence of amplified target sequences. This may require the addition of reagents to the cells to develop the signal of an indirectly detectable label such as AT., biotin or dig. When the detectable label is an enzyme, microscopic analysis of the cells is preferred. The microscopic analysis can be by means of the visual observation of the cells or tissues (fluorescence or luminous microscopy), or the automated analysis of the image using instruments such as the DISCOVERY (Becton Dickinson Image Cytometry, Leiden, Holland) to evaluate the amount and signal strength of positive cells. When the label is a radioactive label, the cells can be suspended in the scintillation fluid and the signal can be detected by means of scintillation counting. The use of a directly detectable fluorescent label allows fluorescence analysis of the cells in suspension by flow cytometry (e.g.
FACSAN, Becton Dickinson Immunocytometry Systems, San José, C.A.). In a plot of the number of cells against the fluorescence intensity, a shift in maximum fluorescence to the right indicates an increase in the number of cells containing the target sequence. Conversely, a shift in maximum fluorescence to the left, in the graph, indicates a reduced number of cells that contain the target sequence. Alternatively, the products of the amplification can be released from the cells before detection as described above or can be visualized after gel electrophoresis as bands of the amplification products, for example, by staining with EtBr, the hybridization of a detector probe or the extension of the primer. When radiolabel is used for the primer or sensing probe, the amplification products can be visualized by autoradiography of the gels. Conventional methods for preparing cells for in-situ amplification and flow cytometry analysis involve the isolation of peripheral blood whole-blood mononuclear cells (PBMC, for example, by means of a FICOLL gradient centrifugation) before staining. the antibodies and / or the amplification. An additional step may be necessary to isolate T cells from PBMCs. These conventional protocols require approximately two days to obtain the flow cytometric results for a whole blood sample. It has now been discovered that a whole blood sample can be fixed and amplified in-situ with flow cytometric analysis in a single day using FACS® Lysing Solution (Becton Dickinson Immunocytometry Systems, San Jose, California) to prepare the sample. This Usante reagent contains diethylene glycol, heparin, buffer or buffer solution of citrate and formaldehyde, has a pH of 7.2. Due to the presence of formaldehyde, the FACS® Lysing Solution provides the advantage of reducing the biological risk of biological samples. In the new protocol for sample preparation, the sample is simply smoothed with FACS® Lysing Solution, then fixed, permeabilized and amplified in itself as described above. When cells in suspension or in tissues are to be analyzed by both SDAt in you and with immunostaining, it is preferable that the antibody be bound to the epitope or antigen of interest before lysis and binding, and that the antibody is conjugated to an indirectly detectable brand, such as biotin. The antibody-conjugate is then stabilized by fixation on the cells. Then, from the SDAt in si tu, the bound antibody is detected by reaction with the appropriate reagents that develop the signal, for example, streptavidin conjugated to a fluorochrome or antibiotin conjugated to a fluorochrome. The fluorochrome for the detection of the white amplification and the fluorochrome of the antibody can be detected independently by flow cytometry, allowing the practitioner to simultaneously determine the presence of the target in a cell and identify the type of cell in which the target is found. . In addition, the new sample preparation protocol can be reduced by including a signal primer labeled with fluorescence in the amplification reaction. As described above, the signal primer extends and becomes double-stranded during the amplification reaction in a specific form of the target amplification, eliminating the need for additional steps subsequent to amplification to detect the products of the amplification. The design of the amplification primers for the SDA usually requires the synthesis of multiple primers targeting the target region of interest, followed by the pairing of the primer pairs in the SDA reactions. to determine the efficiency of the amplification of each pair. This is because the parameters that affect the performance of the SDA primer are not well understood. We have found that apparently insignificant changes in the target binding regions of the amplification primers for SDA can have important and unpredictable effects on the efficiency of the amplification and that the melting point of the target binding region is not necessarily related to the efficiency of the activity of the primers. During the development of the SDAt in si tu tu, a pair of amplification primers that were specific