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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

• This invention relates to methods of immobilizing nucleic acid molecules by binding them to a solid support.
• it relates to an intermediate coupling or coupler molecule which maximizes the usual inherent advantages of immobilization.
• this invention provides for selective and reversible binding through complementary restriction enzyme half sites, a subset of DNA molecules within a heterogeneous population.
• DNA or deoxyribonucleic acid
• RNA ribonucleic acid
• DNA and RNA are composed of monomeric units called nucleotides.
• a nucleotide consists of a pentose sugar molecule (in DNA, deoxyribose; in RNA, ribose) attached to a phosphate group, and a nitrogenous heterocyclic base linked to the glycosidic carbon of the sugar. The base characterizes the nucleotide.
• DNA there are four bases: adenine ("A”), guanine (“G”), cytosine ("C”) and thymine (“T”).
• the bases in RNA are A, G, C, and uracil ("U”).
• nucleic acids To form the nucleic acids, nucleotides are connected by phosphodiester bonds between the 3' and 5' carbons of adjacent pentoses. The result is a chain from which the bases project perpendicularly outward.
• the code for protein resides in the sequence of bases in DNA.
• a particular triplet of DNA bases (or "codon") specifies a particular one of the 20 amino acids which are the normal constituents of protein.
• Natural DNA is formed as two anti-parallel strands.
• the bases on each strand extend toward those on the other. Hydrogen bonds between pairs of opposed bases hold the two strands together.
• the base sequences of the two strands are complementary; that is, an A on either strand is always opposed by a T on the other, and a C on either strand by a G on the other.
• the combination of a base on one strand and its complementary opposed base from the other strand is called a base pair; the possible base pairs of DNA are thus AT, TA, CG, and GC.
• DNA is replicated in nature by the simultaneous synthesis of complementary DNA on both original strands.
• RNA In RNA the thymine base is replaced by uracil ("U”), but the same complementarity between bases exists.
• U uracil
• a cell When a cell is to synthesize a particular protein, coded on a region of its DNA, it begins by synthesizing an RNA strand complementary to that region. This process, called “transcription”, takes place at the DNA molecule, which is used as a template.
• the synthesized messenger RNA (“mRNA”) then, becomes a kind of template for "translation”, i.e., the synthesis of protein according to the code carried by the mRNA.
• nucleic acids and polynucleotides, so as to ascertain and to alter the sequences in vivo and in vitro.
• the methods of manipulation of nucleic acids include treatment with enzymes and with less complex chemicals, biological manipulations in which living cells are employed, and physical manipulations.
• enzymes -- proteins which catalyze specific biochemical reactions -- are used to form or break bonds in nucleotides. Many of these enzymes have been derived from bacteria or from bacteriophages (bacterial viruses) . Among the most useful enzymes in modern genetic engineering are the ligases, which join polynucleotides end-to-end, and the restriction endonucleases, which cleave them (Roberts, R.J. , CRC Crit. Rev. Biochem. 1, 123 (1976); Fuchs, R. and Blakesley, R., Methods in Enzymology 100, 3 (1983)).
• restriction endonuclease recognizes a specific base sequence (called its "recognition site") and cleaves DNA at that site, hydrolyzing phosphodiester bonds on both strands. Many restriction endonucleases cleave the two strands at bonds removed by a few nucleotides from each other, thus producing short single stranded regions at each of the cleaved ends. These self- complementary ends, now called half-sites, are then cohesive and may be re-joined. Since all the cleaved ends produced by that particular restriction endonuclease are identical, heterologous DNA sequences which have been cleaved by the same restriction endonuclease may be joined to each other at their half-sites.
• oligonucleotides and nucleic acids In manipulations of oligonucleotides and nucleic acids, it has often been useful to bind the macromolecules to solid supports. (Gilham, P.T., J. Amer. Chem. Soc. 86, 4982 (1964)). Efforts have generally been of two kinds. One kind of activity has been directed to the synthesis of single-stranded polynucleotides by attaching one nucleotide to a support and then adding mono-or oligonucleotides as needed to achieve the desired sequence (Matteucci, M.D. and Caruthers, M.H., Tetrahedron Letters, 21, 719-722 (1980); U.S. Letters Patent No. 4,373,071 to Itakura) .
• a distinctly different objective is that of binding an already existing polynucleotide to a solid or insoluble support, preferably covalently.
• This process which has been called “immobilization” , presents many advantages in working with the bound polynucleotide. For example, it can reduce the number of manipulations , facilitate the separation of reaction components , and reduce losses of the polynucleotide or nucleic acid as compared to soluble methods .
• Immobilized DNA reacts more reliably and reproducibly with enzymes because the attachment density can be controlled and the number of manipulative steps minimized.
