US20020165175A1 - Fast and enzymeless cloning of nucleic acid fragments - Google Patents

Fast and enzymeless cloning of nucleic acid fragments Download PDF

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Publication number
US20020165175A1
US20020165175A1 US09/836,436 US83643601A US2002165175A1 US 20020165175 A1 US20020165175 A1 US 20020165175A1 US 83643601 A US83643601 A US 83643601A US 2002165175 A1 US2002165175 A1 US 2002165175A1
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Prior art keywords
nucleic acid
vector
acid fragment
resistance gene
adapter sequences
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Xiaowu Liang
Andy Teng
Shizhong Chen
Dongyuan Xia
Philip Felgner
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Gene Therapy Systems
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Gene Therapy Systems
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Priority to US09/836,436 priority Critical patent/US20020165175A1/en
Assigned to GENE THERAPY SYSTEMS, INC. reassignment GENE THERAPY SYSTEMS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHEN, SHIZHONG, TENG, ANDY, FELGNER, PHILIP L., LIANG, XIAOWU, XIA, DONGYUAN
Priority to PCT/US2002/012334 priority patent/WO2002083871A2/fr
Priority to US10/125,789 priority patent/US6936470B2/en
Priority to AU2002256280A priority patent/AU2002256280A1/en
Publication of US20020165175A1 publication Critical patent/US20020165175A1/en
Priority to US11/045,686 priority patent/US20050233422A1/en
Abandoned legal-status Critical Current

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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

  • the present invention relates to methods, systems and kits for fast and enzymeless cloning of nucleic acid fragments into vectors and for forced cloning selection for successful transformation.
  • DNA fragments are isolated from cDNA libraries or chromosomes and subcloned into a vector utilizing various enzymes.
  • the DNA fragment-containing vector is introduced into a host cell according to various methods of transformation.
  • a selection marker is usually included in the vector to increase the probability that the host cell has the DNA fragment-containing vector.
  • the DNA fragment within the vector can then be replicated along with the host cell DNA.
  • the DNA fragment-containing vector then can be isolated and purified from the host cell and transfected into animal cells or tissues for functional analysis of the encoded gene product.
  • the present invention provides simple and rapid methods, systems and kits for cloning nucleic acid fragments.
  • the present invention relates to a method for cloning a nucleic acid fragment into a vector by flanking the fragment with first and second adapter sequences.
  • the fragment can be contacted with the vector having sequences homologous to the first and second adapter sequences under conditions such that the nucleic acid fragment is incorporated into the vector by homologous recombination in vivo in a host cell.
  • the nucleic acid fragment can be generated by polymerase chain reaction (PCR).
  • the first and second adapter sequences can be incorporated to the nucleic acid fragment by PCR.
  • the resulting nucleic acid fragment can be a transcriptionally active PCR fragment.
  • the first and second adapter sequences further can include a functional element.
  • the functional element can include a promoter, a terminator, a nucleic acid fragment encoding a selection marker gene, a nucleic acid fragment encoding a known protein, a fusion tag, a nucleic acid fragment encoding a portion of a selection marker gene, a nucleic acid fragment encoding a growth promoting protein, a nucleic acid fragment encoding a transcription factor, a nucleic acid fragment encoding an autofluorescent protein (e.g. GFP), and the like.
  • an autofluorescent protein e.g. GFP
  • the nucleic acid fragment can include an additional element.
  • the additional element can be, for example, an operably linked promoter, a termination sequence, an operon, a fusion tag, a signal peptide for intracellular or intercellular trafficking, a peptide, a protein, an antisense sequence, a ribozyme, a protein binding site, and the like.
  • the promoter can be, for example, a promoter from a plant or a plant pathogen, such as cauliflower mosaic virus, from a mammal or a mammalian pathogen, such as CMV, SV40, MMV, HIV, from a fungus, such as yeast (Gal 4 promoter), from a bacterium or a bacterial phage, for example, lambda, T3, T7, SP6 and the like.
  • the terminator sequence can be derived from a plant, a procaryotyic source or a eukaryotic source, such as SV40, bovine growth hormone, rabbit beta-globin i, and the like.
  • the operon can be, for example, lac operon, Tet/on operon, Tet/off operon, trp operon.
  • the fusion tag can include 6x or 8x his-tag, GST tag, fluorescent protein tag, Flag tag, HA tag, and the like.
  • the protein can include an enzyme, a receptor, a transcription factor, a lymphokine, a hormone, an antigen, and the like.
