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  • 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
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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  • 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
  • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
• • 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

Definitions

• This invention relates to removal of undesired nucleic acid (NA) , either DNA or RNA. from desired NA. It is especially applicable to removal of contaminating vector sequences from recombinant cloned NA probes.
• NA nucleic acid
• Probes containing less than about 50-100 bases are usually produced synthetically. Larger probes usually are produced by cloning recombinant DNA comprising a probe insert in a DNA cloning vector.
• the procedure for making a cloned, labeled single stranded DNA (ssDNA) probe involves (a) cloning recombinant DNA comprising probe DNA insert in a DNA cloning vector, (b) excising the probe DNA from the vector by treatment with endonuclease and separating the probe DNA from the vector by gel electrophoresis, (c) labeling the probe, for example, by nick translation in presence of each of the four deoxyribonucleoside triphosphates (dNTP's), at least some of which are labeled (d) denaturing the probe and vector DNA.
• dNTP's deoxyribonucleoside triphosphates
• the procedure for making a cloned, labeled single stranded RNA (s ⁇ RNA) probe involves (a) cloning recombinant DNA comprising a DNA insert, corresponding to the desired s ⁇ RNA probe, in a DNA cloning vector having a transcription promoter upstream of the insert, (b) cutting the recombinant DNA with endonuclease to produce a linear DNA template containing the promoter and the insert, (c) contacting the linear DNA template with each of the four ribonucleoside triphosphates (rNTP's).
• rNTP's ribonucleoside triphosphates
• RNA polymerase to synthesize labeled ssRNA on the template and (d) separating the DNA and unincorporated rNTP's from the ssRNA.
• contaminating, labeled vector sequences result from the fact that gel electrophoresis does not completely separate the probe DNA from the vector DNA, even when electrophoresis is repeated several times.
• contaminating, labeled vector sequences result either from incomplete linearization of the recombinant DNA. such that transcription continues into the vector region, or from adventitious transcription due to presence in the vector of RNA polymerase initiation sites other than the promoter. Buffone et al. distribute Clin. Chem.. 31:2043-4 (1985).
• vector ssNA is adsorbed to capture vector ssNA immobilized on a nitrocellulose or nylon membrane.
• a probe DNA is first partially purified by electrophoresis, then labeled and denatured. After denaturation the probe, membrane and a buffer are placed in a plastic bag (hybridization pouch) and held for an extended period under hybridization conditions. Contaminating vector sequences in the probe are captured by the membrane-adsorbed vector ssNA.
• this method gives improved removal of vector sequences compared to electrophoresis alone, it is cumbersome and time consuming. Hybridization on the membrane is slow. The entire procedure requires about 16 hours. The membrane cannot be reused because of loss of capture ssNA from the membrane. Consequently, the procedure requires large amounts of capture vector.
• Pla ⁇ mid DNA is 32 P labeled and fragmented by nick tran ⁇ lation in pre ⁇ ence of DNa ⁇ el. Pla ⁇ mid DNA fragments are denatured and renatured with an excess of vector DNA. Probe DNA remains single stranded while vector DNA becomes double stranded. The s ⁇ DNA is then separated from the vector dsDNA on hydroxylapatite. The procedure i ⁇ cumbersome and time consuming and has not been demonstrated to remove trace levels of vector contamination.
• DNA and RNA have been covalently attached to bead and membrane supports and used in affinity purifications to capture desired NA.
• Examples of literature disclosing this are Moss et al., J. Biol. Chem.. 256:12655-8 (1981), Langdale et al.. Gene 36:201-210 (1985). B ⁇ nemann et al.. Nucleic Acids Research. 10:7163-7180 (1982). and Bunemann et al.. Nucleic Acids Research 10:7181-7196 (1985). It is believed that bead-based hybridization has not been used prior to this invention to remove undesired NA from a sample containing desired and undesired NA. in particular, to remove contaminating vector sequences from recombinant probe NA.
• This invention is a method of removing undesired ssNA from a ⁇ a ple containing desired and undesired ssNA in an aqueous medium.
