JP2003500013A - Cleavage of nucleic acids from solid supports - Google Patents

Abstract

(57) Abstract: The present invention relates to a method comprising the step of selectively cleaving a nucleic acid molecule at a specific nucleotide site at a specific site in the nucleic acid molecule, the method comprising: A method is provided for separating the nucleic acid molecule from a solid support to which the molecule is attached. The nucleic acid molecule may be a chimeric molecule comprising the nucleic acid (nucleotide sequence) and other molecular components having different chemical properties. According to the present invention, there is provided a chimeric molecule (construct) comprising a nucleotide linker sequence having a specific nucleotide capable of selective cleavage, which is bound to a functional group which may be an affinity binding group or a reporter group at a predetermined position. You. The invention is useful in a number of applications, including molecular biological manipulations such as nucleic acid synthesis, amplification, sequencing, nucleic acid separation, affinity-based separation and assay manipulation.

Description

Detailed Description of the Invention

The present invention relates to the cleavage of nucleic acids and molecules or constructs containing nucleic acids from a solid support.

[0002] Many current methods commonly used in biochemistry / biotechnology and their related fields today bind biological or chemical entities (eg biomolecules) to a solid support (ie It is often desirable to immobilize) and then detach from the support. Such reversible immobilization can be used in many techniques known in the art for separation, purification, and isolation procedures, specifically cell isolation (eg, immunomagnetic separation), affinity chromatography, and the like. It finds significant utility. Immobilization, especially immobilization of oligonucleotides and nucleic acids, is frequently used in many molecular biological procedures. Furthermore, not only nucleic acid purification, but also many commonly used techniques such as sequencing, in vitro amplification, cDNA preparation, template preparation, DNA related assays, mutagenesis, etc. are also suitable for solid phase use. Adaptation is being made. This will facilitate handling, increase sample processing efficiency, and enable automation and the like. The ability to operate in the solid phase is often regarded as beneficial.

However, immobilization will introduce its own problems. For this reason, many immobilization systems used today rely on binding between a pair of mating partners to achieve the desired binding of the target to the support. For example, this may be based on the binding of antigen-antibody pairs, specifically the cell surface antigen, or the binding of the immobilized antibody to the protein or other molecule desired to be immobilized. In molecular biology, the binding of nucleic acids or oligonucleotides is usually done by biotin-streptavidin (or avidin) ligation. This is due to the immobilized protein (streptavidin or avidin) binding to the biotinylated molecule (eg, biotinylated oligonucleotide).

Reversing (cutting) such a connection is difficult and one may face the problem of breaking the joined parts. Thus, for example, cleavage of the linkage may require harsh conditions, specifically high pH or salt conditions, and / or high temperatures, which must be detrimental to the attached moieties. Other ligations / couplings that could be used instead of affinity binding, ie covalent ligations, would be difficult to cleave (ie immobilization covalently bound to a solid support) Nucleic acid binds to the modified oligonucleotide capture probe and is not easily cleaved from its support).

Another disadvantage of commonly used prior art immobilization systems is that due to the separation of the immobilized moieties, often part of the "immobilizing linkage" is the problematic, specifically Is to remain attached to the binding ligand or part thereof, eg biotin or part of the arm of the linker. This hinders subsequent manipulations or processing steps downstream of the immobilization moiety, specifically nucleic acid cloning or other processing, or cell viability studies, protein conformation studies, further purification, etc. Will.

Accordingly, there is a continuing need in the art for improved methods of immobilizing and then detaching biological or chemical objects. The present invention attempts to address this need. Although methods for immobilization have been developed in this way, the method is based on the inclusion of a nucleic acid sequence having a site to be selectively cleaved in the immobilizing portion. This site is provided by a nucleotide not normally present in the nucleotide sequence and is subject to selective cleavage using, for example, an enzyme that is specific for the presence of the nucleotide. Furthermore, the cleavage of the immobilized part can be easily achieved by selectively cleaving the nucleotide sequence at its specific site. Therefore, by such a novel method, it becomes possible to efficiently separate a certain nucleic acid or a molecule containing a certain nucleic acid from a solid support in a manner that envisions a precise site.

Since such resolution depends on the presence of nucleotides, the novel method of the present invention has particular application to the immobilization of nucleic acids (ie any nucleotide sequence) and to the manipulation and preparation of nucleic acid / nucleotide sequences. It is suitable. However, the method is applicable to any object or molecule containing a nucleotide sequence that incorporates a cleavable site, such as a conjugate of a nucleic acid with a moiety other than a nucleic acid, such as a protein or other organic molecule. .

The invention therefore provides, in one aspect, a method of separating a nucleic acid molecule from a solid support bound to the nucleic acid molecule. The method is characterized in that a specific nucleotide is incorporated at a predetermined site in the nucleic acid molecule, and the nucleic acid molecule is selectively cleaved at the specific nucleotide site. The nucleic acid molecule may be of any nucleotide sequence (ribonucleotide or deoxyribonucleotide sequence, ie DNA or RNA or any modification thereof), or contains or incorporates a nucleotide sequence. It can be any molecule that is present. For this reason, not only molecules consisting only of certain nucleic acid (s) but also nucleic acid components and other components, such as proteins or peptides or other organic molecules, labels, ligands (one of a pair of affinity binding partners), enzyme substrates, etc. Chimeric molecules consisting of other molecular components such as, i.e., any other molecular or biological or chemical entity are also included.

Accordingly, the term “nucleic acid molecule” refers to one nucleic acid (nucleotide sequence) as well as another molecular component (eg, a protein, having different chemical properties to a nucleic acid
Lipids, carbohydrates, small organic molecules, inorganic molecules, radioactive markers, etc.) are also included. The term "specific nucleotide" means a nucleotide not normally found in a nucleic acid molecule that it incorporates. That is, for a given nucleotide sequence (ie, nucleic acid molecule), the nucleotide is not native to that sequence. Thus, for example, for DNA, uracil bases (hereinafter "
U ″) can be referred to as a specific nucleotide. U is R
This is because it is not usually found in DNA even in NA. Similarly for DNA, ribose-containing nucleotides would be specific nucleotides (in contrast, deoxyribonucleotides would be specific if they were RNA). Other specific nucleotides include modified nucleotides not normally found in nature, and specifically include chemically derivatized nucleotides. Thus, with respect to the DNA nucleotide sequence, the specific nucleotide means any nucleotide other than A, T, C or G (however, a modified product of A, T, C or G is included in the specific nucleotide), RNA For, the specific nucleotides are A, U, C
Alternatively, it may be any nucleotide other than G (but similarly including modifications thereof).

Specific nucleotides thus include those with “unique” bases, which are the usual bases (A, T, C or G for DNA; A, U for RNA).
, C or G), derivatives, or analogs. In addition to “non-native” (ie, intentionally modified) nucleotides, the native form of the nucleotides is also included. For example, uracil and hypoxanthine are bases in nature, but for the purposes of the present invention, their corresponding nucleotides are specific nucleotides if incorporated into DNA.

“Non-native” modified nucleotides also include alkylated nucleotides and nucleotides modified by alkyl hydroxylation or other chemical modification or derivatization. Typical examples are N-7-methylguanine, 8-oxoguanine, deoxyuridine, deoxyinosine, deoxy.
5 ', 6'-dihydroxy-thymine (from O ₅ O ₄ treated DNA), 5', 6'-dihydroxy-thymine, deoxy 3'-methyl adenosine, and 3'-methyl adenosine and the like. Other specific nucleotides also include other bases that are observed to occur in damaged DNA, such as ring-opened pyrimidine-derived derivatives and other oxidation products (see, for example; Sancar and Sancar, (1988) Annu. Rev. Biochem. 57,
29-67).