for the HIV gag gene was designed. A target region of the gag gene that did not contain a recognition site for the restriction endonuclease of the SDA was selected. The white region was also selected on the basis of having two regions of approximately 50 bp each of which had approximately 100 bp separated and relatively constant. A software was then used for the design of the primer to determine the melting point of the target binding of each candidate primer and to evaluate the formation potential of the primer dimers. The primers were modified or discarded according to these preliminary results. Using the experimental tests, a final list of 8 candidate "left" amplification primers (directed towards the 3 'end of the target on the first strand) and 9 candidate "right" amplification primers (directed to the 3' end of the target) was compiled. the second strand). Buffer primers and detector probes were designed taking into account only the melting point and the proper placement with respect to the amplification primers. The efficiency of the amplification of the pair combinations of the "left" and "right" primers was determined experimentally in the SDAt in vi tro using a plasmid clone of the target gag, Bca polymerase and BsoBI. The amplicons were detected and quantified by hybridization and extension of a 32 P-labeled probe, followed by gel electrophoresis. For further development of the SDAt in if you selected the pair of amplification primers with the best efficiency of in vi tro amplification. In an optimized buffer system, as described above, in this series of primers less than 10 copies of the white gag sequence were detected in the SDAt in vi tro (the BsoBI sites are in italics, and the binding sequences of the underlined white): Left amplification primer (SEC ID NO: 1)ACCGCATCGAATGCATGTC CGGGTGGTAAAAGTAGTAGAAG TM 1 ° C Right-side amplification primer (SEQ ID NO:2) CGATTCCGCTCCAGACTTCTCGGGGTGTTTAGCATGGTGTT TH 55 ° C Shock primers AAATGGTACATCAGGCC TM 57 ° C (SEQ ID NO: 3) GCAGCTTCCTCATTGAT TM 58 ° C (SEQ ID NO: 4) Detector probe (SEQ ID NO: 5) GGTGGCTCCTTCTGATAATG TM 63 ° C A pair of amplification primers specific for exon 3 of the HLA-DQa gene was designed in a similar manner, using human placental DNA for the evaluation of the pair of candidate primers of amplification in vi tro. When HLA genes are present in all cells, this target is used as a positive control for SDAt in si tu. Because leakage of the amplicons was not found to be a major problem, slightly smaller white regions were selected for the initial identification of the candidate primers (about 75-100 bp). Initially, 3"left" amplification primers and 3"right" amplification primers were designed and experimentally tested in pairwise combinations. In an optimized buffer system, as described above, the next set of primers gave the best results of the amplification, detecting less than 5 copies of the target sequence of the HAL-TQa exon 3 in the SDAt in vi tro. Left amplification primer (SEQ ID NO: 6) ACCGCATCGAATGCATGTC CGGGTGGTCAACATCACATGGC TM 60 ° C Right amplification primer (SEQ ID NO: 7) CGATTCCGCTCCAGACTTCTCGGGTGAGAGGAAGCTGGTC TM 53 ° C Buffer primers GTCTTGTGGACAACATCTTTCC TM 6 ° C (SEQ ID NO: 8) TAACTGATCTTGAAGAAGGAATGATC TM 59 ° C (SEQ ID NO: 9) Detector probe (SEQ ID NO: 10) AATGGGCACTCAGTCACAGA TM 65 ° C The white binding sequence confers target specificity on the amplification primer. Thus, the target binding sequences of the amplification primers of the invention are also useful in nucleic acid amplification protocols other than SDA, eg, PCR and 3SR. Specifically, any amplification protocol that uses cyclic hybridization, specific to the primers for the target sequence, the extent of the primers that use the target sequence as a template and the displacement of the extension products from the target sequence can employ the White binding sequences of the amplification primers of the invention. For amplification methods that do not require any specialized target binding sequence, such as the restriction endonuclease recognition site of the amplification primers shown in the attached SEQUENCE LIST (eg, PCR), the amplification primers they can only contain white binding sequences. Amplification methods that require binding sequences at sites other than the target, specialized, other than those shown in the SEQUENCE LIST (eg, 3SR) may employ amplification primers that contain the target binding junctions of the primer primers. amplification enumerated and the sequence or structure that is required in the selected amplification method as is known in the art. In addition, a restriction endonuclease recognition site different and suitable for SDAt may also be substituted for the restriction endonuclease recognition site shown in the SEQUENCE LIST, using methods known in the art. The following experimental examples are provided to illustrate certain embodiments of the invention, but are not proposed as limiting the invention as defined in the appended claims.