• the method comprises (a) contacting the sample under hybridization conditions with capture ssNA which is complementary to the undesired ssNA and which is covalently bound to solid, water insoluble beads or to one member of a specific binding pair, (b) in the case where the capture ⁇ NA is covalently bound to one member of a specific binding pair, contacting the ⁇ ample with solid, water-insoluble beads to which i ⁇ covalently bound the other member of the specific binding pair, and (c) separating the beads carrying.
• the capture ssNA comprises ⁇ sDNA sequences of a DNA cloning vector, the desired ssNA compri ⁇ es a labeled s ⁇ DNA or ⁇ sRNA probe derived from a DNA in ⁇ ert which has been cloned in the vector, and the undesired ⁇ sNA compri ⁇ es labeled ssDNA or ssRNA contaminating sequences derived from the vector.
• the invention includes solid, water insoluble capture beads to which is covalently attached DNA sequences of a DNA cloning vector, and kits compri ⁇ ing the beads along with buffer ⁇ and printed instructions for using the contents to remove contaminating vector sequences from labeled recombinant cloned ssNA probes.
• the invention also includes, compounds consisting of sequences of a DNA cloning vector covalently attached to one member of a specific binding pair, and kits comprising such DNA sequences * along with solid, water insoluble capture beads to which is covalently attached the other member of the ⁇ pecific binding pair, b ⁇ ffer ⁇ , and printed instruction ⁇ for u ⁇ ing the contents to remove contaminating vector sequence ⁇ from labeled recombinant cloned ssNA probes.
• the method of this invention provides several advantages over the membrane-based hybridization method described above for removing contaminating vector sequences from NA probes.
• the hybridization i ⁇ faster, requiring only about one hour.
• the procedure is less cumber ⁇ ome becau ⁇ e the capture reagents can be manufactured and stored for use at a later date, and the hybridization can be readily carried out in a microcentrifuge tube rather than a hybridization pouch.
• the captured DNA can be removed by denaturation and the beads can be reused several times, thus requiring much les ⁇ capture DNA.
• the hybridization can be carried out in ⁇ olution offering potentially even fa ⁇ ter hybridization and more complete removal of contaminating vector sequences.
• the bead ⁇ must be water insoluble and stable to the physical and chemical conditions to which they are subjected during linking of the capture DNA or ⁇ pecific binding substance (e.g.. avidin) and hybridization and denaturation of the capture and undesired NA. They must also be capable of being covalently linked to the capture NA or specific binding substance in a manner which is stable to the hybridization and denaturation conditions. It is desirable that the beads exhibit low nonspecific adsorption of nucleic acids in the hybridization conditions.
• the beads must be capable of being separated readily from the aqueous medium following hybridization, e.g.. by settling, centrifugation or application of magnetic field.
• Beads in the size range 1-300 microns are satisfactory for separation by settling or centrifugation. Beads in the size range 10-100 microns are preferred.
• beads which have been used heretofore in affinity purifications by hybridization of desired nucleic acids as described in B ⁇ nemann et al.. Mos ⁇ et al.. Langdale et al. and B nemann et al., supra, can be used in this invention.
• a preferred class of beads are composed of organic polymers having terminal amine groups, such as P-100 Bio-Gel aminoethyl polyacrylamide beads of Bio-Rad Co.
• beads which can be used include Sephadex ⁇ G-25, Sephacryl®S-500, Sephacryl® S-1000. and Sepharose®CL-2B, CL-4B and CL-6B, all products of Pharmacia Fine Chemicals, Upsala, Sweden.
• Sephadex® i ⁇ a bead-formed dextran gel prepared by crosslinking selected dextran fractions with epichlorohydrin.
• Sephacryl® beads are formed by crosslinking allyl dextran with N.N'-methylene bisacrylamide.
• Sepharo ⁇ e® CL beads are agarose crosslinked with 2.3-dibromopropanol.
• Cellex®410 a dry cellulose powder available from Bio-Rad Laboratories.
