Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
  • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
  • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Definitions

• the present invention is in the fields of molecular and cellular biology
• the invention is particularly directed to methods useful for the production and isolation of nucleic acid molecules
• the invention concerns isolation of mRNA molecules and the production and isolation of cDNA libraries (single- and double-stranded)
• the invention concerns selection and isolation of particular nucleic acid molecules of interest from a sample which may contain a population of molecules
• the invention concerns the use of affinity- labeled primer adapter molecules which allow improved isolation and production of such nucleic acid molecules, increasing both product recovery and speed of isolation
• RNA messenger RNA
• cDNA complementary DNA
• the mRNA molecules from an organism are isolated from an extract of the cells or tissues of the organism This isolation often employs solid chromatography matrices, such as cellulose or Sepharose, to which o gomers of thymidine (T) have been complexed Since the 3 1 termini on all eukaryotic mRNA molecules contain a string of adenosine (A) bases, and since A binds to T, the mRNA molecules can be rapidly purified from other molecules and substances in the tissue or cell extract From these purified mRNA molecules, cDNA copies may be made using an enzyme having reverse transc ⁇ ptase (RT) activity, which results in the production of single-stranded cDNA molecules complementary to all or a portion of the mRNA templates Incubating the single-stranded cDNA under appropriate conditions allows synthesis of double-stranded DNA which may then be inserted into a plasmid or
• cDNA cloning This entire process, from isolation of mRNA to insertion of the cDNA into a plasmid or vector to growth of host cell populations containing the isolated gene, is termed "cDNA cloning " If cDNAs are prepared from a number of different mRNAs, the resulting set of cDNAs is called a "cDNA library," an appropriate term since the set of cDNAs represents the different populations of functional genetic information (genes) present in the source cell, tissue or organism Genotypic analysis of these cDNA libraries can yield much information on the structure and function of the organisms from which they were derived In traditional production methods, the cDNA molecules must be size fractionated and multiple phenol/chloroform extractions and ethanol precipitations performed Each of these requirements has inherent disadvantages, such as product loss and limitations in cDNA yield due to multiple extractions/precipitations (Lambert, K N , and Williamson, V M , Nucl. Acids Res.
• each of these methods relies on PCR amplification prior to cloning of the cDNA molecules, often resulting in biased cDNA libraries (i.e., highly expressed sequences predominate over those that are expressed in lower quantities)
• these methods often are less efficient than conventional cDNA synthesis methods which use solution hybridization of the primer-adapter to the template (/. e. , rotational diffusion is required for increased hybridization rates, see Schmitz,
• RNA total RNA or poly A+ mRNA
• the present invention is directed to methods useful for the production and isolation of nucleic acid molecules (single- and double- stranded) from small amounts of input nucleic acid molecules More particularly, the invention provides methods for the production of a cDNA molecule (single- or double-stranded) from an RNA template (e g , single-stranded mRNA or polyA+ RNA) by using gand- coupled primer-adapter molecules Such primer-adapter molecules may also be used in accordance with the invention to isolate mRNA or polyA+ RNA molecules from an RNA-containing sample
• the invention is directed to a method for producing a nucleic acid molecule comprising mixing a nucleic acid template, preferably a mRNA or a polyA+ RNA molecule, with a polypeptide having polymerase and/or reverse transc ⁇ ptase activity and a primer-adapter nucleic acid molecule, wherein the primer-adapter nucleic acid molecule comprises one or more gand molecules and one or more cleavage sites (preferably a restriction endonuclease cleavage site or an endonuclease cleavage site)
• This primer-adapter may be designed to hybridize to any portion of the template
• a first nucleic acid molecule e g , a single-stranded cDNA
• This first nucleic acid molecule contains the primer-adapter (preferably at or near its termini) which facilitates isolation of the first nucleic acid molecule and/or any nucle
• mRNA- or polyA+ RNA-specific primer-adapters is used Such a primer- adapter is hybridized to the mRNA and/or polyA+ RNA to form a p ⁇ mer- adapter/polyA+ RNA hybrid
• the primer-adapter can then facilitate isolation of the mRNA and/or polyA+ RNA from a sample
• the primer-adapter since the primer-adapter is hybridized to the molecule of interest and can be removed by denaturation, cleavage sites in the primer-adapter are not needed
• the primer-adapter molecules of the invention may also be used to isolate specific nucleic acid sequences
• the invention allows selection and isolation of specific nucleic acid molecules (e g , genes or portions thereof) from a population of nucleic acid molecules
• the use of two or more such target- specific primer-adapters allows selection of more than one different sequence of interest
• two or more target-specific primer-adapters directed to different portions of a sequence of interest facilitates selection of such sequences by reducing background contamination
• the target-specific primer-adapter hybridizes to the desired molecule and can be removed by denaturation, cleavage sites in the target-specific primer- adapter are not needed
• the primer-adapter molecules facilitate isolation of molecules comprising such primer-adapters by relying on the ligand portion of the primer-adapter After the primer-adapter is