EXAMPLE 1 The target HLA-DQa exon 3 was amplified and detected in si tu in cells with acute myelogenous leukemia (AML) (KG-la) using the series of selected primers described above. First the cells were fixed for 30 minutes in 4% paraformaldehyde and washed three times in phosphate buffered saline solution IX (PBS). They were then permeabilized with 0.01% saponin for 20 minutes and washed three times with PBS IX. Five microliters of cells that were permeabilized and fixed (10 cells / ml in KP, 35mM, pH 7.6) were added to 40μL of 35mM KPi; pH 7.6, 6.3 mM MgCl2, dGTP, TTP or dATP 50 μL each, 1.4 mM dCTPaS, 500 nM amplification primers, 50 nM buffering primers and 15% glycerol. After lightly mixing the samples were incubated at 95 ° C for 2 minutes and transferred to a THERMAL-LOK® temperature block which was maintained at the reaction temperature (52 ° C). 5μL of the cocimatic cocktail were then added (0.5μL 10NEB 2,0. 36μL of Bca, 22 units / ml, l.OμL of BsoBI of 160 units / ml and 3.14μL of water) and were mixed to initiate the amplification reaction. The final volume of the reaction was 50μL. After 30 minutes the reaction was stopped by placing an ice and the amplification products were detected. Radiolabeled detector probes (2X10b cpm) were added to the amplification reaction and hybridized for the amplification products in situ for 3 minutes at 95 ° C, followed by 60 minutes at 37 ° C. After washing twice for 25 minutes in 200μL of IX SSC, the cells were counted in scintillation fluid. The results of one of these experiments was as follows:In Table 1, the amplified KGla cells were probed with the HLA-DQa exon 3 specific detector probe. Table 2 was a negative control in which the restriction endonuclease was omitted in the amplification reaction, preventing SDAt. Tubes 3 and 4 corresponded to tubes 1 and 2 but were probed with a specific gag detector probe that is not related to the target sequence. All the samples that were amplified in the presence of all the necessary enzymes and were detected with the HLA-DQa probe exon 3 showed specific amplification of the target proposed in si tu. The experiment was repeated including the incubation of the non-amplified cells with amplicons generated in si tu. This was evaluated to transfer the amplicons to the negative cells either by adhering the amplicons to the cell surface or by collecting the amplicons by means of the negative cells. After amplification in itself, the reaction was centrifuged to pellet the cells. The supernatant was incubated for 15 minutes at 70 ° C to eliminate BsoBI activity and added to the permeabilized and fixed KGla cells which had previously been heated at 95 ° C for 2 minutes. The mixture was incubated at 52 ° C for 30 minutes and the cells were then hybridized, washed and counted as above as specific and non-specific (unrelated) detection probes. The results are shown below: IN SITUATION HYBRID SIGNAL TUBE (cpm) 1 2924 negative control, specific probe 2 16248 SDAt in si tu, specific probe 3 17548 polymerase 3X, specific probe 4 5582 cell incubation, specific probe 5 871 control negative, unrelated probe 6 311 SDAt in si tu, unrelated probe 7 501 3X polymerase, unrelated probe 8 453 cell incubation, unrelated probeThe restriction endonuclease was omitted in the reaction for the negative control in Tube 1. Tubes 2 and 3 represented the complete amplification reaction with specific detection. Tube 3 shows the signal resulting from SDAt in si tu under the same conditions of Tube 2 but with a triple increase in polymerase concentration. In Tube 4 the SDAt amplicons generated in-house were incubated with non-amplified cells / which were then hybridized, washed and detected as in the amplified samples. Tube 4 was included to determine how much positive signal, in Tube 2, is due to non-specific adhesion of the amplicons to the non-amplified cells. Tubes 5-8 correspond to the reaction conditions of Tubes 1-4, except that the non-related gag probe was hybridized as a negative control. As in the previous experiment, the amplification in if your specific target was clearly presented in those samples that contained all the necessary enzymes and were detected with the HLA-DQa probe exon 3. The generation of the false positive signal caused by a mechanism in that the amplicons that can be found in the supernatant are taken by the non-amplified cells, can occur to some extent, but the substantially high signal in Tubes 2 and 3 clearly shows that the SDA was carried out in itself. Most of the signal is specific to the target and is not due to the transfer of the amplicon. In an alternative detection system, the experiments were repeated using signal primers labeled with fluorescein. The signal primers were added to the amplification reaction at a concentration of 100 nM. By means of this, the fluorescent probe was extended by means of the polymerase and moved from the target by extension of the upstream amplification primer. After stopping the amplification reaction with ice, the samples were washed twice with 