• Magnetic beads can also be used, such as the particles described in Hersh, U.S. 3.933.997. Ithaki ⁇ sio ⁇ U.S. 4,115.534, Forrest et al. U.S. 4.141.687. Mansfield et al.. U.S. 4.197.337 and Chagnon. Danish application DK 2374/84. The latter are commercially available under the trade name Biomag® from Advanced Magnetics. Preferred magnetic beads are the coated CrO_ particles described in Lau U.S. Application S.N. 06/841,107, allowed October 3. 1986, issue fee paid December 30. 1986.
• M gne ic particles having an outer layer of a silane compound with functional groups, such as 3-aminopropyl-triethoxy ⁇ ilane, can be utilized for covalent attachment of DNA or ⁇ pecific binding substance, either directly or through liner compounds, as taught in the above-cited references.
• Preferred ⁇ pecific binding pairs are biotin and avidin or streptavidin.
• Other pairs which can be used include antibodies and their antigens or haptens; intrin ⁇ ic factor and vitamin B12; folate binding protein and folic acid; thyroxine binding globulin and thyroxine; and many others.
• Covalent attachment of capture NA to beads composed of organic polymer containing terminal a ine groups or coated with compounds having free amine groups, such as 3-aminopropyl-triethoxysilane. is accomplished by the condensation of the 5*-phosphate of the NA with the amine of the bead using the water soluble carbodi- imide, l-eth ⁇ l-3,3-dimethylaminopropylcarbodiimide (EDAC) in 2-morpholinoethanesulfonic acid (MES).
• EDAC water soluble carbodi- imide
• MES 2-morpholinoethanesulfonic acid
• Capture NA can be coupled to bead ⁇ such a ⁇ Sephadex®G-25. Cellex®410, and Sephacryl®S-500 via diazotization, or to bead ⁇ ⁇ uch a ⁇ Sephadex®G-25. Sepharose®CL-2B and CL-6B, and Sephacryl®S-500 and S-IOOO via cyanogen bromide activation, as taught in the B ⁇ nemann et al. and B ⁇ nemann references, supra. Nucleic acids can be coupled to Celiex®410 via epoxy activation as taught in the Mos ⁇ et al. reference, ⁇ upra.
• Capture NA can be covalently bonded to amine groups of ⁇ pecific binding ⁇ ub ⁇ tance ⁇ by the ⁇ ame chemistries used to bond NA to amine groups of bead ⁇ , e.g., condensation of NA 5 1 -phosphate groups with amine groups using EDAC in MES. as described above.
• Capture DNA can be covalently attached to biotin by nick translation, end-filling or end-labeling using biotinylated deoxyuridyl residues a ⁇ de ⁇ cribed in Chan et al.. Nucleic Acid Research. 13:8083-8901
• Capture DNA can be attached to protein by the method described in Renz et al.. Nuclei Acids Research 12:3435-3444 (1985) and used with bead ⁇ covalently attached to antibodies to the protein.
• Thi ⁇ invention i ⁇ applicable to removal of unde ⁇ ired, contaminating NA from any sample. Accordingly, any NA which will hybridize with contaminating NA can be used as capture NA.
• a large number of DNA cloning vectors can be used for making labeled DNA and RNA probes and all of these vector ⁇ can be used to provide capture DNA for use in this invention.
• the invention i ⁇ particularly applicable to capture DNA co pri ⁇ ing ⁇ equence ⁇ of DNA vector ⁇ of bacterial origin, such as pBR322 and vectors containing all or part of pBR322. such as ⁇ BR325 and pSP64. The latter is described in Melton et al.. Nucleic Acids Research 12:7035-7056 (1984).
• ⁇ capture DNA It is possible to use complete linearized vectors a ⁇ capture DNA. but it i ⁇ preferable to cut the vector ⁇ into fragments of about 100 to 1000 base pairs using one or more restriction endonuclease ⁇ and attach the fragments to beads or ⁇ pecific binding substance ⁇ . Larger NA sequences can be used, but are not preferred becau ⁇ e they are more ⁇ ubject to degradation during u ⁇ e.