• Preferred solid supports for use in the invention include, but are not limited to, nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchlo ⁇ de, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, latex beads, magnetic beads, paramagnetic beads, superparamagnetic beads or microtitre plates and most preferably a magnetic bead, a paramagnetic bead or a superparamagnetic bead, that comprises one or more hapten molecules specifically recognizing and binding to the ligand molecule
• Particularly preferred hapten molecules according to this aspect of the invention include without limitation (i) avidin and streptavidin, (ii) protein A, protein G, a cell-surface Fc receptor or an antibody- specific antigen, (iii) an enzyme-specific substrate, (iv) polymyxin B or endotoxin-neutralizing protein (ENP), (v) Fe ++ , (vi) a transferrin
• growth factor interleukin or colony-stimulating factor receptor
• CD4 CD4, (x) spect ⁇ n or fodrin, (xi) ICAM-1 or ICAM-2, (xii) C3bi, fibrinogen or Factor X, (xi ) anky ⁇ n, (xiv) integ ⁇ ns , ⁇ -, ⁇ 2 ⁇ ,, ⁇ 3 ⁇ ,, 6 ⁇ -, 7 ⁇ , and ⁇ 6 ⁇ 5 , (xv) integ ⁇ ns ⁇ - ⁇ ,, ⁇ 2 ⁇ ,, ⁇ ⁇ , and ⁇ v ⁇ 3 , (xvi) integrins ⁇ 3 ⁇ ,, ⁇ 4 ⁇ *, ⁇ 4 ⁇ 7 , ⁇ 5 ⁇ ,, ⁇ v ⁇ b ⁇ nb ⁇ 3 , ⁇ v ⁇ 3 and v ⁇ 6 , (xvii) integrins ⁇ v ⁇ ! and ⁇ v v !
• fibronectin (xx) collagen, (xxi) laminin, (xxii) glycophorin, (xxiii) Mac-1 , (xxiv) LFA-1, (xxv) ⁇ -actin, (xxvi) gpl20, (xxvii) cytokines (growth factors, interleukins or colony-stimulating factors), (xxviii) insulin, (xxix) ferrotransferrin, (xxx) apotransferrin, (xxxi) lipopolysaccharide, (xxxii) an enzyme, (xxxiii) an antibody, and (xxxiv) biotin
• Particularly preferred ligand molecules for use according to the invention include without limitation (i) biotin, (ii) an antibody, (iii) an enzyme, (iv) lipopolysaccharide, (v) apotransferrin, (vi) ferrotransferrin, (vii) insulin, (viii) cytokines (growth factors, interleukins or colony-stimulating factors),
• cytokine e.g., growth factor, interleukin or colony-stimulating factor
• an insulin receptor e.g., insulin receptor, a transferrin receptor, (xxx) Fe +++ , (xxxi) polymyxin B or endotoxin- neutralizing protein (ENP), (xxxii) an enzyme-specific substrate, (xxxiii) protein A, protein G, a cell-surface Fc receptor or an antibody-specific antigen, and (xxxiv) avidin and streptavidin
• the invention thus relates to a method for making a nucleic acid molecule comprising (a) mixing a polypeptide having polymerase and/or reverse transc ⁇ ptase activity with a nucleic acid template and a primer-adapter of the invention, and
• the primer-adapter may be located at or near the 3' terminus, while if a reverse transc ⁇ ptase is used the primer-adapter may be located at or near the 5' terminus of the synthesized nucleic acid molecule
• the first nucleic acid molecule may be used as a template to make a second nucleic acid molecule complementary to all or a portion of the first nucleic acid molecule If a primer- adapter is used in this synthesis, a double-stranded nucleic acid molecule is produced which comprises a primer-adapter at or near each terminus, although on different strands of the molecule However, the primer-adapter may be omitted from this second synthesis thereby providing for a double-stranded nucleic acid molecule having a primer-adapter at one terminus
• primer-adapters of the invention may be used in methods for amplifying a nucleic acid molecule Such methods comprise
• Such amplification methods may specifically comprise (a) contacting a double-stranded nucleic acid molecule to be amplified with a polypeptide having polymerase and/or reverse transcriptase activity, a first primer-adapter complementary to a portion of the first strand of the double- stranded molecule and a second primer-adapter complementary to a portion of the second strand of the double-stranded molecule,
• the first primer-adapter or the second primer- adapter may be replaced with any oligonucleotide primer to prime synthesis of a nucleic acid molecule
• RNA e.g. , mRNA or polyA+ RNA
• This preferred method comprises mixing the RNA template with one or more polypeptides having reverse transcriptase activity and a primer and incubating the mixture under conditions sufficient to make a DNA (e.g., a cDNA) molecule complementary to all or a portion of the RNA template
• the synthesized DNA molecule may then be used as a template for additional DNA synthesis or DNA amplification
• a cDNA library may be produced when using a population of RNA molecules (for example, RNA isolated from a cell or tissue)
• the method may specifically comprise
• the invention may specifically comprise
• the sample containing the desired molecules is a population of double-stranded or single-stranded cDNA molecules
• the invention relates to a method of isolating one or more desired nucleic acid molecules comprising (a) obtaining a sample containing a population of cDNA molecules which contain (or are thought to contain) one or more desired cDNA molecules, (b) contacting the sample with one or more target-specific primer- adapters capable of specifically binding to one or more of the desired cDNA molecules, and (c) isolating the desired cDNA molecules from the sample
• the target-specific primer-adapters may be used in selection of a specific cDNA molecule after the cDNA molecule is synthesized from the RNA template (binding to the RNA/cDNA double-stranded molecule or binding to the single-stranded cDNA molecule after removing the RNA strand)
• the target-specific primer-adapters may be used to bind the double-stranded cDNA molecule
• Such target-specific primer-adapters may also be used in accordance with the invention to select one or more desired molecules from a population of amplified nucleic acid molecules
• the invention is also directed to vectors, including expression vectors, comprising the cDNA molecules or nucleic acid molecules produced in accordance with the invention, and to host cells comprising these cDNA molecules, nucleic acid molecules or vectors.