150-200 μL of phosphate buffered saline for 5 minutes each washed. When washing, the smaller and non-extended signal primers were removed from the cells, while the larger, fluorescent and specific signal primers of the target amplification were retained by those cells in which there was amplification. In these experiments, the washed cells were visualized by fluorescent microscopy where a strong fluorescent signal was observed in the positive cells and the negative cells were not fluorescent. However, the extension of the signal primer is also compatible with the detection and / or counting of the fluorescent cells by flow cytometry. In the third series of similar experiments, the amplification products were detected in a colorimetric assay. Cells were fixed in 4% paraformaldehyde for 20 minutes, washed 3 times in IX PBS and permeabilized in 0.01% saponin for 10 minutes. After washing in SDA buffer solution (35 mM KPO4 pH 7.5, 15% glycerol, 4mM MgOAc), 5 μL of cell suspension (5X105 cells) was added to 40 μL of SDA buffer with the primers and dNTPs as in FIG. last case. The blank was amplified using the series of primers HLA-DQa exon 3 as in the previous case, adding 5 μL of the enzyme mixture after the denaturation of the blank, to make a final volume of the reaction of 50 μL. The mouse cells served co or a negative cell line because, in spite of the homology between the human HLA and the mouse MHC, the mouse MHC target is not amplified in this amplification system, the base analysis Data from the genetic sequence revealed that the homology between the mouse and the human linker regions of the primers used in the present is poor, and was negatively confirmed by the fact that they failed to amplify the purified mouse DNA in PCR using these primers. After denaturing for 2 minutes at 9 ° C, the detector probes labeled with digoxigenin (1-lOnM) were hybridized for two hours at 33 ° C. Hybridization was followed by 3 washes subsequent to hybridization in IX SSC at room temperature and exchange of buffer solution with TRIS lOOmM pH 7.5 / 150mM NaCl. An anti-digoxigenin antibody (Fe fragment) conjugated to alkaline phosphatase (AP) was then incubated with the cells for 2-4 hours at room temperature. After washing with TRIS / NaCl to remove the unbound antibody to the negative cells and exchange the alkaline phosphatase buffer solution (TRIS 100mM pH 9.5 / 100mM NaCl / 50mM MgCl2) NBT / BCIP was used to develop the color in those cells in which there was amplification. They were deposited on slides and visualized by microscopy. Human cells gave a strong colorimetric signal when the HLA-DQa probe exon 3 hybridized in the amplified cells. Hybridization of the HLA-DQa detector probe exon 3 in non-amplified cells gave a negative or very pale colorimetric response but still clearly negative. Other negative controls included the absence of amplification (ie there was no addition of enzymes in the SDA), absence of detector probe and non-specific detector probe (ie, gag or chick-actin detector probe in amplified cells), all of which were also negative in the trial. In mouse cells alone, none of the HLA-DQa exon 3 or gag probes produced significant color, but, when mixed with human and mouse cells during the amplification reaction, the colorimetric signal in the mouse cells was increased. Nevertheless, the evidence suggests that this is a trick of the colorimetric detection system itself and that the negative cells are colored in a non-specific way.
EXAMPLE 2 Venous blood was collected in tubes to receive VACUTAINER® blood with EDTA (Becton Dickinson Vacutainer Systems).
To 50 μL of whole blood was added anti-CD4 antibodies < L120) conjugated with DNP and anti-CD3 (Leu4) treated with biotin and allowed to stain for 20 minutes at room temperature. To lyse the erythrocytes 1.0 ml of IX FACS® Lysing Solution (Becton Dickinson Immunocytometry Systems) was added to each tube for 10 minutes. The cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature and permeabilized by the addition of 0.5 ml of IX FACS® Lysing Solution and TWEEN-20 0.025%. The cells were washed twice with 35 mM KP04, resuspended in 5 μL of 35 mM KP04 and transferred to 0.5 ml microcentrifuge tubes. For the SDAt in if you added to the prepared blood sample 35 mM KP04, 1.4 mM dCTPaS, 200 μM each of dATP, dGTP and dTTP, Mg acetate 4 mM, buffer primer 0.05 μM and amplification primer each 0.5 μM. The samples were heated at 95 ° C for 3 minutes and then at 52 ° C at 55 ° C for 3 minutes to anneal the primers.
After priming the primers, 5 μL of an enzyme mixture (0.5 μL 10X NEB 2 buffer, 160 BsoBI units, 8 Bca polymerase units) was added to initiate the amplification reaction. The tubes were incubated at 55 ° C for 30 minutes. The amplification products were detected in-situ by the hybridization of the fluorescein-labeled detector probes. Hybridization was performed at 95 ° C for 5 minutes followed by 33 ° C at 37 ° C for 60 minutes in 25 μL of SDA reaction buffer containing 100 to 150 mg of specific or unrelated detector probe.