• Capture DNA can be denatured to provide ⁇ DNA either before or after attachment to the beads or specific binding sub ⁇ tance ⁇ .
• the capture DNA in the product ⁇ of thi ⁇ invention can be either dsDNA or ssDNA.
• Probes DNA and RNA probe starting materials for this invention can be prepared by the conventional procedures outlined in the Background portion of thi ⁇ specification. Synthesi ⁇ and cloning of recombinant DNA are described " in Maniati ⁇ et al.. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor
• the cloned probe insert Prior to labeling a DNA probe the cloned probe insert is excised from the vector by endonuclease digestion, then the probe DNA is ordinarily separated from the vector DNA by gel electrophoresis and eluted from the gel. a ⁇ de ⁇ cribed in Vogel ⁇ tein et al., Proc. Nat'l Acid. Sci. USA. 76:615-619 (1979) and Yang et al.. Method ⁇ in Ezny oloqy. 68:176-182 (1979). It is presently preferred to carry out at least one such gel electrophoresis prior to affinity removal of vector sequence ⁇ , according to this invention, although it is contemplated that the method of the invention may in some cases permit elimination of the electrophoresis step.
• the DNA i ⁇ denatured to produce s ⁇ DNA by conventional means, e.g., by heating at a temperature of 90-100°C for several minute ⁇ or by mixing with dilute sodium hydroxide, about pHIO or a higher. Bloomfield et al.. Physical Chemistry of Nucleic Acids, pages 332-339.
• the ssDNA is then ready for affinity purification in accordance with thi ⁇ invention.
• the labeling step can be performed after the denaturation step, and can even be performed after the affinity purification steps of this invention.
• RNA i ⁇ ready for removal of contaminating vector ⁇ equence ⁇ by the affinity purification method of thi ⁇ invention.
• the removal of vector related or other undesired NA sequences is accomplished by the addition of capture N (homologous to the vector sequence ⁇ ) to the NA probe where the capture NA i ⁇ either attached to a bead ⁇ upport or attached to one member of a ⁇ pecific binding pair.
• Both the capture NA and probe NA mu ⁇ t be in a eingle ⁇ tranded form to allow efficient hybridization between the capture NA and the undesired probe HA.
• Hybridization conditions are ⁇ elected which allow the undesired NA ⁇ equence ⁇ to hybridize to the capture DNA.
• Preferred hybridization conditions include aqueous medium, a high salt buffer temperature of 65 ⁇ C and reaction time of 1 hour or longer a ⁇ illu ⁇ trated below.
• hybridization condition ⁇ can be used, e.g., conditions disclosed in U.S. Patent 4.358.535. col. 5 and in Maniatis, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory, 1982. page ⁇ 208-209. 324-333 and 387-389.
• the hybridization will be carried out in aqueou ⁇ buffered medium containing 0-60 volume percent of polar organic solvent ⁇ uch as forma ide, at a temperature in the range of about 30 to 80°C, usually 40-70 ⁇ C, with a reaction time of about 15 minutes to 5 hours, preferably 1-2 hours.
• the hybridization i ⁇ carried out in a microcentrifuge tube such as an Eppendorf tube, but it can be carried out in a plastic bag or other suitable container.
• the support can be pelleted by centrifugation or removed by the presence of a magnetic field when the bead ⁇ are magnetic.
• the removal of the capture NA is achieved by the addition of the other member of the ⁇ pecific binding pair which is attached to a ⁇ upport, separation of the unde ⁇ ired NA hybridized to the capture NA is accompli ⁇ hed by the a ⁇ ociation of the binding pair followed by the removal of the capture support.
• the capture beads can be denatured to remove the hybridized vector ssNA and recycled. The process must be rigorous enough to remove the vector NA without destroying the beads or the crosslinking of the DNA to the beads.. Treatment of the beads with heat, dilute alkali or organic ⁇ olvent ⁇ can accomplish this recycling.