• the invention also provides methods for producing a recombinant polypeptide comprising culturing these host cells under conditions favoring the expression of a recombinant polypeptide and isolating the polypeptide, and provides recombinant polypeptides produced according to these methods.
• kits for the production of a nucleic acid molecule or a cDNA molecule comprising a carrier means such as a box, carton, or the like being compartmentalized to receive in close confinement therein one or more containers, such as tubes, vials, bottles, ampules and the like, wherein a first container comprises a primer-adapter molecule comprising one or more ligand molecules, preferably biotin, and which comprises one or more cleavage sites, preferably one or more restriction endonuclease cleavage sites.
• additional containers which may contain one or more polypeptides having reverse transcriptase activity and/or polymerase activity.
• kits comprising additional containers which may contain a solid support having one or more haptens capable of specifically binding the ligand or ligands of the primer-adapters of the invention.
• additional containers which may contain one or more endonucleases which recognize and cleave the cleavage sites in the primer- adapters of the invention.
• Figure 1 is a depiction of the production and isolation of a double- stranded cDNA molecule and its ligation into a plasmid vector (pCMVSPORT), according to the methods of the present invention
• B denotes biotin molecules (and thus sites of biotinylation of the cDNA molecule)
• RE denotes location of restriction endonuclease cleavage sites used to facilitate removal of the cDNA from the solid phase support following isolation
• the present invention is particularly suited for the rapid production and isolation of cDNA libraries from small amounts of poly A+ RNA or mRNA in a high-throughout manner
• a population of single-stranded poly A+ RNA or mRNA is hybridized in solution with a ligand- coupled primer adapter (non-specific or gene-specific).
• primer-adapter refers to a nucleic acid molecule which is capable of specifically binding (e.g., hybridizing) to a template nucleic acid molecule (e.g., a mRNA or polyA+ RNA molecule).
• the primer-adapter allows priming of the transcription, reverse transcription, polymerization or elongation of a nucleic acid molecule complementary to all or a portion of the template nucleic acid molecule
• the first and second strand cDNA reactions are preferably performed in one tube, introducing the ligand at or near the 3' end of the double-stranded cDNA produced
• the ligand-coupled cDNA may then be isolated by binding to a solid support coupled with a hapten to which the cDNA will bind through ligand-hapten interactions, thereby allowing the concentration of the cDNA and exchange of the buffer without organic extraction and precipitation
• the bound cDNA is released from the solid phase support by restriction enzyme digestion
• This asymmetric cDNA is then cloned directionally into a vector that contains the appropriate termini (one terminus matches the restriction site used to release the cDNA and the other terminus is blunt ended) Subsequent or prior to clon
• genes or gene fragments may be selectively isolated using target- specific primer-adapters of the invention
• the methods of the invention eliminate the need for DNA adapters and cDNA fractionation (normally a necessary step to remove excess unligated adapters)
• the invention thus facilitates rapid production and isolation of larger amounts of cDNA and the construction of cDNA libraries from nanogram amounts of poly A+ RNA or mRNA without the need for PCR amplification.