The specific nucleotide incorporated into the nucleic acid molecule provides a selective cleavage site for the incorporated nucleic acid molecule. Specific nucleotides are therefore nucleotides that can be incorporated into a nucleotide sequence. Specifically, it is capable of participating in a polymerase reaction and specifically forming a pair with an original (ordinarily found) nucleotide, and further capable of selective cleavage.

The terms “selective cleavage” or “selectively cleavable” mean that the nucleic acid molecule is specifically cleaved at the site of the specific nucleotide, rather than at another site. Thus, cleavage may be "directed," in other words, controlled cleavage to occur at selective sites. In this way, the selective sites may optionally incorporate one or more, and will incorporate one or more specific nucleotides.

It will be appreciated, however, that some "tolerance" is observed in the system as the absolute specificity of cleavage cannot always be guaranteed in any biological system. Thus, it is acceptable that a few or even few cases of non-specific cleavage occur at sites other than the specific nucleotide site. The requirement is that the cleavage occur essentially only at the site of the specific nucleotide.

For convenience, such selective cleavage may be carried out enzymatically by using an enzyme which is specific for the particular specific nucleotide in question (in this sense the term "specific" refers to an enzyme And its corresponding substrate, conversely, represents the relationship with the substrate recognized by the corresponding enzyme). What is required is that the enzyme recognizes and cleaves the specific nucleotide in question in a manner that distinguishes it from other nucleotides (ie, the normal nucleotides present in a nucleic acid molecule).

Such a specific enzyme may typically be a DNA glycosylase having specificity for a particular base or a plurality of particular bases. Such a glycosylase enzyme exists as an enzyme that recognizes and acts on only one of the bases or only a specific group. It is believed that the enzyme is involved in the DNA repair process that cleaves bases, and the enzyme separates normal and improper bases (ie improper base pairing) or defective (damaged) bases. Can be recognized and distinguished.
DNA glycosylases act by cleaving the specific bases recognized from the deoxyribose-phosphate backbone of DNA by cleaving the N-glycosidic bond. The non-basic (non-purine / non-pyrimidine) sites generated by this reaction are labile and can be easily cleaved primarily at high pH or high temperature. However, various cleavage methods for non-basic sites are available, including methods using chemical reagents or enzymes that cleave the phosphodiester bond of the nucleotide sequence. These are discussed further below. This provides the mechanism of selective cleavage according to the present invention.

Many such enzymes have been studied and described in the literature (Cleaver and Layh
er, (1995) Cell, 80, 825-827; Sancar and Sancer, supra). Any such enzyme can be used in the present invention, along with the corresponding substrate, such as a specific nucleotide. However, the invention is not limited to the use of enzymes to effect selective cleavage, but may utilize any suitable mechanism or system for cleavage.
Thus, for example, ribonucleotides incorporated into DNA nucleotide sequences are sensitive to conditions such as high pH, ^ions such as Mg ²⁺ , Mn ²⁺ , and may simply provide sites for selective cleavage by such treatment. . Sequences consisting exclusively of deoxyribonucleotides (eg dT, C, G, A, U) are unaffected by such treatment. The presence of ribose instead of deoxyribose thus provides a way to selectively cleave at one position. Example 3 below
The use of such a cleavage system in the method of cDNA synthesis is described.

In a preferred embodiment of the invention, the nucleic acid molecule is a DNA molecule, the specific nucleotide is U, and the selective cleavage is the uracil DNA glycosylase (UDG) enzyme (also known as uracil N-glycosylase or UNG). By using. It may be an intact enzyme or a modified enzyme and is known in the art for various UDG enzymes, including both native and modified enzymes, and described in the literature. (Eg Cleaver and L
ayher, supra and US-A-5,035,996; US-A-5,536,649, Longo et al., Gene (1990), 93.
, 125-128. It describes the use of UDG in controlling "carry-over" contamination in PCR and provides a source for UDG enzymes. WO 92/01814 also describes PCR "carry-over" control and specific nucleotides using other glycosylase enzymes, not just UDG.
Further, a range of different UDG enzymes and their sources that can be used in the present invention are also described). Thermostable morphology (Koulis, A. et al., FEMS Microbiology L
etters (1996), 143,267-271; Kaboev, OK et al., Letters (1981) 132 (2), 337-34.
0) and thermolabile morphology (HK ^™ UNG, Epicenter Technology, Inc.). UDG is available from commercial suppliers such as Amersham Life Sciences (UK), Boehringer Mannheim (Germany) and Epicenter Technology (USA).

Glycosylase enzymes having the desired properties may be isolated from natural sources. For example, an enzyme having a good activity even at a low temperature, specifically at 4 ° C., may be selected from organisms in a cold environment, for example, arctic sea shrimp. The glycosylase gene was identified and cloned (J. Biol. Chem., 1984, (25
9): 13723-13729 [E. coli], J. Biol. Chem., 1989, 264: 9911-9914 [human]). UDG
Alternatively, other glycosylases are available and available. Among them are: hypoxanthine-DNA glycosylase, 3-methyladenine-D
NA glycosylase I, 3-methyladenine-DNA glycosylase II, hydroxymethyluracil-DNA glycosylase, and formamide-pyrimidine DN
A glycosylase. A description of glycosylases and their corresponding substrates is given in the table below.

[0020]

[Table 1]

The specific nucleotide is incorporated and cleaved at a predetermined position. This simply means that the site of incorporation (ie the position of cleavage) can be preselected (or controlled or designated) so that cleavage can occur at the exact site of the desired location of the nucleic acid molecule. It is meant. The specific nucleotide may be incorporated into a limited position of the nucleic acid molecule.

As mentioned above, the method of the present invention has utility in any method of immobilization for nucleic acid molecules. That is, the present invention provides a method of reversibly immobilizing a nucleic acid molecule from another aspect, the method comprising: (a) a step of incorporating a specific nucleotide into the nucleic acid molecule at a predetermined site (b) Binding the nucleic acid molecule to a solid support, followed by (c) selectively cleaving the nucleic acid molecule at the specific nucleotide site.

Step (c) of selective cleavage will separate the nucleic acid molecule from the support. It will be appreciated that steps (a) and (b) may be performed sequentially or simultaneously. Thus, for example, the nucleic acid molecule may be bound to a solid support before incorporating the specific nucleotide into the nucleic acid molecule. That is, by linking nucleotides or nucleotide sequences containing the specific nucleotide.

Alternatively, as described in detail below, the specific nucleotides may be incorporated into the nucleic acid molecule prior to attachment to the solid support. This is done, for example, by solid phase synthetic methods, by enzymatic or chemical coupling or other binding reactions (specifically binding by affinity). As will be described in detail later, there is also a method of causing “uptake” and “binding” at the same time so as to form a nucleic acid molecule by primer extension, for example, by using an immobilized primer sequence containing a specific nucleotide. .

For convenience, specific nucleotides may be introduced in the methods of the invention as part of a polynucleotide or oligonucleotide sequence to be introduced or incorporated into a nucleic acid molecule. This nucleotide sequence is viewed as a "mooring line" or "linker sequence" consisting of nucleotides, and it incorporates a specific nucleotide at one or more positions which may or may not be adjacent. This "linker sequence" may specifically be 4-100 nucleotides in length, eg 5-50, conveniently 10-30.
Or 12 to 21. Furthermore, it may consist entirely of specific nucleotides, or it may contain one or more specific nucleotides at different locations depending on the selection.