Alternatively, the amplification products were detected by incorporating a fluorescent-labeled signal primer during amplification. In this case, the buffer solution of the amplification reaction additionally included 100 nM of the fluorescein-labeled signal primer at the 5'-specific or non-related position. The initial heating step, the addition of the enzymatic mixture and the amplification were performed as described above. After in-situ hybridization of the detector probe or the termination of the SDAt in the presence of the signal primer, the cells were washed with IX SSC at room temperature for 30 minutes. The staining of the antibodies of the markers on the cell surface was developed with anti-DNP-phycoerythrin (PE) and streptavidin labeled with Cy5 / PE. Cells were washed once with IX PBS and resuspended in IX PBS for flow cytometric analysis. In the plotted points of the lateral disperser compared to the frontal disperser (SSC vs. FSC) the white blood cells showed not to have been affected by the experimental treatments. That is, the cells that had undergone SDAt in si tu of the white HLA-DQa exon 3 (using the series of primers described in the above) with the determination of the CD4 / CD3 immunophenotype showed populations of normal lymphocytes, granulocytes and monocytes. The population of lymphocytes also appeared normal in the fluorescent points plotted of CD4 vs. CD3. These experiments demonstrated that the FACS® Lysing Solution and the immunophenotype determination were both compatible with the SDAt in si tu. For the detection of HIV by SDAt in si tu, a cell line containing the HIV genome (H9 +) was mixed with normal whole blood and proceeded as described above and the gag white sequences were amplified using the series of selected primers described in the foregoing. Additional oligonucleotides that can be used as detector probes or signal primers were also designed for use as alternatives to the SEQ ID. NO: 5: AGCCACCCCACAAGATTT (SEC ID, NO: 11) GTAATACCCATGTTTTCAGCAT (SEQ ID NO: 12) AAATCTTGTGGGGTGGCT (SEQ ID NO: 13) ATGCTGAAAACATGGGTATTAC (SEQ ID NO: 14) On the plotted points of the SSC vs. FSC, the H9 + cells were clearly distinguishable as a population of lymphocytes, monosites and granulosites independently with superior frontal dispersion than any of the populations of white blood cells. The whitesHLA-DQa exon 3 gag of HIV were amplified by SDAt in si tu using the series of primers described in the above and were detected by hybridization of the detector probe or the incorporation of a signal primer. The graphs of the histogram of FL1 vs. Cell counts are shown in Figure 1. The HLA-DQa positive control exon 3 showed a substantial shift of maximum fluorescence to the right (approximately 100 channels) compared to the negative control reactions in which no enzymes were added or they used unrelated probes or signal primers for detection. The amplification reaction of HIV showed a similar shift in the fluorescence peak in the graphs of the histogram of FL1 compared to the cell count. These experiments confirmed that the amplification was presented in si t u and demonstrated that an amplified HIV target can be detected by full-blood flow cytometry used. The magnitude of the peak displacements for the detector probe and the detection methods of the signal primer were similar.
Alternatively, venous blood was collected in tubes to receive VACUTAINER® blood with EDTA and the PBMCs were isolated by centrifugation by a FICOLL-PAQUE®. Collected cells were washed with IX PBS, and monocytes and B cells were removed using Human T cell enrichment columns (R & D Systems). The enriched fraction of T cells was stained with anti-CD4 antibody conjugated with DNP and anti-CD3 treated with biotin for 2Q minutes at room temperature. After 20 minutes of fixation in 4% paraformaldehyde in PBS, the cells were washed with PBS and counted. The cells were permeabilized with 10 μg / mL of saponin and washed twice with 35 mM KP04. 5 X 105 cells were resuspended in 5 μL of KP04 buffer and transferred to 0.5 mL microcentrifuge tubes. The SDAt in si tu, the detection of the products of the amplification and the determination of the immunophenotype were performed as described above for the sample preparation method of FACS® Lysing Solution. The experimental results were almost identical for the two methods of sample preparation.