• the invention also provides a simple selection technique which allows isolation of desired genes or gene fragments from the constructed cDNA library
• nucleic acid molecules and in particular cDNA molecules may be prepared from a variety of nucleic acid template molecules
• Preferred nucleic acid molecules for use in the present invention include single-stranded or double-stranded RNA More preferred nucleic acid molecules include polyadenylated RNA (polyA+ RNA), messenger
• RNA mRNA
• tRNA transfer RNA
• rRNA ribosomal RNA
• nucleic acid template molecules that are used to prepare nucleic acid or cDNA molecules according to the methods of the present invention may be prepared synthetically according to standard organic chemical synthesis methods that will be familiar to one of ordinary skill More preferably, the nucleic acid template molecules may be obtained from natural sources, such as a variety of cells, tissues, organs or organisms Cells that may be used as sources of nucleic acid molecules may be prokaryotic (bacterial cells, including those of species of the genera Escherichia, Bacillus, Serratia, Salmonella, Staphylococcus, Streptococcus, Clostridium, Chlamydia, Neisseria, Treponema, Mycoplasma, Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia, Agrohacterium, Rhizohium, and Streptomyces) or eukaryotic (including fungi
• insects especially Drosophila spp. cells
• nematodes particularly Caenorhahditis elegans cells
• mammals particularly human cells
• Mammalian somatic cells that may be used as sources of nucleic acids include blood cells (reticulocytes and leukocytes), endothelial cells, epithelial cells, neuronal cells (from the central or peripheral nervous systems), muscle cells (including myocytes and myoblasts from skeletal, smooth or cardiac muscle), connective tissue cells (including fibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes and osteoblasts) and other stromal cells (e.g., macrophages, dendritic cells, Schwann cells).
• blood cells reticulocytes and leukocytes
• endothelial cells epithelial cells
• neuronal cells from the central or peripheral nervous systems
• muscle cells including myocytes and myoblasts from skeletal, smooth or cardiac muscle
• connective tissue cells including fibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes and osteoblasts
• stromal cells
• spermatocytes and oocytes may also be used as sources of nucleic acids for use in the invention, as may the progenitors, precursors and stem cells that give rise to the above somatic and germ cells.
• nucleic acid sources are mammalian tissues or organs such as those derived from brain, kidney, liver, pancreas, blood, bone marrow, muscle, nervous, skin, genitourinary, circulatory, lymphoid, gastrointestinal and connective tissue sources, as well as those derived from a mammalian (including human) embryo or fetus.
• prokaryotic or eukaryotic cells, tissues and organs may be normal, diseased, transformed, established, progenitors, precursors, fetal or embryonic Diseased cells may, for example, include those involved in infectious diseases (caused by bacteria, fungi or yeast, viruses (including AIDS) or parasites), in genetic or biochemical pathologies (e.g., cystic fibrosis, hemophilia, Alzheimer's disease, muscular dystrophy or multiple sclerosis) or in cancerous processes.
• infectious diseases caused by bacteria, fungi or yeast, viruses (including AIDS) or parasites
• genetic or biochemical pathologies e.g., cystic fibrosis, hemophilia, Alzheimer's disease, muscular dystrophy or multiple sclerosis
• Transformed or established animal cell lines may include, for example, COS cells, CHO cells, VERO cells, BHK cells, HeLa cells, HepG2 cells, K562 cells, F9 cells and the like
• Other cells, cell lines, tissues, organs and organisms suitable as sources of nucleic acids for use in the present invention will be apparent to one of ordinary skill in the art
• nucleic acid molecules such as mRNA
• nucleic acid molecules may be isolated therefrom by methods that are well-known in the art (See, e.g., Maniatis, T , et al., Cell 15 687-701 (1978), Okayama, H , and Berg, P , Mol. Cell. Bwl. 2 161-170 (1982), Gubler, U , and Hoffman, B , Gene 25 263-269 (1983))
• the invention provides an improvement in isolating mRNA and/or polyA+ RNA from samples
• primer-adapters of the invention which specifically recognize and bind polyA+ RNA or mRNA, allows for such selection
• the primer-adapter recognizes and hybridizes to the polyA tail of the mRNA or polyA+ RNA
• primer-adapters may include an primer-adapters comprising oligo(dT)
• use of the ligand portion of the primer-adapter allows isolation of the desired RNA molecule
• the polyA+ RNA or mRNA molecules thus isolated may then be used to prepare cDNA molecules and cDNA libraries using the methods of the present invention
• nucleic acid molecules and in particular cDNA molecules or cDNA libraries comprising one or more ligand molecules are produced by mixing a nucleic acid template obtained as described above, which is preferably a mRNA molecule or a polyA+ RNA molecule, with one or more polypeptides having polymerase activity and/or reverse transcriptase activity and with a one or more primer-adapters of the invention Under conditions favoring the reverse transcription and/or polymerization of the input nucleic acid molecule, synthesis of a nucleic acid molecule complementary to all or a portion of the template is accomplished Preferred polypeptides (e.g.
• enzymes having reverse transcriptase and/or polymerase activity to be used in the present invention include, but are not limited to, Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase, Rous Sarcoma Virus (RSV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Rous Associated Virus (RAV) reverse transcriptase, Myeloblastosis Associated Virus (MAV) reverse transcriptase, Human Immunodeficiency Virus (HIV) reverse transcriptase, retroviral reverse transcriptase, retrotransposon reverse transcriptase, hepatitis B reverse transcriptase, cauliflower mosaic virus reverse transcriptase, bacterial reverse transcriptase, Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotoga maritine
• Particularly preferred for use in the invention are the variants of these enzymes that are substantially reduced in RNase H activity.