Techniques for synthesizing polynucleotide and oligonucleotide sequences for use in this regard are well known in the art and described in the literature, and any known method can be used. The specific nucleotides can be incorporated into the linker in any convenient manner, for example by solid phase synthetic methods with the appropriate nucleotides or enzymatically, for example by incorporating the approximate nucleotide provided using a polymerase enzyme. Alternatively, specific nucleotides may be incorporated into the linker using specific enzymes. For example, a transferase enzyme such as a terminal deoxynucleotidyl transferase that can add a mononucleotide such as UMP from the corresponding NTP (in this case UTP) precursor.

Similarly, linker sequences may be introduced into the nucleic acid molecule in any convenient or desired manner known or described in the literature. It may for example be in the form of a primer for a chain extension reaction which is ligated or template directed and polymerase catalyzed, whereby the primer (ie linker) will be incorporated into the product of the chain extension reaction. In other words, the nucleic acid molecule of the present invention is the product of a primer extension reaction. This will be discussed further below.

Methods for coupling nucleic acids to other molecules are also known in the art and will be described in detail below. The solid support may be any known support or matrix currently widely used or proposed for immobilization, separation and the like. These take the form of particles, sheets, gels, filter papers, membranes or microtiter strips, tubes or plates, fibers or capillaries, preferably, for example, agarose, cellulose, alginates, Teflon®, latex. , Or may be made of a polymeric material such as polystyrene. Biochips may be used as a solid support to provide a system for small scale experiments, eg, as described by Nilsson et al. (Anal. Biochem. (1995), 224, 400-408). Particulate materials, especially beads, are generally preferred, especially polymeric beads, a wide variety of which are known in the art. Magnetizable ("magnetic") beads are preferred to aid handling and separation. Preferably such magnetic particles should be superparamagnetic to prevent remanence and thus agglomeration, advantageously monodisperse to provide uniform mobility and separation. EP by Sintef for manufacturing superparamagnetic monodisperse particles
-A-106873.

Superparamagnetic beads of monodisperse polymer are Dynal as “Dyna beads (trade name)”
It is commercially available from AS (Oslo, Norway) and is particularly suitable for use in the present invention. The nucleic acid molecule may be attached or attached to the solid support by any convenient means. For example, any chemical or physical means may be used as long as the specific nucleotide is easily cleaved. This can be done, for example, by capacitive coupling
Specific examples include hydrogen bonding, capture, or more conveniently by covalent bonding. For example by covalent attachment of a group on the solid support to a "linker" or a functional linking group at the 5'end of the nucleic acid molecule. The nucleic acid molecule may also be directly or indirectly bound to another molecule which is also bound to the solid, insoluble support by any means described above.

Methods have been described in the literature for attaching nucleic acids to solid supports, and also for modifying and adapting the surface of solid supports to attach or carry nucleotide sequences. For a general review, see Greg T. Hermanson, "Bioconjugate Technology," Academic Press, Inc. 1996 ISBN 0-12-342335.
, Chapter 17, Nucleic Acid and Oligonucleotide Modification and Binding, and Mohammed As
"Bioconjugation-Protein Coupling Technology for Biomedical Sciences" by Lam and Alastair Dent, Macmillan Reference L
td., 1998 ISBN 0-333-583752, Chapter 7, Preparation of protein-nucleic acid conjugates).

Binding of oligonucleotides to magnetic beads is described, for example, in EP-A-0640626.
It is described in. In addition to direct covalent coupling, nucleic acids may be indirectly bound to the solid support by affinity binding pairs. That is, one of the binding partners is on the solid support and the other is on the nucleic acid molecule to be immobilized. The use of the biotin-streptavidin (avidin) system is well known in molecular biology as a means for immobilizing nucleic acids, biotinylated nucleotides are readily available, and so are solid supports carrying streptavidin. is there. For example, streptavidin-coated magnetic beads are available from Dynal AS (Oslo, Norway), streptavidin-coated tubes and microwell plates from Roche Molecular Biochemicals.

Instead of biotin / streptavidin, different affinity binding partners may be used, for example antigens / haptens and antibodies, enzymes and substrates.
Again, there are a series of commercially available products, such as Roche
Magnetic particles carrying anti-digoxigenin antibody, anti-fluorescein antibody, and anti-biotin antibody are available from Molecule Biochemicals. The nucleic acid molecule may also incorporate a nucleotide sequence (target site) recognized by a specific DNA binding probe carried on a support. For example, the lac repressor that binds to the lac operon
It's Lac I.

Incorporation of specific nucleotides as primers is a preferred aspect of the invention. As mentioned above, this provides a ready-to-use approach for preparing specific nucleic acid molecules that incorporate specific nucleotides. That is, a nucleic acid molecule is formed by extending a primer in a chain extension reaction, and a specific nucleotide is provided by being incorporated into the primer. This only requires inclusion of a specific nucleotide (NTP) in the primer sequence, ie replacing the normal nucleotide with a specific nucleotide at one or more defined positions in the synthetic DNA primer. Such manipulations form the basis of many uses of the present invention.

The primer extension reaction may be either DNA or RNA synthesis, or any such reaction known in the art, eg, a simple linear extension reaction. Therefore, not only DNA synthesis from a DNA template but also DNA synthesis (reverse transcription) from an RNA template, RNA synthesis from a DNA template, etc. are covered.
In vitro amplification reaction based on the principle of primer extension, whether it is exponential or not, for example, PCR and its modification are also included. Primer extension reactions form the basis of many molecular biology procedures in today's usage. Thus, the nucleic acid molecule may be a PCR product, a sequencing product, a cDNA synthesis product, a template production product, or the like.

For convenience, linker sequences (eg, primers) may be used that provide a means for immobilization on a solid support. Such means may be linked to the solid support and bound to the corresponding binding partner, for example an affinity binding partner such as biotin, or a carboxy group on the solid support. Or CN
It may be a functional group capable of interacting with a Br-activated hydroxy group and capable of undergoing a chemical ligation reaction such as an amino group or a terminal nucleoside.

Although biotinylated polynucleotides or oligonucleotides incorporating specific nucleotides form a particularly preferred aspect of the invention, this aspect of the invention provides specific means provided with some means for immobilization. Also included are poly- or oligonucleotides incorporating various nucleotides. In the PCR (or similar) aspect of the invention, the PCR reaction may be performed before or after immobilizing the primers on the solid support. That is, PCR primers may be used already attached to the solid support, rather than merely providing a means for immobilization.

Therefore, immobilized poly- or oligonucleotides incorporating specific nucleotides are also within the scope of the invention. Such immobilized poly- or oligonucleotides are preferably attached to the magnetic beads, preferably via a terminus conjugation, and preferably at the 5'end. The method of the examples will describe a typical PCR reaction that takes advantage of the present invention.

Biotinylated synthetic oligonucleotides with dUMP in place of dTMP at one or more positions (eg biotin-GTAATACGACTCACTA U AGGGC) can be used for PCR and also in the same way as dTMP containing oligonucleotides. And can be incorporated into dsDNA products. After PCR and binding to the solid support, the internal dUMP site is the UDG
Be the target of activity. In addition, abasic sites (sites that are no longer basic) can be cleaved by high temperature or high pH, releasing the PCR product from the solid support. Instead, the non-biotinylated complementary strand is first released by heat denaturation, leaving the biotinylated strand immobilized on the solid support. Then, the single-stranded DNA is
It may be cleaved with DG, released, and both strands collected separately and purified. This will be useful if the sense and antisense strands are needed for hybridization probes or for the preparation of cDNA subtraction libraries.