• immobilized DNA also provides the opportunity for an affinity matrix for nucleic acid enzymes (Alberts, B. M. and Herrick, G. in Methods of Enzymology XXI, 198-217 (1971)) and other nucleic acids (Shih, T.Y. and Martin, M.A., Biochemistry 13, 3411 (1974)).
• Binding by these methods is unsatisfactory for many applications. For example, because of steric hindrance one or more portions of the immobilized DNA may be relatively inaccessible for manipulation.
• DNA molecules are immobilized selectively, reversibly and specifically, by binding them to a support via restriction endonuclease half- sites.
• a "coupler" molecule comprising a single or double-stranded oligonucleotide with an endonuclease restriction half site at one end, and covalently attaching that coupler to the solid support at the coupler's other end.
• the result is a nucleic acid-matrix system utilizing the selective affinity of nucleic acids having complementary restriction half-sites.
• a coupler is covalently bound to a solid support to form a coupler matrix system.
• a coupler comprises a DNA molecule having at one end a restriction enzyme half site and at its other end a reactive chemical moiety for covalent coupling to a solid support.
• Such a coupler matrix system can be used to immobilize DNA.
• the DNA to be immobilized should have a restriction enzyme half site end complementary to that of the coupler. If this DNA does not have the appropriate half-site on its end, it may be cleaved with a suitable restriction enzyme. Further, a desired orientation of the DNA can be achieved by cleavage with different restriction endonucleases, one that generates a half-site complementary to the half site of the coupler.
• DNA can also be attached non-covalently through the terminal restriction site by hydrogen bonding to the terminal restriction site of the coupler matrix.
• a further aspect of the invention involves the provision of a coupler bound to DNA.
• the juncture of the coupler and DNA employs complementary restriction enzyme half sites. These bound components can be immobilized by covalent reaction of the coupler's other end to a solid support.
• the DNA molecule is freely accessible to a wide variety of uses , for example in hybridization or as a substrate for a number of molecular reactions.
• the coupler provides a "spacer arm" permitting maximal access to the DNA with minimal steric hindrance from the solid support.
• the coupler-matrix an affinity resin for only those DNA molecules having an end complementary to the exposed coupler restriction enzyme half-site.
• the DNA molecule covalently immobilized through a restriction site can be released intact by restriction endonuclease cleavage.
• the DNA molecule is asymmetric in its restriction enzyme half site ends, the DNA will attach to the coupler matrix by only the homologous end. This forces an orientation or alignment of all DNA molecules bound in the nucleic acid matrix, making opportunities for selectivity of reaction with the bound DNA. For instance, only one specific end will be labelled when subjected to polynucleotide kinase and ⁇ - 32 P-ATP. This eliminates the need for subsequent strand separation, or restriction cleavage and subfragment purification when used for a restriction site mapping experiment. In fact, the restriction site mapping could be executed while the DNA remains immobilized.
• a further object of the invention is to provide a means for selectively attaching a DNA molecule to a solid support through any desired terminal endonuclease restriction site. Another object is to immobilize DNA in a selected orientation.
• Still another object is to provide an affinity matrix for the separation of DNA molecules containing a specified terminal restriction site.
• FIGURE 1 shows the base pair sequence of a specific coupler according to the invention
• FIGURE 2 illustrates the preparation of that coupler
• FIGURE 3 illustrates one way of attaching the coupler to a matrix or solid support
• FIGURE 4 illustrates studies on the activation and verification of the coupler matrix
• FIGURE 5 illustrates the immobilization of DNA from plasmid pSP14 on the coupler matrix
• FIGURE 6 illustrates the reaction of various restriction endonucleases with the immobilized labelled pSP14 DNA
• FIGURE 7 illustrates the preparation of a second specific coupler and its base sequence
• FIGURE 8 illustrates the binding of that coupler to DNA and the subsequent immobilization of the DNA-coupler system to a matrix.
• a coupler was bound to a solid support. Then DNA was immobilized by attachment to the coupler matrix. The effectiveness of the coupler matrix in immobilizing a 3986 bp (base pair) plasmid DNA fragment was then studied.
• Coupler I 182 bp double-strand DNA fragment
• FIGURE 1 shows the base pair sequence in its entirety.
• FIGURE 2 illustrates the preparation of Coupler I.
• 1.5 mg of purified plasmid pUC9 was sequentially digested with restriction endonucleases Bgl I, then Hind III. Then the phenol extracted reaction products were subjected to NACS chromatography (Thompson, J. A., Blakesley, R. W. , Doran, K.
• Coupler I was concentrated by ethanol precipitation, then resuspended in TE buffer. The progress of the Coupler I purification was monitored by subjecting samples to 1% agarose gel electrophoresis.