• an enzyme “substantially reduced in RNase H activity” is meant that the enzyme has less than about 20%, more preferably less than about 15%, 10% or 5%, and most preferably less than about 2%, of the RNase H activity of a wildtype or "RNase H + " enzyme such as wildtype M-MLV or AMV reverse transcriptases.
• the RNase H activity of any enzyme may be determined by a variety of assays, such as those described, for example, in U.S. Patent No. 5,244,797, in Kotewicz, M.L., et al, Nucl. Acids Res.
• Suitable ligands for this purpose include, but are not limited to (i) biotin, (ii) an antibody, (iii) an enzyme, (iv) lipopolysaccharide, (v) apotransferrin, (vi) ferrotransferrin, (vii) insulin, (viii) cytokines (growth factors, interleukins or colony-stimulating factors), (ix) gpl20, (x) ⁇ -actin, (xi) LFA-1, (xii) Mac-1, (xiii) glycophorin, (xiv) laminin, (xv) collagen, (xvi) fibronect
• cytokine e.g., growth factor, interleukin or colony-stimulating factor
• an insulin receptor e.g., insulin receptor, a transferrin receptor, (xxx) Fe +++ , (xxxi) polymyxin B or endotoxin-neutralizing protein (ENP), (xxxii) an enzyme-specific substrate, (xxxiii) protein A, protein G, a cell-surface Fc receptor or an antibody-specific antigen, and (xxxiv) avidin and streptavidin
• the ligand-coupled primer-adapter nucleic acid molecules in which one or more ligand molecules are attached (preferably covalently) to one or more nucleotides of the primer-adapter molecule (see, for example, Figure 1), may be produced using conventional organic synthesis methods that are familiar to one of ordinary
• a primer-adapter molecule comprising one or more, two or more, three or more or four or more ligand molecules, most preferably biotin molecules, is prepared
• the primer-adapter molecule also preferably comprises one or more endonuclease cleavage sites, preferably restriction endonuclease cleavage sites. These sites facilitate the release of the newly synthesized nucleic acid molecule comprising the primer-adapter from the hapten-coupled solid support.
• endonucleases which can be used in accordance with the invention include, but are not limited to, Genell.
• restriction endonucleases which can be used in accordance with the invention include, but are not limited to, Alu ⁇ , Eco41 III, EcoRN, Fspl, Hpal, Mscl, Nrul, Pvull, Rsal, Seal, Smal, Sspl, Stul, Tha , Aval, Bam ⁇ l, Banll, Bg/ll, CM, Eco ⁇ , Hindlll, Hpall, Kpnl, Msel, Ncol, Ndel, Notl, Pstl, Pvul, SacllSstl, Sail, Xbal, Xhol and l-Ceul.
• restriction endonuclease sites engineered into the primer-adapter molecule are preferably chosen to result in either blunt ends or sticky ends.
• blunt-end restriction enzymes the recognition sites for which may be engineered into the primer-adapter molecules of the invention, include without limitation AM, Eco41 III, EcoRV, Fspl, Hpal, Mscl, Nrul, Pvull, Rsal, Seal, Smal, Sspl, Stul and Thai.
• sticky-end restriction enzymes examples include without limitations, val, Bam l, Banll, Bgl ⁇ l, CM, EcoRI, Hindlll, Hpall, Kpnl, Msel, Ncol, Ndel, Notl, Pstl, Pvul, SacllSstl, Sail, Xba, Xhol and I-Cewl.
• the primer-adapter molecule is engineered to contain a site recognized by rare cutting restriction endonucleases, for example, those recognizing 8 or more bases (e.g. , a 8- basepair cutter, etc.).
• restriction sites may include a Notl restriction site, a l-Ceul restriction site, a ⁇ l-Pspl restriction site, an l-Ppol restriction site, a PI- 7/1 restriction site and a PI-Ecel restriction site.
• the above-mentioned restriction enzymes, and others that may be equivalently used in the methods of the present invention, are available commercially, for example from Life Technologies, Inc. (Rockville, MD). See also Roberts, R.J., Nucl. Acids Res. 77(Suppl.):r347-r387 (1989), for other examples of restriction enzymes and their cleavage sites.