Similar procedures may be performed utilizing other specific nucleotide / cleavage systems and / or other primer extension reactions such as, for example, sequencing primer-system reactions. Another important use of the present invention is in the preparation of cDNA. Methods for isolating mRNAs for cDNA synthesis by using oligonucleotide capture probes capable of binding the desired mRNA are now well established. In addition, the bound capture probe can be labeled with a subsequent transcriptase using reverse transcriptase (eg, see EP-A-0444119 of Dynal AS, which describes approaches for such solid-phase-based cDNA synthesis). It may be used as a primer in a cDNA synthesis reaction. Such capture probes / primers can form the "linker sequence" of the present invention.

To isolate total mRNA, it is conventional to use an oligo dT capture probe / primer
It may be used to bind to the poly A tail present on all mRNAs. Such oligo dTs can be adapted for use in the present invention by incorporating one or more unique nucleotides such as U. Suitably poly or oligo dU may be used. This has the advantage of simplifying the preparation.

Oligo (dU) base pairing is more potent than that of oligo (dT) to the poly (A) tail of mRNA and will improve capture of mRNA and is therefore significant for its use. It should be noted that it brings benefits. Therefore, a further preferred aspect of the invention is the oligo dU or poly which has been provided to the solid support with means for immobilisation, preferably biotin, or attached to a solid support, preferably magnetic beads. to provide dU.

Therefore, removal of the cDNA product from the solid support represents a preferred embodiment of the invention. The template of mRNA immobilized on a solid support carrying oligo (dT) is cD
Can be replicated in NA. During this reverse transcription reaction, the produced cDNA will be covalently bound to the solid support via the oligo (dT) primer. Therefore, cDNA cleavage is difficult using current methods. dUMP to oligo (dT) (eg 5'TTTTTTTT
TTTTTTTTTTTTTTTTU) forms the target site for UDG activity. Subsequent cleavage of the abasic site by high temperature or high pH separates the cDNA from the solid support. Alternatively, multiple target sites can be introduced (eg:
5'UUUUUUUUUUUUUUUUUUUUUUU). After reverse transcription, it is advantageous to remove the RNA strand before release of the bound cDNA product by UDG cleavage, for example by RNase digestion such as RNase H or by any other means such as high pH. , Would be effective.

In addition to primer extension based methods, the methods of the invention may be combined with other in vitro amplification methods such as the ligase chain reaction (LCR) and the Qβ replicase system (WO87 / 06240). . Thus, nucleic acid molecules may be linked, for example, to a liga.
formation). Unique nucleotide is LCR
It can be included in one or more oligonucleotides that are ligated in the reaction. Therefore, the LCR oligonucleotide may be treated as the linker sequence of the present invention.

In the above aspects of the invention, the nucleic acid molecule may be primarily a DNA molecule or a DNA-RNA molecule. However, other forms of construction, in particular a means for immobilizing one end, are provided at the other end, for example additional DNA sequences, RNA sequences, RNs.
It will be appreciated that it is also possible to have an A-DNA sequence or a "linker sequence" linked to the double-stranded DNA. Thus, simply various forms of "nucleic acid-containing" nucleic acid molecule are possible. Such nucleic acid molecules will be prepared by any conventional or convenient method according to techniques well known in the art. For example, RNA-DNA constructs will be readily synthesized using an oligonucleotide synthesizer. Alternatively, RNA and DNA may be linked using RNA ligase (R
NA ligase will link DNA to DNA and RNA to RNA). Individual dNTPs or rNTPs can be introduced enzymatically into the nucleotide chain using a suitable polymerase enzyme, for example a mutant of a DNA polymerase such as T7 DNA polymerase (Sausa and Padilla (1995) EMBO J. 14, 4609-4621).

Furthermore, as mentioned above, other constructs or chimeras of nucleic acid molecules are possible. Thus, the nucleic acid molecule may suitably include a linker sequence linking the protein (or peptide or polypeptide). Such proteins include, for example, antibodies or fragments thereof (including antibody derivatives such as single chain antibodies and synthetic antibodies), enzymes or receptor proteins or other binding proteins or binding regions thereof or fragments thereof ( For example, streptavidin,
Protein A, G protein, protein L, or a fragment thereof, or an actually known synthetic or modified (eg, genetically modified) antibody,
Affinity binding proteins such as lectins). Alternatively, the linker sequence may be linked to an enzyme substrate, receptor ligand, antigen / hapten, or fragment thereof, and the like. Thus, preferably, the "second" component of the chimeric nucleic acid molecule is an affinity binding group, ie one of a pair of affinity binding partners.

Such a chimeric nucleic acid molecule may specifically include any solid phase step or procedure based on affinity binding, such as in procedures or assays for separation and purification of cells or proteins or other molecules. Has utility in. Therefore, a further aspect of the present invention is to bind to a solid support and subsequently
A method of preparing a construct for cleavage therefrom, the method comprising the step of incorporating into said construct a nucleotide sequence comprising a specific nucleotide capable of selective cleavage at a predetermined site. I am trying.

In another aspect, the invention also comprises a specific nucleotide linked to a functional group, preferably an affinity binding group or a reporter group, which allows selective cleavage at a predetermined site. A chimeric molecule (or construct) containing a nucleotide linker sequence is provided. As mentioned above, preferably the linker sequence in such a chimeric molecule is further immobilized (ie bound to a solid support) or means for immobilizing it on a solid support. Is given.

The functional group may be, for example, any group having properties such as binding activity, or detectable or signal-giving activity useful in an assay or separation procedure. Suitably, as mentioned above, the functional group is an affinity binding group. Such affinity binding groups may be affinity binding proteins, or derivatives thereof, as described above. Alternatively it may be any ligand exhibiting affinity binding properties, for example a hapten / antigen or biotin or any organic molecule.
In fact, every new generation of affinity binding groups is being developed. For example, synthetic or chimeric antibodies or other antibody derivatives and antibody mimetics (eg,
Single chain antibodies or their derivatives) or any binding molecule generated randomly in the library or generated in the display. Alternatively, it may be a reporter group. It is defined herein to include any group that can provide a detectable signal or can participate in a signal generating reaction. Thus, the group may include, for example, an enzyme substrate, or a marker or label, specifically a radioactive label.

The present invention may include, for example, constructs in which the group for affinity binding is provided with a reporter group, such as a label, per se. This can be, for example, a radioactive label or other detectable label, such as a colored, pigmented, chromogenic, fluorescent, or chemiluminescent label or enzyme marker, It may be any of the labels known in the art or described in the literature.

Typical functional groups will include binding proteins such as antibodies or streptavidin, which may optionally be labeled; eg horseradish peroxidase (HRP) or alkaline phosphatase (AP). Enzyme; for example, an enzyme substrate such as a peptide, a substrate for an enzyme under study such as an HIV protease or a phosphorylation site for a tyrosine kinase, or indeed any substrate for an enzymatic assay (eg : Testosterone or other biomolecules); carbohydrates; lipids; haptens (eg small organic molecules); and labels will also be included.

In such a construct, the linker sequence is preferably an oligo dU or poly dU sequence, and further, it is preferable to carry a biotin group as a means for immobilization. Preferred functional groups are antibodies or fragments or derivatives thereof, or haptens.