• This invention includes kite containing reagents and instructions for carrying out the method of the invention.
• kit will contain solid * , water- soluble beads to which is covalently attached sequences of a DNA cloning vector, hybridization buffer and in ⁇ truction ⁇ .
• a ⁇ econd type of kit will contain: solid, water insoluble beads to which is covalently attached one member of a specific binding pair; capture ssNA to which is covalently attached the other member of the ⁇ pecific binding pair; hybridization buffer; and in ⁇ truction ⁇ .
• a preferred kit of the fir ⁇ t type include ⁇ : a vial containing about 20 mg of aminosilane coated Cr0 2 beads to which is covalently attached about 30 g of s ⁇ DNA ⁇ equences of pBR322 and about 1 ml of a storage buffer (1.0 M Tris»HCl.
• A) Add 1 ml of ⁇ torage/hybridization buffer, ⁇ pin bead ⁇ out. Remove ⁇ pernatant and di ⁇ card.
• B) Add 400 u.1 of storage/hybridization buffer to bead ⁇ . Bead ⁇ are now ready for probe addition.
• Tri ⁇ tris(hydroxy ethyl)aminomethane hydrochloride.
• SDS sodium dodecyl sulfate
• IX TE stands for 10 mM Tris and 1 mM EDTA. pH 7.5.
• the coupling agent l-ethyl-3,3-dimethylamino propylcarbodimide was weighed out. 200 mgs of EDAC was added to the bead. DNA mixture. The coupling reaction was allowed to run overnight at room temperature with agitation.
• the uncoupled DNA was removed and the DNA attached to the support was denatured by first removing the reaction buffer and then washing the bead ⁇ four times with O.IN NaOH 0.1% SDS, 5 ml ⁇ each wash.
• the wa ⁇ he ⁇ and reaction buffer were ⁇ aved for quantitation of the coupling efficiency. 84% of the pBR322 wa ⁇ coupled to the bead ⁇ .
• the pBR322 was immobilized in the same manner in a concentration range from 10 9, 108, 107, 106 copies/well.
• HIND III T CMV fragment cloned into pBR322. transformed and grown in an E. coli cell line.
• HIND III T plasmid wa ⁇ i ⁇ olated from the growth, digested with HIND III restriction enzyme and i ⁇ olated on an agaro ⁇ e gel.
• the membrane were washed to remove unhybridized probe. This was carried out by: (1) washing 10 minutes at room temperature in
• pBR322 vector contamination can also be removed from recombinant NA by using solution hybridization of the NA sample with complementary pBR322 DNA containing one member of a specific binding pair (e.g. biotin) followed by the selective removal of the hybridized pBR322 sequence ⁇ with the ⁇ econd member of the specific binding pair (e.g. ⁇ treptavidin) which has been covalently attached to a ⁇ olid ⁇ upport.
• a specific binding pair e.g. biotin
• the ⁇ econd member of the specific binding pair e.g. ⁇ treptavidin
• the HIND III L CMV DNA probe was produced from the HIND III L fragment of CMV DNA cloned into pBR322, transformed and grown in an E. coli cell line.
• a ⁇ uitable control would be to treat the contaminated CMV-L DNA probe with 2 ⁇ g of pBR322 (non-biotinylated) in the same manner a ⁇ de ⁇ cribed for the biotinylated pBr322 (above) for hybridization with target DNA on a nylon membrane.
• the nylon membranes were wa'shed twice at room temperature for 10 minutes with 2X SSC. twice at 68° C for 30 minutes with 2X SSC. 1% SDS. and twice at room temperature for 10 minutes with 0.2X SSC.
• the washed membranes were dried and autoradiographed at -70° C for 16 hours and compari ⁇ on of the treated and untreated probe showed a 5 fold reduction in the PBR322 crossreactivity for the treated probe and equal ⁇ ensitivity for detection of the CMV-L target DNA.

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• 1988
  • 1988-06-22 JP JP63506107A patent/JPH02503983A/ja active Pending
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