  • the vector can be, for example a plasmid, a cosmid, a bacterial artificial chromasome (BAC), and the like.
  • the plasmid can be CoE1, PR100, R2, pACYC, and the like.
  • the vector can also include a functional selection marker.
  • the functional selection marker can be, for example, a resistance gene for kanamycin, amplicillin, blasticidin, carbonicillin, tetracycline, chloramphenicol, and the like.
  • the vector further can include a dysfunctional selection marker that lacks a critical element, and wherein the critical element is supplied by said nucleic acid fragment upon successful homologous recombination.
  • the dysfunctional selection marker can be, for example, kenamycin resistance gene, kanamycin resistance gene, ampicillin resistance gene, blasticidin resistance gene, carbonicillin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, and the like. Further, the dysfunctional selection marker can be, for example, a reporter gene, such as the lacZ gene, and the like.
  • the vector can include a negative selection element detrimental to host cell growth.
  • the negative selection element can be disabled by said nucleic acid fragment upon successful homologous recombination.
  • the negative selection element can be inducible.
  • the negative selection element can be, for example, a mouse GATA-1 gene.
  • the vector can include a dysfunctional selection marker and a negative selection element.
  • the host cell can be a bacterium.
  • the bacterium can be capable of in vivo recombination. Examples of a bacterium include JC8679, TB1, DH5a, DH5 ⁇ , HB101, JM101, JM109, LE392, and the like.
  • the plasmid can be maintained in the host cell under the selection condition selecting for the functional selection marker.
  • the first and second adapter sequences can be at least 11 bp.
  • the first and second adapter sequences can be at least 40 bp.
  • the first and second adapter sequences can be at least 50 bp.
  • the first and second adapter sequences can be at least 60 bp.
  • the first and second adapter sequences can be greater than 60 bp.
  • the contacting can include transforming a host cell with the vector and the nucleic acid fragment.
  • the transformation can include electroporation, chemical transformation, and the like.
  • the host cell can be a bacterium bearing the vector.
  • the bacterium can be capable of in vivo recombination.
  • the bacterium can be, for example, JC8679, TB1, DH5 ⁇ , DH5, HB101, JM101, JM109, LE392, and the like.
  • the contacting of the vector and the nucleic acid fragment can include transforming the host cell bearing the vector with the nucleic acid fragment.
  • the vector can be a plasmid.
  • the plasmid can include a functional selection marker.
  • the functional selection marker can be, for example, a resistance gene for kanamycin, ampicillin, blasticidin, carbonicillin, tetracycline, chloramphenicol, and the like.
  • the plasmid can include a dysfunctional selection marker that lacks a critical element, and wherein the critical element is supplied by said nucleic acid fragment upon successful homologous recombination.
  • the dysfunctional selection marker can be, for example a resistance gene for kanamycin, kenamycin, ampicilin, blasticidin, carbonicillin, tetracycline, chloramphenicol, and the like.
  • the dysfunctional selection marker can be, for example, a reporter gene, such as the lacZ gene, and the like.
  • the vector can include a negative selection element detrimental to host cell growth, and the negative selection element can be disabled by the nucleic acid fragment upon successful homologous recombination.
  • the negative selection element can be inducible, for example.
  • the negative selection element can be, for example GATA-1 gene.
  • the vector can include a dysfunctional selection marker and a negative selection element.
  • One embodiment of the present invention relates to a method for selecting for successful transformation of a vector by a nucleic acid insert.
  • the method can provide a nucleic acid insert flanked by first and second adapter sequences that is adapted for recombining with homologous sequences in a vector.
  • the vector can have a dysfunctional selection marker lacking a critical element and the nucleic acid insert contains the critical element.
  • the nucleic acid insert can be contacted with the vector to effect recombination at homologous sites such that the critical element is supplied to the vector by the nucleic acid insert and the dysfunctional selection marker is restored to a functional one.
  • the successfully restored selection marker can be selected for based upon growth of a host containing the successfully recombined vector that allows the host to grow or be identified in a selective environment.
  • the recombining can be by homologous recombination.
  • a further embodiment of the present invention relates to a method for selecting for successful transformation of a vector by a nucleic acid insert.
  • the method can include providing a nucleic acid insert flanked by first and second adapter sequences that is adapted for recombining with homologous sequences in a vector.
  • the vector can include a negative selection element detrimental to cell growth.
  • the nucleic acid insert can be contacted with the nucleic acid insert to effect recombination at homologous sites such that the negative selection element is disabled.