Methods for linking nucleotide sequences to other molecules are well known in the art, and such constructs are described, for example, by Hermanson (supra) and Aslam.
It may be prepared according to the method described in Dent (supra). General guidance on procedures and methods for linking oligonucleotides (which may be modified) to other molecules is also given in R. Helmuth (1990) PCR Protocol: Method and Application Guidance, Chapter 15. Academic Press, Inc. and YM Dennis Lo. Et al. (19
90) PCR Protocol: Method and Application Guide, Chapter 14, Academic Press, Inc.
Will be found in.

“Modified” oligonucleotides include additional chemical groups on the oligonucleotide during synthesis, such as, for example, reporter groups, specifically labels such as TAMRA, rhodamine, or any other known fluorescent label. It is well known that it can be prepared by adding. Such chemical groups may be, for example, digoxigenin, estradiol, or a hapten such as dinitrophenol (DNP), which provides a target for binding to the antibody, biotin (bound to streptavidin / avidin), or , An entity that is both a reporter group and an antigen / hapten for binding to an antibody, such as fluorescein, and the like, as well as an affinity binding partner as described above. Many such modified oligonucleotides are described, for example, in Oswell research Products Ltd., (Southampton
(UK) commercially available. As explained above, in the chimeric molecule of the present invention, the single oligonucleotide specifically comprises an immobilizing moiety (ie, means for immobilization) such as biotin and an affinity binding group (eg: Haptens), or one or more additional “chemical groups” (or “modification units”) such as reporter groups (eg labels). For example, the chimeric molecule of the present invention may take the form of an oligonucleotide carrying a biotin molecule at the 5'end and an amino group at the 3'end, the amino group being an amino-reactive molecule. It may be used, for example for labeling, as it may provide a means for attachment to the oligonucleotide. Alternatively, at its 3'end, the oligonucleotide may carry a hapten such as digoxigenin. For labeling, digoxigenin is bound to horseradish peroxidase (HRP) via an anti-digoxigenin antibody.
) May be linked to an enzyme such as

The construct can be utilized in a separation procedure using standard known techniques. Thus, for example, in the case of antibody functional groups, the construct may be used to attach to any target entity, such as a cell or protein, for which separation is desired. Immobilization of the construct, either before or after binding to the target entity, allows for separation of the target entity, after which, according to the invention, the target entity will be cleaved by cleavage of the linker sequence. .

For example, in a protein purification procedure, an affinity binding group (ie, ligand) for the protein of interest can be linked to a solid support via an oligonucleotide “linker” sequence of the invention. In this construct, the chimeric molecule of the present invention comprises an immobilization means, a cleavable "linker sequence", and an affinity capture ligand for the desired target protein.

A typical affinity capture ligand is for purifying thyroxine binding protein.
L-thyroxine (Pensky and Marshall (1969) Arch. Biochem. Biophys. 135: 304.
), Folic acid for capturing folate (or salt) binding proteins (Salter et al.,
(1972) FEBS Lett. 20: 302) and cholesteryl hemisuccinate (HDL and LDL) for capturing lipoproteins (Wichman (1979) Biochem. J. 181:
691) is included. In each case, the ligand is cleaved from the support, allowing recovery of the protein and bound ligand. The ligand could also be a small molecule such as L-lysine, a protein such as protein A, a carbohydrate such as D (+) melibiose or a lectin such as concanavalin A. One-third of all enzymes require nucleotide coenzymes, so nucleotide ligands
In particular, it is suitable for enzyme purification and thus provides a means for purification (Barker et al. (1972) J. Biol. Chem. 247: 7235). Ligand-protein interactions will be enhanced by the use of a spacer arm between the cleavable oligonucleotide linker sequence and the ligand to reduce steric hindrance. For example, nucleotide ligands are often immobilized by ligation to spacer arms of 8, 9 or 11 atoms and adenosine 5'monophosphate via a base such as N-6 (Catalogue No. A3019, Sigma-Aldrich Compa
ny) (Chaffotte et al., (1977) Eur. Biochem. 78: 309).

In an assay, a similar or similar principle, eg an ELISA assay, or
Other assay principles known in the art may be used. The ability of the present invention to cleave an antibody-enzyme complex from a solid support finds utility in an ELISA assay. Usually in assays such as ELISA, only one ligand (ie, assay target) can be detected,
It can be quantified per well. However, improvements would allow some ligands to be detected by a multiplex approach. For example, more than one ligand (in this case, the assay target) will be captured in the wells of an ELISA plate at the same time with an antibody specific for each ligand. In addition, a secondary antibody specific for the first ligand may be added, whereby the enzyme or poly (dU
To other detection methods via cleavable linker sequences according to the invention, such as After normal well treatment, the enzyme may be separated from the complex and washed away by using a cleavable linker. This allows a second secondary antibody specific for the second ligand to be added to the wells, allowing the enzyme assay to be performed on the first. The entire process may be repeated several times, allowing the multiplicity of the ELISA assay.

In the method of isolating cells, the use of the reversible immobilization method of the invention is preferred,
It represents a valid surface. That is, this aspect of the invention provides a method for separating target cells from a sample, the method comprising attaching the target cells to a solid support by means of a chimeric molecule as previously defined. And is characterized in that the functional group is an affinity binding group that specifically binds to the cell. The cells separated by this method can be separated from the solid support by cleaving the nucleotide linker sequence.

Thus, more particularly, this aspect of the invention provides: (a) binding the target cell to a solid support by means of an affinity binding partner that specifically binds to the cell, The bond with the support contains a nucleotide sequence containing a specific nucleotide that is selectively cleavable at predetermined sites, (b) separating the support-bound cells from the sample, (c) And) releasing the target cells from the support by selectively cleaving the nucleotide sequences at specific nucleotide sites.

The term cell is used herein for all prokaryotic (including archaeal) and eukaryotic cells, as well as other viable or membrane-coated entities such as viruses and mycoplasmas. , Used to contain subcellular components such as organelles. Representative "cells" therefore include all types of mammalian and non-mammalian cells, plant cells including algae, protoplasts, fungi, bacteria including cyanobacteria, mycoplasmas, protozoa, bacteriophages. Viruses, and organelles.

Thus, the sample may be any material containing such cells,
For example, food, similar products, medical and environmental samples. therefore,
The sample may be a biological sample and may include any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasma, protoplasts, and organelles. Thus, such biological material is suitable for all types of mammalian and non-mammalian cell,
It may include plant cells, algae including cyanobacteria, fungi, bacteria, protozoa and the like. Typical samples are therefore whole blood and blood-derived products such as plasma or buffy coat,
It includes urine, stool, cerebrospinal fluid or any other body fluid, tissue, cell culture, cell suspension and the like, as well as environmental samples such as soil, water or food samples.

Samples may also include relatively pure or partially purified material, such as semi-pure preparations obtained by other cell separation methods. When enzymatic cleavage with a DNA glycosylase is used to release bound cells, in some circumstances, more vigorous cleavage conditions are conventionally used to cleave the non-basic site generated by glycosylase action. It may be desirable to avoid (eg high temperature and / or pH). Therefore, in order to effect cleavage under more physiological conditions that are favorable to living cells, mechanical stress to destroy enzymatically digested (ie, degraded) nucleotide linkers and enzymatic conditions under physiological conditions are required. DNA glycosidases may be used in the cell separation process based on a combination with cleavage. Such mechanical stress may be provided externally, for example by stirring, mixing, pipetting, vibrating, etc. However, the mechanical stresses envisioned for the binding may be sufficient simply by the linked entities, ie the cells and the solid support.