SEQUENCE LIST (1) GENERAL INFORMATION: (i) APPLICANT: Lohman, Kenton L. Ostreroba, Natalie V. Van Cleve, Mark Reid, Robert A. (ii) TITLE OF THE INVENTION: DETECTION OF NUCLEIC ACIDS IN CELLS THROUGH AMPLIFICATION OF THE HEBRA BY DISPLACEMENT IN A THERMOFILIQUE RECORD (iii) NUMBER OF SEQUENCES: 14 (iv) CORRESPONDENCE DIRECTION: (A) CONSIGNEE: Richard j. Rodrick, Becton Dickinson and Company (B) STREET: 1 Becton Dríve (C) STATE: NJ (E) COUNTRY: EU (F) CP: 07417 (v) LEGIBLE FORM OF COMPUTATION: (A) TYPE OF MEANS: Flexible Disk ( B) COMPUTER: PC compatible with IBM (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentIN version # 1.0, version 1.25 (vi) DATE OF CURRENT APPLICATION (A) NUMBER OF REQUEST: ( B) DATE OF PRESENTATION: (C) CLASSIFICATION (viii) ATTORNEY / AGENT INFORMATION i (A) NAME: Fugit, Donna R. (B) REGISTRATION NUMBER: 32,135 (C) REFERENCE NUMBER / FILE: P-3 62 (2) INFORMATION OF SEC. DO NOT. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( ix) CHARACTERISTICS: (A) NAME / KEY: link mise (B) LOCATION: 25.-42 (D) OTHER INFORMATION: / name of the standard «=" WHITE UNION SEQUENCE ", (ix) FEATURE: (A) NAME / KEY: link mise (B) LOCATION: 19..24 (D) OTHER INFORMATION: / name of the standard = "SITE OF RECOGNITION OF ENDONUCLEASE RESTRICTION" (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 1: ACCGCATCGA ATGCATGTCT CGGGTGGTAA AAGTAGTAGA AG (2) INFORMATION SEC. DO NOT. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( ix) FEATURE: (A) NAME / KEY: link mise (B) LOCATION: 25..41 (D) OTHER INFORMATION: / name of the standard = "WHITE UNION SEQUENCE", (ix) FEATURE: (A) NAME / KEY: characteristic mise (B) LOCATION: 19..24 (D) OTHER INFORMATION: / name of the standard = "SITE OF RECOGNITION OF ENDONUCLEASE RESTRICTION" (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 2: CGATTCCGCT CCAGACTTCT CGGGGTGTTT AGCATGGTGT T (2) INFORMATION OF SEC. DO NOT. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( Xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 3: AAATGGTACA TCAGGCC (2) INFORMATION SEC. DO NOT. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 4: GCAGCTTCCT CATTGAT (2) INFORMATION OF SEC. DO NOT. 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 5: GGTGGCTCCT TCTGATAATG (2) INFORMATION OF SEC. DO NOT. 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 42 base pairs (B) TYPE: nucleic acid (C) HEBRAS: individual (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( ix) CHARACTERISTICS: (A) NAME / KEY: link mise (B) LOCATION: 25..42 (D) OTHER INFORMATION: / name of the standard = "WHITE LINK SEQUENCE", (ix) FEATURE: (A) NAME / KEY: characteristic mise (B) LOCATION: 19..24 (D) OTHER INFORMATION: / name of the standard = "SITE OF RECOGNITION OF ENDONUCLEASE RESTRICTION" (Xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 6: ACCGCATCGA ATGCATGTCT CGGGTGGTCA ACATACATG GC (2) ID SEC. DO NOT. 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( ix) FEATURE: (A) NAME / KEY: link mise (B) LOCATION: 25..40 (D) OTHER INFORMATION: / name of the standard * "WHITE LINK SEQUENCE", (ix) FEATURE: (A) NAME / KEY: characteristic mise (B) LOCATION: 19..24 (D) OTHER INFORMATION: / name of the standard = "SITE OF RECOGNITION OF ENDONUCLEASE RESTRICTION" (xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 7: CGATTCCGCT CCAGACTTCT CGGGTGAGAG GAAGCTGGTC (2) INFORMATION SEC. DO NOT. 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 8: GTCTTGTGGA CAACATCTTT CC (2) ID SEC. DO NOT. 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 9: TAACTGATCT TGAAGAAGGA ATGATC (2) INFORMATION SEC. DO NOT. 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( Xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 10: AATGGGCACT CAGTCACAGA (2) INFORMATION OF SEC. DO NOT. 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. eleven; AGCCACCCCA CAAGATTT (2) ID SEC. DO NOT. 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( Xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 12: GTAATACCCA TGTTTTCAGC AT (2) INFORMATION OF SEC. DO NOT. 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 13: AAATCTTGTG GGGTGGCT (2) INFORMATION SEC. DO NOT. 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( xi) DESCRIPTION OF THE SEQUENCE: SEC ID NO. 14 ATGCTGAAAA CATGGGTATT AC