• the primer-adapter molecule is used to produce nucleic acid molecules from the input nucleic acid using any of a number of well-known techniques
• Such synthetic techniques involve hybridization of the primer-adapter to the nucleic acid template and extending the primer-adapter to make a nucleic acid molecule complementary to all or a portion of the template
• Such synthesis is accomplished in the presence of nucleotides (e g , deoxy ⁇ bonucleoside t ⁇ phosphates (dNTPs), dideoxy ⁇ bonucleoside t ⁇ phosphates (ddNTPs) or derivatives thereof) and one or more polypeptides having polymerase and/or reverse transcriptase activity
• the primer-adapters of the invention may be used in any nucleic acid synthesis reaction including cDNA synthesis, nucleic acid amplification and nucleic acid sequencing, using well- known techniques For synthesis of cDNA, the primer-adapter molecules of the invention may be used in conjunction with methods of cDNA
• the primer adapter molecules of the invention may be used in single-tube synthesis of cDNA molecules according to the invention
• the input nucleic acid molecule preferably a mRNA or polyA+ RNA molecule
• the primer-adapter molecule of the invention is hybridized in solution with the primer-adapter molecule of the invention, and the hybridized complex is contacted with a polypeptide (e g , an enzyme) having reverse transcriptase activity (which is preferably any of those described above) in the presence of dNTPs and cofactors needed for cDNA synthesis
• the second cDNA strand may then be synthesized in the same reaction vessel by a modified Gubler-Hoffman reaction (D'Aless
• nucleic acid molecules or libraries comprising one or more primer-adapters
• nucleic acid molecules or libraries may then be rapidly isolated from solution by binding the nucleic acid molecules to a solid support comprising one or more hapten molecules that will bind the gands
• solid support comprising one or more hapten molecules that will bind the gands
• any solid support to which a gand- specific hapten molecule can be bound may be used
• solid phase supports include, but are not limited to, nitrocellulose, diazocellulose, glass, polystyrene, polyvinylchlo ⁇ de, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, beads and microtitre plates
• beads made of glass, latex or a magnetic material and particularly preferred are magnetic, paramagnetic or superparamagnetic beads Linkage of the hapten molecule to the solid support can be accomplished by any method of hapten
• a biotin-binding hapten such as avidin or streptavidin may be linked to the solid support
• the solid support used is avidin- or streptavidin-coupled magnetic, paramagnetic or superparamagnetic beads which are commercially available, for example, from Dynal A S (Oslo, Norway) or from Sigma (St Louis, Missouri)
• hapten will depend upon the choice of ligand used in the production of the primer-adapter molecule; appropriate haptens for use in the methods of the invention will thus be familiar to one of ordinary skill in the art
• the solution comprising the nucleic acid molecules which comprise the primer-adapters of the invention is contacted with the hapten-coupled solid support under conditions favoring binding of the ligand by the hapten
• these conditions include incubation in a buffered salt solution, preferably a TRIS-, phosphate-, HEPES- or carbonate-buffered sodium chloride solution, more preferably a TRIS-buffered sodium chloride solution, still more preferably a solution comprising about 10-100 mM TRIS-HCI and about 300-2000 mM NaCI, and most preferably a solution comprising about 10 mM TRIS-HCI and about 1 M NaCl, at a pH of about 6-9, more preferably a pH of about 7-8, still more preferably a pH of about 7 2-7 6, and most preferably a pH of about 7 5
• Incubation is preferably conducted at 0°C to about 25 °C, and most preferably at about 25
• nucleic acid molecules may be bound to the solid phase support, unwanted or contaminant materials (such as buffers and enzymes from first and second strand synthesis reactions, untransc ⁇ bed input RNA molecules, etc ) may be eliminated by simply removing them in the supernatants
• the contaminants may be removed by gently aspirating and discarding the supernatants
• avidin- or streptavidin-coupled magnetic, paramagnetic or superparamagnetic beads are used as the solid support
• the nucleic acid (e.g., cDNA)-containing beads are segregated from the supernatants using a magnet (such as a Magna-Sep Magnetic Particle Separator, Life Technologies, Inc ) and the supernatants are withdrawn using a pipette Prior to their release from the solid support, the immobilized nucleic acid molecules are withdrawn using a pipette Prior to their release from the solid support, the immobilized nucleic acid molecules are
• the nucleic acid (e.g., cDNA) molecules may be released from the solid support by contacting the support with an endonuclease, which may be a restriction endonuclease, that specifically recognizes the sequence engineered into the primer-adapter molecule as described above, under conditions favoring the cleavage of the recognition sequence
• an endonuclease which may be a restriction endonuclease, that specifically recognizes the sequence engineered into the primer-adapter molecule as described above, under conditions favoring the cleavage of the recognition sequence
• an endonuclease which may be a restriction endonuclease, that specifically recognizes the sequence engineered into the primer-adapter molecule as described above, under conditions favoring the cleavage of the recognition sequence
• an endonuclease which may be a restriction endonuclease, that specifically recognizes the sequence engineered into the primer-adapter molecule as described above, under conditions favoring the cleavage of the