However, if desired, other methods can also be used to facilitate the cleavage of the abasic site caused by the action of DNA glycosylase, eg, exonuclease III or C, which cleaves at the abasic site. E. Coli endonuclease IV
Including the use of "cleaving reagents" such as enzymes or chemical reagents such as. This also allows a gentle cutting procedure to be adopted. Such cutting methods are further described in the examples below.

For example, “cold-adapted” or “cold-adapted” that can function well at low temperatures (eg, 4 ° C.) that contribute to cell separation and handling, such as enzymes derived from organisms in the cold environment, such as the Arctic shrimp described above. The use of "resistant" enzymes is particularly preferred for cell separation procedures. The main advantage of the present invention is that it can be easily multiplexed. More specifically, the present invention is capable of conferring the ability to selectively cleave one type of bond when one or more (and indeed many) other types are present. This ability is in many areas
Has potential value. Thus, different specific nucleotides may be incorporated into different nucleic acid molecules, eg, different linker sequences used to generate the nucleic acid molecule. This results in a diversity of nucleic acid molecules, each nucleic acid molecule (or “population” of each population or molecule, etc.) differing in the nature of the unique nucleotide / linker sequences. This means that something like a nucleic acid molecule (eg a linker sequence) can (in the presence of a different nucleic acid molecule / linker sequence), in particular a suitable cleavage system (ie glycosylase) specific for the particular nucleotide of interest. It means that it can be selectively cleaved using an enzyme). Thus, this also opens up possibilities for a large number of different nucleic acid molecules (or different "types" or populations of molecules) immobilized on a solid support (which may be the same or different). .
Each such molecule (or "type" or "population") is selectively separated from the solid support by the presence of a selectively cleavable specific nucleotide, such as a selectively separable linker sequence. It In other words, "different" linker sequences (which differ in the nature of the specific nucleotides) are used to anchor or link different nucleic acid molecules to a solid support so that they are of different "tethering" or conjugation systems. Represents a range. And this allows each such linker sequence to be selectively cleaved in the presence of the other linker sequence, thereby providing a moiety (ie, a nucleic acid molecule) attached through the linker sequence. Are selectively separated.

It is understood that this aspect of the invention thus provides, in a general sense, a method for selectively separating a variety of different nucleic acid molecules from a bound solid support. Unique nucleotides are incorporated at defined sites in each nucleic acid molecule, and different nucleic acid molecules are characterized by incorporating different specific nucleotides. In addition, the method includes the step of selectively cleaving each nucleic acid molecule at specific nucleotide sites.

One or more diverse nucleic acid molecules (ie, one or more different types) are thus selectively separated, depending on the selection. As mentioned above, the solid support may be the same or different for each different nucleic acid molecule, and the selective cleavage of such different nucleic acid molecules may be separately (e.g., at different moieties). , Or may be spatially separated) or continuous.

As mentioned above, different “nucleic acid molecules” may be different types or populations of nucleic acid molecules. The term "multiple" as used herein means two or more. Similarly, the invention also provides a method of reversibly immobilizing a large number of different nucleic acid molecules (as described above), characterized by incorporating different specific nucleotides into each different nucleic acid molecule. I am trying.

Multiplexing in this manner has found useful applications in the field of affinity-based separations or assays. This is because it can selectively release targets to which different affinity moieties (ie, different affinity binding groups) are attached. Thus, in the context of cell separation, for example, a plurality of different cell types may be constructed with a construct containing an affinity moiety (or other affinity binding group) attached to a cleavable linker sequence of the invention. It may also be bound to a solid support (eg beads) via Each type of antibody (ie, each affinity binding group) may have a different cleavable linker sequence (eg, poly U, methyladenine, etc.). Thus, the entire population of cells can be incubated with the antibody-bead mixture and attached to the beads via a selectively cleavable label (ie, linker). In addition, the user may choose to separate certain types of cells (eg, cell type "A") as follows. By adding a glycosylase specific for the linker sequence to which the antibody that binds to cell type "A" is tethered (termed tether "1"). On the other hand, it does not separate any other cell type (eg, cell type “B”) until a glycosidase specific for the other linker sequence (eg, for tether “2”) is added. This is attractive when cell samples are valuable and only a limited number of cells are available.
For example, CD4-cells can be purified with CD8 + cells in the same tube, and each cell population can be separated by the addition of glycosidases 1, 2, etc., in turn.

Further applications of the multiplexing approach exist in the field of separation or purification of PCR products. Thus, for example, a specific linker may be added to each biotinylated PCR primer. As a typical example, the forward and reverse P
The CR primers could each incorporate different unique nucleotides. For example, primer T3 may incorporate U, and primer T7
It may be incorporated into the methyl adenine (MA) in (i.e., T3 ^U and T7 ^MA).
After growth, binding to streptavidin beads and denaturation to further separate the strands, each strand of the PCR product was labeled with glycosylase UDG or methyladenine (MA).
Glycosylase can be used to separate in sequence. This simplifies the need to prepare separate purification tubes for purifying each strand.

Alternatively, multiplex RT-PCR reactions could be co-purified to compare gene expression levels. In other words, expression levels or patterns of different genes can be compared by using RT primers for different genes. The primers each have different specific nucleotides, which will result in relative cleavage of each different RT product. As a typical example of such a method, an assay can be envisioned. For example, an assay for GAPDH would have an orthograde (or retrograde) biotinylated primer containing U (eg, GAPDH-F ^U ). In addition, assays for mRNAs of interest, such as p53, will have biotin p53-F ^MA . The PCR reaction will incorporate fluorescent deoxynucleotides into both GAPDH and p53 PCR products. They are,
For example, both could be purified together in the same test tube containing a biotin-conjugated solid support such as streptavidin-beads (M-280 Dynabeads from Dynal ASA as an example). After washing, etc. to remove unincorporated nucleotides, the amount of GAPDH PCR product could be measured by adding UDG and analyzing the amount of fluorescence emitted from the beads. Similarly, p53 could be measured by the addition of methyladenine glycosylase.

The only limitation of this multiplexing approach is the number of different glycosylases. 10
In the case of 1 glycosylase enzyme and 4 fluorescent nucleotides, multiplexing would allow the release of 40 different PCR products derived from beads in the same tube. Another application of the multiplexing approach will be in sequencing and, in particular, in the purification of multiplex sequencing reactions.

The present invention offers the potential for several sequencing reactions to be purified together. This is possible by using different sequencing primers that incorporate different specific nucleotides. To facilitate purification, different primers may be the same or different, but preferably,
The same immobilization means such as biotin (bound to a solid support carrying streptavidin, eg beads) may be provided. Specifically, biotinyl present somewhere in the sequence having uracil (T3 ^U) (1) or, (T3 ^MA) with methyl adenine in the sequence (2), such as, in some embodiments A modified T3 sequencing primer can be used. The first sequencing reactions, using primers T3 ^U, the second separation reaction using primers T3 ^MA. After the sequencing reaction is complete, the first and second reactions are mixed together and purified on streptavidin beads. After washing, the release of reaction 1 is brought about by the addition of UDG, which reaction may then be "read" (eg loaded on a sequencing gel) with methyladenine glycosylase. The release of Reaction 2 by the addition of is read. Clearly, the number of sequencing reactions that will be released is limited only by the number of different glycosylases available. Such a multiplexing approach may be suitable for high throughput sequencing laboratories. beads
-For 96-well plates containing a mixture of sequencing reactions, UDG all 96-
It is possible to add to the wells and remove the first 96 reactions, then add methyladenine glycosylase to the same 96-wells, omit the second batch of 96 reactions, and so on. The products required would simply be T3 ^U and UDG, T3 ^MA and methyladenine glycosylase. The release of DNA from the beads alone would not require any changes to the purification sequencing reaction.