• a ribonucleotide was then added to the 3'-0H end of the Bgl I half-site of Coupler I, then periodate oxidized and linked to hydrazide cellulose.
• the specific process is shown in FIGURE 3.
• a 2.0 pg aliquot of the purified 182 bp Coupler I was incubated with 16 units of terminal deoxynucleotidyl transferase (TdT) and 96 ⁇ M rATP, selectively introducing a short oligoribonucleotide to the 3' extended Bgl I terminus.
• TdT terminal deoxynucleotidyl transferase
• 96 ⁇ M rATP selectively introducing a short oligoribonucleotide to the 3' extended Bgl I terminus.
• the resulting adenylylated Coupler I was oxidized by treatment with 1.0 mg of
• the coupler could be ligated by RNA ligase to a pAp already coupled to cellulose.
• the coupler can be modified by various chemical methods at its 3' or 5' ends to allow covalent attachments to a solid support (Chu, B. C. F., Wahl, G. M., Orgel, L.E., Nucleic Acids Res. 11, 6513 (1983); Shabarora, Z. A., Ivanovskaya, M. G. , and Isaguliants, M. G. , FEBS Lett. 154, 288 (1982); Shih supra and Potuzak supra.) .
• solid supports may be employed.
• solid support refers to any of the water-insoluble matrices described in U.S. Letters Patent No. 4,342,833 to Chirikjian, column 3, lines 15-58.
• Table I shows the results of studies on the efficiency of Coupler I attachment, as compared to attachment of tRNA by the same process.
• OX periodate oxidation
• Coupler I Matrix Further studies on the coupler-matrix combination, hereinafter "Coupler I Matrix,” were performed as shown in Table II, and in FIGURES 4 and 5.
• FIGURE 4 illustrates an experiment to examine the orientation and suitability of the prepared Coupler I Matrix for reaction. Aliquots of labelled Coupler I Matrix were treated by various techniques, as follows , the results of which are seen in Table II.
• Coupler I Matrix was placed in a microfuge tube, rinsed several times with buffer (50 mM Tris-HCl [pH 8], 50 mM NaCl and 10 mM
• restriction endonuclease digestion products in the soluble volume were characterized by subjecting samples of each to electrophoresis in a 12% polyacrylamide gel, followed by autoradiography.
• FIGURE 5 shows the immobilization process, which used phage T4 DNA ligase to bind an EcoR/BamH I restriction fragment to Coupler I Matrix.
• FIGURE 6 illustrates studies involving labelling, endonuclease reaction, and reaction products of the immobilized pSP14 DNA. As seen in FIGURE 6, approximately 50 ⁇ g of immobilized pSP14 was 5 '-end 32 P labelled by incubation with 55 units of T4 polynucleotide kinase and 0.5 mCi ⁇ - 32 P-ATP for one hour at
• a DNA was bound to an activated coupler.
• the coupler nucleic acid system was immobilized by attachment to a solid support.
• Coupler II a tetradeoxynucleotide, hereinafter called "Coupler II".
• the sequence of the coupler is 5'-d(TpCpGpG)-3' as seen in FIGURE 7, and this sequence is complementary to one of the half-site sequences generated by digesting
• FIGURE 7 illustrates the activation of the coupler.
• 33 mg of the coupler was 32 P- labeled at the 5 ' end with T4 polynucleotide kinase and ⁇ - 32 P-
• the 32 P- labeled coupler was incubated with 300 units of terminal deoxynucleotidyl transferase (TdT) and 96 ⁇ M rATP, selectively introducing an oligoribonucleotide to the 3'- end of the coupler.
• TdT terminal deoxynucleotidyl transferase
• rA* dialdehyde
• FIGURE 8 illustrates the method whereby Activated Coupler II is bound to a DNA molecule to generate a coupler-nucleic acid system which can be subsequently immobilized to a solid support.
• FIGURE 8 further illustrates that the coupler-nucleic acid system was immobilized by attachment to a solid support utilizing the specific high affinity between biotin and streptavidin (Green, N.M. , Advances in Protein Chemistry, 29, pp. 85-133, 1975).
• a 10 mg aliquot of the coupler-nucleic acid system was mixed with 0.49mg of streptavidin covalently attached to agarose (solid support) in 20 mM Tris-HCl (pH 7.2), 0.1 ml of lmM Na 2 EDTA. After 5 minutes at 22°C, the mixture was filtered through glass wool.
• a 260 measurements and radioactive counting determined that 92% of the coupler-nucleic acid system was immobilized by this procedure.
• the invention has been described with particular emphasis on the preferred embodiments , but it should be understood that variations and modifications within the spirit and scope of the invention may occur to those skilled in the art to which the invention pertains.

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• 1984
  • 1984-04-05 EP EP19840901838 patent/EP0177497A4/de not_active Withdrawn
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