• cD ⁇ A or cD ⁇ A libraries from the solid support include incubation at about 20 °C to about 40 °C, preferably at about 25 °C to about 39°C, more preferably about 30°C to about 37 °C, and most preferably about 37°C, for about 30-180 minutes, preferably about 60-150 minutes, and most preferably about 120 minutes
• the nucleic acid molecules e.g., cD ⁇ A molecules or cD ⁇ A libraries
• Kits The present invention also provides kits for use in production and isolation of nucleic acid molecules (e.g., cD ⁇ A molecules or libraries)
• Kits according to this aspect of the invention comprise a carrier means, such as a box, carton, tube or the like, having in close confinement therein one or more containers, such as vials, tubes, ampules, bottles and the like, wherein a first container contains one or more primer-adapter nucleic acid molecules, which are preferably biotinylated primer-adapter nucleic acid molecules
• the kits of the invention may further comprise one or more additional containers containing a hapten- coupled solid support, which may be any of the above-described solid supports and which is most preferably avidin- or streptavidin-coupled magnetic, paramagnetic or superparamagnetic beads
• the kits of the invention may further comprise one or more additional containers containing, for example, one or more nucleotides (e.g., dNTPs, ddNTPs or derivatives
• nucleotides or derivatives thereof may include, but are not limited to, dUTP, dATP, dTTP, dCTP, dGTP, dITP, 7-deaza-dGTP, ⁇ -thio-dATP, ⁇ -thio-dTTP, -thio-dGTP, ⁇ -thio-dCTP, ddUTP, ddATP, ddTTP, ddCTP, ddGTP, ddlTP, 7- deaza-ddGTP, ⁇ -thio-ddATP, ⁇ -thio-ddTTP, ⁇ -thio-ddGTP, ⁇ -thio-ddCTP or derivatives thereof, all of which are available commercially from sources including Life Technologies, Inc (Rockville, Maryland), New England BioLabs (Beverly, Massachusetts) and Sigma Chemical Company (Saint Louis
• kits may comprise one or more additional containers containing one or more endonucleases or restriction enzymes used for release of the nucleic acid molecules (e.g., cDNA molecules or cDNA libraries) from the solid support
• the kits encompassed by this aspect of the present invention may further comprise additional reagents (e.g., suitable buffers) and compounds necessary for carrying out nucleic acid reverse transcription and/or polymerization protocols
• the present invention can be used in a variety of applications requiring rapid production and isolation of nucleic acid molecules
• the invention is particularly suited for isolation of mRNA or polyA+ RNA molecules, for isolation of desired nucleic acid molecules from a population of nucleic acid molecules, and for production of nucleic acid molecules (particularly full-length cDNA molecules from small amounts of mRNA)
• the invention is also directed to methods for the amplification of a nucleic acid molecule, and to nucleic acid molecules amplified by to these methods
• a nucleic acid molecule may be amplified (/ e , additional copies of the nucleic acid molecule prepared) by amplifying the nucleic acid molecule (e g , a cDNA molecules) of the invention according to any amplification method that is known in the art
• Particularly preferred amplification methods according to this aspect of the invention include PCR (U S Patent Nos 4,683,195 and 4,683,202), Strand Displacement Amplification (SDA, U S Patent No 5,45
• the invention is also directed to methods that may be used to prepare recombinant vectors which comprise the nucleic acid molecules or amplified nucleic acid molecules of the present invention, to host cells which comprise these recombinant vectors, to methods for the production of a recombinant polypeptide using these vectors and host cells, and to recombinant polypeptides produced using these methods
• Recombinant vectors may be produced according to this aspect of the invention by inserting, using methods that are well-known in the art, one or more of the nucleic acid molecules or amplified nucleic acid molecules prepared according to the present methods into a vector (see Figure 1)
• the vector used in this aspect of the invention may be, for example, a phage or a plasmid, and is preferably a plasmid
• Appropriate tr ⁇ ns-actmg factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host
• the vectors are expression vectors that provide for specific expression of the cDNA molecule or nucleic acid molecule of the invention, which vectors may be inducible and/or cell type-specific. Particularly preferred among such vectors are those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
• Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids or bacteriophages, and vectors derived from combinations thereof, such as cosmids and phagemids, and will preferably include at least one selectable marker such as a tetracycline or ampicillin resistance gene for culturing in a bacterial host cell.
• the nucleic acid molecules (e -g-, cDNA molecules) or amplified nucleic acid molecules of the invention should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters. Other suitable promoters will be known to the skilled artisan.
• vectors preferred for use in the present invention include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors,
• Bluescript vectors pNH8A, pNHl ⁇ a, pNH18A, ⁇ NH46A, available from Stratagene; pcDNA3 available from Invitrogen; pGEX, pTrxfus, pTrc99a, pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia; and pSPORTl, pSPORT2 and pSV'SPORTl, available from Life Technologies, Inc.
• Other suitable vectors will be readily apparent to the skilled artisan.
• Representative host cells that may be used according to the invention include, but are not limited to, bacterial cells, yeast cells, plant cells and animal cells.