The components or means required to carry out the method of the invention may be supplied, preferably in the form of a kit. Thus, a further aspect of the invention provides a kit for use in the method of the invention as previously defined, which kit comprises: (a) means for introducing a specific nucleotide into a nucleic acid molecule. And (b) a means for selectively cleaving the specific nucleotide.

Means (a) may suitably be an oligonucleotide sequence containing the specific nucleotide, eg the linker sequence described above, preferably a primer. Means (b) may suitably be a DNA glycosylase enzyme. The kit may additionally include components such as a solid support, or means for immobilization on the solid support. Preferably the kit also comprises oligo dU or poly dU provided with means for immobilisation on a solid support, preferably biotin, or attached to a solid support, preferably magnetic beads. good.

Applications and applications of the invention include the following: The first and second strands of the cDNA can be made on a solid support and then for cloning purposes such as library preparation, or for example,
For the preparation of probes for use in hybridization procedures with biochips (gene chips), released by selective cleavage and after sequencing reaction,
But before loading, remove the non-embedded dNTP. 2. Both sense and antisense strand probes can be prepared from the same ds cDNA preparation, simplifying the process. 3. Reverse subtraction hybridization is possible, and the first strand cDNA is
It can be removed from the support and hybridized with the mRNA immobilized on the solid support. 4. Usually, since there is a biotin molecule at the 5'end that inhibits ligation, DNA such as a PCR product can be dominantly cloned. 5. Cells may be subjected to affinity binding-based solid support procedures in a manner such that the "foreign" material remaining bound to the cells is readily released from the support while remaining within a minimal range (eg, It can be easily separated by immunomagnetic separation (IMS). Thereby, for use in therapy and / or for subsequent stimulation,
It finds particular application in cell separation. Thus, for example, therapeutic T cells (eg, subsequent reintroduction into a patient) are isolated and stimulated by IMS (eg:
It will also be separated from the IMS support for reintroduction, again by procedures involving IMS). 6. The cleavable nucleotide sequence is a new generation of affinity binding molecules (eg, single-stranded) being developed in such a way that the cleavage site is positioned as close as possible to the site of affinity binding. Easily linked to antibodies and their derivatives (eg paratope-reduced), or peptide library production molecules,
Alternatively, they may be combined. This allows cleavage to occur such that as little "foreign" material bound to the separated entity (ie, the binding "partner" of the affinity binding group) is left as far as possible.

[0076]

【Example】

The invention is explained in more detail on the basis of the following examples, which do not limit the invention and with reference to the figures.

[0077]

Example 1 Method for removing cDNA product from solid support 1. Jakobsen, KS et al., Advances in Biomagnetic Separation, Uhlen, M. Ed.
aton Publishing, (1994) pp 61-71, or Dynal mR
Dynabeads, oligo 5'TTTTTTTTTT according to the instructions for NA capture
TTTTTTTTTTTTTU or 5'UUUUUUUUUUUUUUUUUU
UUUUU is used to capture, wash and prepare 1 μg of mRNA.

2. The complex of mRNA and beads was mixed with 1X reverse transcription buffer (Life Technologies, Inc.) 50
Wash twice in μl. 3. Add 5 μl of 20 μl reverse transcriptase containing 200 units of Super Script II reverse transcriptase.
Nm dNTPs ^{such, 33 P dATP (3000mCi / mmol} ), mixed in 1X reverse transcriptase buffer containing 100 mM DTT and _{2.5mM MgCl 2 (Life Technologies, Inc} ).

4. Incubate at 37 ° C for 20 minutes, 42 ° C for 20 minutes, 55 ° C for 20 minutes. 5. Incubate with 1 μl RNase H for 15 minutes at 37 ° C and 10 minutes at 70 ° C. 6.50 μl UDG reaction buffer (10 mM Tris-HCl, pH 8.9, 50 mM KCl and 25 mM MgC
l ₂ ) Wash twice. 7. One unit of uracil-DNA glycosylase (in 20 μl UDG reaction buffer)
Boehringer Mannheim) is added and incubated at 37 ° C. for 10 minutes.

8. Incubate at 95 ° C for 3 minutes to cleave DNA at nonbasic sites. 9. Using a magnet, collect the supernatant containing the released cDNA. Results FIG. 1 shows that the release of first-strand labeled cDNA is significantly increased when UDG is incubated with oligo (dTU) compared to oligo (dT). Respectively
Approximately 80% of the cDNA is released compared to less than 20%.

[0081]

Example 2 Method for Removal of PCR Product from M-280 Streptavidin Beads 1. Unmodified PCR with biotinylated primer, for example biotin-GTAATACGACTCACTA U AGGGC, having dUMP inside, and suitable template. PCR is carried out containing the primers and the reaction components (if using biotinylated PCR primers containing dUMP, no changes are necessary compared to the same unmodified primer).

2. On a Centricon-50 spin column (Amicon Inc.) using the manufacturer's instructions,
Unincorporated biotinylated primer can be removed. 3. Purified PCR product (100ng ~ 5μg) in 100μl B & W buffer (2M NaCl, 10mM T
Immobilize in 100 μl of M-280 streptavidin magnetic beads (Dynal) in ris-HCl (pH 7.5) and 1 mM EDTA) at 50 ° C. for 3 hours with rotation.

4. Proceed with steps 6 to 9 of Example 1 above. 5. Free double-stranded or single-stranded PCR products can be collected and used for downstream applications such as cloning. Results Figure 2 shows radiolabeled biotinylated PC from M-280 streptavidin beads.
Release of R product is shown. bioT3U is, biotin -AATTAACCCTCAC U AA
It is AGGG. bioT7U is, biotin -GTAATACGACTCACTA U A
It is GGGC.

[0084]   In the presence of UDG a significantly increased amount of PCR product is released than in the absence.

[0085]

Example 3 Removal of cDNA Products from Solid Supports Using Ribonucleotides as Specific Nucleotides 1. Using Daynabeads oligo 5'end biotin TTTTTTTTTTUTTTTTTTTTTT as described by Jakobsen et al. Capture, wash and prepare 1 μg of mRNA.

2-4. Same as in Example 1. 5. Add 2 lots of 100 μM NaOH (20 μl) and add RNA containing oligonucleotide (rU
) Is cleaved to release the attached cDNA. 6. Using a magnet, collect the supernatant containing the released cDNA. 7. 40 μl of 100 mM HCl if necessary, or an appropriate amount of 1M Tris-HCl buffer solution (
pH7) is added to neutralize the alkali.

[0087]

Example 4 Removal of cDNA Product from Solid Support Using Ribonucleotides as Specific Nucleotides 1-4. Same as Example 3 above. 5. Add 10 μl of 25 mM MgCl ₂ or MnCl ₂ solution to a final Mg ²⁺ or Mn ²⁺ concentration of at least 10 mM to cleave RNA (rU) containing oligonucleotides and attach the attached cDNA. To release. Heat at 94 ° C for 5 minutes.

[0088]   6. Using a magnet, collect the supernatant containing the released cDNA.

[0089]

Example 5 Method of Using Exonuclease III to Cleave Nonpurine Base Sites 1-7 steps are as described in Example 1. 8. Add 150 units of Exonuclease III (Promega Corp. USA)
Incubate at 30 ° C for 30 minutes.

9. Using a magnet, collect the supernatant containing the released cDNA. Record. Exonuclease III belongs to a class of nonbasic site-cleaving enzymes generally known as AP nucleases (Lindahl and Anderson (1972) Biochemistry
11: 3618-3623). Exonuclease III also has a double-strand-specific DNase activity and destroys double-stranded DNA, eg PCR products, but not single-stranded DNA. Therefore, as described in this Example, only in single-stranded DNA, exonuclease III
It is preferable to cleave the non-basic site at. Other suitable enzymes that cleave abasic sites include E. coli endonuclease IV (Demple and Harrison, (1994) A
nnu. Rev. Biochem. 63: 915-948), which may be used in place of exonuclease III in this example.

[0091]

[Example 6] Linker cleavage and cell purification 1. Dynabeads CD4 (cell separation and protein purification, Technical Handbook, No. 1)
Isolation of cells with biotin-polyU-anti-CD4 is performed as described in 2nd edition, Dynal).

2. Resuspend the rosette-shaped cells in 100 ml of cell culture medium. 3. Add 100 units of UDG (Roche Molecular Biochemicals) and 1500 units of exonuclease III. 4. Incubate at 37 ° C for 45-60 minutes. 5. Remove the free beads using a magnetic separator.

[0093]   6. Collect the cell-containing supernatant.

[0094]

Example 7 Linker Cleavage and Protein Purification Method 1. Wash 1 × 10 ⁷ tissue culture cells three times in PBS. 2. Lyse cells in 1 ml of 2% Triton X-100 containing 50 μg / ml TLCK and TPCK, 200 μg / ml PMSF.

3. Cell lysate 40 μl of Dynabeads-cleavable linker-affinity molecule (
For example, incubation with Dynabeads-poly (dU) -8-carbon spacer-adenosine 5'terminal monophosphate). 4. Collect the beads using a magnet and wash 3 times with PBS. 5.40 μl UDG reaction buffer (10 mM Tris-HCl, pH 8.9, 50 mM KCl and 25 mM MgC
Resuspend the beads in l ₂ ).

6. Add 2 units uracil-DNA glycosylase (Boehringer Mannheim). 7. Incubate at 37 ° C for 30 minutes. 8. Add 15 units of Exonuclease III (Promega Corp. USA), 37 ℃
Incubate for 30 minutes.

[0097]   9. Using a magnet, collect the supernatant containing free protein and ligand.

[Brief description of drawings]

FIG. 1 is a graph showing% DNA separated with dTMP or dUMP oligonucleotide primers treated with UDG.

FIG. 2 is a graph showing the separation of dUMP PCR primer-PCR products (CPM (000's) released in the presence or absence of UDG).

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SL, SZ, TZ, UG, ZW ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, C N, CR, CU, CZ, DE, DK, DM, DZ, EE , ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, K P, KR, KZ, LC, LK, LR, LS, LT, LU , LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, S G, SI, SK, SL, TJ, TM, TR, TT, TZ , UA, UG, US, UZ, VN, YU, ZA, ZW

Claims (28)

[Claims] 1. A solid support having a specific nucleotide incorporated therein at a predetermined site in the nucleic acid molecule, and the step of selectively cleaving the nucleic acid molecule at the site of the specific nucleotide, to which the nucleic acid molecule is bound. A method of separating the nucleic acid molecule from the. 2. A step of (a) incorporating a specific nucleotide into a predetermined site in a nucleic acid molecule, (b) a step of binding the nucleic acid molecule to a solid support, and (a) and (b) both in order or simultaneously. A method of reversibly immobilizing a nucleic acid molecule comprising the step of (c) selectively cleaving the nucleic acid molecule at the specific nucleotide site. 3. The method according to claim 1 or 2, wherein the nucleic acid molecule is a chimeric molecule containing a nucleic acid component and a separate non-nucleic acid component. 4. The specific nucleotide is uracil, hypoxanthine, ribonucleotide, N
-7-methylguanine, 8-oxoguanine, deoxyuridine, deoxyinosine, deoxy 5 ', 6'-dihydroxythymine, 5', 6'-dihydroxythymine, deoxy 3'-methyladenosine, or 3'-methyladenosine The method according to any one of claims 1 to 3, characterized in that there is. 5. The method according to claim 1, wherein the selective cleavage is carried out enzymatically. 6. The method of claim 5, wherein the selective cleavage is performed using a DNA glycosylase enzyme. 7. The nucleic acid molecule comprises DNA, the specific nucleotide is uracil (U), and the selective cleavage is performed using a uracil DNA glycosylase enzyme (UDG). 7. The method according to any one of 6 to 6. 8. The method according to claim 1, wherein the specific nucleotide is incorporated into the nucleic acid molecule as a part of a linker sequence. 9. The method according to claim 8, wherein the linker sequence is a primer. 10. The method according to claim 1, wherein the nucleic acid molecule is a primer extension product. 11. The method according to claim 1, wherein the support is magnetic beads. 12. A method according to any of claims 8 to 11, characterized in that the linker sequence is provided with means for immobilisation on a solid support. 13. The method according to any one of claims 10 to 12, wherein the nucleic acid molecule is a cDNA or a product of an in vitro amplification reaction or a sequencing reaction. 14. The nucleic acid molecule comprises a linker sequence bound to a protein, enzyme substrate, receptor ligand, antigen or hapten, or a fragment thereof, or bound to an affinity binding group or a reporter group. Claims 8 and 11
Or the method according to any one of 12 above. 15. Preparing a construct for attachment to a solid support and subsequent cleavage, comprising the step of incorporating into the construct a nucleotide linker sequence containing a specific nucleotide capable of selective cleavage at a predetermined site. how to. 16. A chimeric molecule comprising a nucleotide linker sequence containing a specific nucleotide capable of binding to a functional group and selectively cleaved at a predetermined site. 17. The chimeric molecule according to claim 16, wherein the functional group is an affinity binding group or a reporter group. 18. The method according to claim 15 or claim 16 or 17, characterized in that the linker sequence is immobilized on a solid support or provided with means for immobilization. Chimeric molecule according to. 19. The chimeric molecule according to claim 16, wherein the affinity binding group is an antibody, a fragment thereof, a derivative thereof, or a hapten. 20. An affinity binding group comprising the step of binding a target cell to a solid support with a chimeric molecule as defined in any of claims 16 to 19, wherein said functional group specifically binds to said target cell. A method for separating the target cells from a sample, wherein 21. A plurality of different nucleic acid or chimeric molecules are attached or bound to a solid support, each of the different molecules incorporating a different specific nucleotide. The method according to either 18 or 20. 22. A kit comprising: (a) means for introducing a specific nucleotide into a nucleic acid molecule; and (b) a means for selectively cleaving the specific nucleotide, wherein the kit is any one of claims 1 to 14. A kit used for the method defined in. 23. A polynucleotide or oligonucleotide incorporating a specific nucleotide, which is immobilized on a solid support or carries an immobilization means. 24. The polynucleotide or oligonucleotide is poly dU or oligo dU.
24. The polynucleotide or oligonucleotide of claim 23, which is 25. The polynucleotide or oligonucleotide is a primer.
The polynucleotide or oligonucleotide according to 23. 26. The polynucleotide or oligonucleotide according to any one of claims 23 to 25, wherein the immobilization means is biotin. 27. The polynucleotide or oligonucleotide according to any one of claims 23 to 25, wherein the solid support comprises magnetic beads. 28. A large number of polynucleotides or oligonucleotides as defined in any of claims 23 and 25 to 27, characterized in that each different polynucleotide or oligonucleotide incorporates a different specific nucleotide.