• Preferred bacterial host cells include Escherichia spp. cells (particularly E. coli cells and most particularly E. coli strains DH10B and Stbl2), Bacillus spp. cells (particularly B. subti/is and B. megaterium cells), Streptomyces spp. cells,
• Preferred animal host cells include insect cells (most particularly Spodopterafrugiperda Sf9 and Sf21 cells and Trichoplusa High-Five cells) and mammalian cells (most particularly CHO, COS, VERO, BHK and human cells) These and other suitable host cells are available commercially, for example from Life Technologies, Inc , American Type Culture Collection and Invitrogen
• a recombinant polypeptide may be produced by culturing any of the above recombinant host cells under conditions favoring production of a polypeptide therefrom, and isolation of the polypeptide
• Methods for culturing recombinant host cells, and for production and isolation of polypeptides therefrom, are well-known to one of ordinary skill in the art
• the methods of the invention may be used to generate a gene-specific cDNA library from a complex population of poly A+ RNA
• the methods of the invention in combination with polymorphism analysis methods such as AFLP, also facilitate rapid and direct identification of transcriptional differences between two different DNA populations
• the primer- adapter used in the invention can be designed to contain a regulatory sequence, such as a promoter, enhancer or other regulatory region
• a promoter for T7 or SP6 RNA polymerase may be engineered into the primer- adapter, thereby enabling the production of additional copies of the original mRNA for use in amplification or subtraction
• the methods of the invention can be used to isolate poly A+ RNA from total RNA, such as from cells, tissues, organs or organisms, or to generate a cDNA library directly from total RNA In the latter application, the invention is particularly useful when the mRNA of interest represents only a minute fraction of the total RNA, by the invention, this low-level mRNA may be rapidly and efficiently isolated from
• the primer-adapter used in cDNA synthesis contained four biotin (B) residues B-GACT (-B) AGT (-B)T(-B)CTAGATCGCGAGCGGCCGCCC(T 15 ) (SEQ ID NO 1)
• streptavidin paramagnetic beads were prepared Briefly, paramagnetic beads (Life Technologies, Inc.) were resuspended and 150 ⁇ l of bead suspension was placed into a microcentrifuge tube for each reaction The tubes were the placed into a Magna-Sep Magnetic particle Separator (magnet) for two minutes, and supernatant removed by aspiration The beads were then washed by adding 100 ⁇ l of TE buffer (10 mM TRIS-HCI (pH 7 5), 1 mM EDTA) to each tube, resuspending beads, and removing supernatant after two minutes as described above Following washing, the beads were resuspended in 160 ⁇ l of Binding Buffer (10 mM Tris-HCI (pH 7 5), 1
• the tubes were placed on ice and the reaction terminated by the addition to each tube of 10 ⁇ l of 0 5 M EDTA
• the biotinylated cDNA molecules were then isolated by contacting the solution with the streptavidin- coupled paramagnetic beads Briefly, 160 ⁇ l of the beads prepared as described above were added to the cDNA reaction mixture tubes, and the tubes gently mixed and incubated for 60 minutes at room temperature.
• Tubes were then inserted into the magnet for two minutes, after which supernatants were removed and discarded The beads were then washed by gentle resuspension with 100 ⁇ l of wash buffer (10 mM TRIS-HCI (pH 7 5), 1 mMEDTA, 500 mM NaCI), followed by re-insertion into the magnet After two minutes, supernatants were removed and discarded and the washing step repeated Following the second wash, beads were resuspended in 100 ⁇ l of wash buffer, transferred into fresh tubes, and washed twice as above (with five minute exposures to the magnet).
• wash buffer 10 mM TRIS-HCI (pH 7 5), 1 mMEDTA, 500 mM NaCI
• a vector e.g., pCMVSPORT
• this ligation introduced into E. coli by transformation as described in the SUPERSCRIPT Plasmid System manual (Life Technologies, Inc ), except the cloning vector was pre-digested with Notl and Sm ⁇ l
• 50 ng of vector was ligated to the cD ⁇ A in a 1 5 ml microcentrifuge tube with 4 ⁇ l of 5X T4 D ⁇ A ligase buffer (250 mM TRIS-HCI (pH 7 6), 50 mM MgCl 2 , 5 mM ATP, 5 mM DTT, 25% (w/v) PEG-8000) and 1 ⁇ l of T4 ligase (1 unit) at 4 °C for 16 hours
• cDNA was produced as described above and the amounts produced were compared to those obtained using an alternative commercially available system (SUPERSCRIPT Plasmid System, Life Technologies, Inc , Rockville, Maryland) Briefly, after introducing the pCMV»SPORT-cDNA ligations into MAX EFFICIENCY DH5 ⁇ TM and ELECTROMAX® DHIOB cells, the cells were plated onto ampicillin-containing plates to determine transformation efficiencies
• the cDNA inserts were sized by using the SP6 and T7 promoter primers and 40 cycles of PCR on 48 randomly chosen colonies for each experiment
• Table 1 shows a comparison of the cDNA yields obtained by the methods of the present invention to those obtained using the Superscript Plasmid System
• the present invention provides methods for the rapid and efficient production of full-length cDNA molecules without the use of time-consuming and yield-reducing cDNA size fractionation steps

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• 1998
  • 1998-05-12 EP EP98922202A patent/EP0981535A4/de not_active Withdrawn
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