  • Successful transformation can be selected for based on the absence of a functional negative selection element.
  • the negative selection element can be inducible, for example.
  • the selection step can include inducing the negative selection element.
  • the embodied method utilizing the negative selection element further can include the method for selecting for successful transformation of a vector by a nucleic acid insert wherein the vector includes a dysfunctional selection marker lacking a critical element and the nucleic acid insert contains the critical element, as discussed above.
  • the negative selection element can be disabled by insertion of a sequence encoding a selection marker.
  • Another embodiment of the present invention relates to a system for cloning a nucleic acid fragment into a vector without restriction enzyme, ligase, gyrase, single stranded DNA binding protein, or other DNA modifying enzymes.
  • the system can include a nucleic acid fragment flanked by first and second adapter sequences and a vector having sequences homologous to the first and second adapter sequences wherein the nucleic acid fragment is adapted to incorporate into the vector by homologous recombination.
  • the nucleic acid fragment flanked by the first and the second adapter sequences can be generated by PCR without the use of a restriction enzyme, a ligase, a gyrase, a single stranded DNA binding protein, or any other DNA modifying enzyme.
  • the nucleic acid fragment flanked by the first and the second adapter sequences can be a transcriptionally active PCR fragment.
  • a further embodiment relates to a system for cloning a nucleic acid fragment into a bacterium without the use of a restriction enzyme, a ligase, a gyrase, a single stranded DNA binding protein, or any other DNA modifying enzyme.
  • the system can include a nucleic acid fragment flanked by first and second adapter sequences and a bacterium bearing a vector, the vector having sequences homologous to the first and second adapter sequences, wherein the nucleic acid fragment is adapted to incorporate into the vector within the bacterium by homologous recombination.
  • the present invention relates to a kit for cloning a nucleic acid fragment into a vector.
  • the system can include reagents for amplification of the nucleic acid fragment, wherein the reagents upon amplification can provide for a nucleic acid fragment flanked by first and second adapter sequences, a vector, a competent cell, or a competent cell bearing the vector, and the like.
  • the competent cell can be ready to be transformed by electroporation, chemical transformation, and the like.
  • the competent cell or the competent cell bearing the vector can be a bacterium.
  • the bacterium can be capable of in vivo recombination.
  • FIG. 1 depicts an embodiment of the present invention related to fast and enzymeless cloning into a vector.
  • FIG. 2 illustrates one embodiment related to generating and cloning a nucleic acid fragment into a vector.
  • FIG. 3 depicts an exemplary receptor plasmid vector.
  • FIG. 4 illustrates one embodiment relating to selection of a successfully transformed host.
  • the present invention in at least certain embodiments, overcomes many of the above-described drawbacks of traditional cloning.
  • the present invention includes methods, systems and kits for fast and simple molecular cloning of a nucleic acid fragment directly into a vector without the use of enzymes, such as restriction endonuclease, DNA ligase or any other DNA modifying enzyme.
  • the present invention also includes simple and fast methods for selecting host cells containing the desired DNA fragment and vector.
  • the present invention generally provides methods, systems and kits for cloning a nucleic acid fragment into a vector by homologous recombination within a host cell.
  • the common sequences on both the 5′ and 3′ ends of the nucleic acid fragment are complimentary with terminal sequences in a linearized empty vector, and the fragment and linearized vector are introduced, by electroporation, for example, together into a host cell, they recombine resulting in a new expression vector with the fragment directionally inserted.
  • the host cell can include the linearized empty vector so that only the nucleic acid fragment is introduced into the host cell.
  • the vector can be circularized, and as used herein a vector can be either linearized or circular.
  • the host cell is converted into an expression vector through homologous recombination. In principle this approach can be applied generally as an alternative to conventional cloning methods.
  • One embodiment of the present invention includes a method for cloning a nucleic acid fragment flanked by first and second adapter sequences into a vector having homologous first and second adapter sequences.
  • the nucleic acid fragment incorporates into the vector by homologous recombination within a host cell.
  • a nucleic acid fragment 10 is flanked by a first adapter sequence 12 and a second adapter sequence 14 .
  • a vector 16 also has a first vector adapter sequence 18 and a second vector adapter sequence 20 , which sequences are respectively homologous to the first and second adapter sequences 12 , 14 of the nucleic acid fragment 10 .
  • the homologous first and second adapter sequences 12 , 14 can be at least 11 bp. In other embodiments the homologous first and second adapter sequences can be at least 15 or 20 bp. Further, in embodiments the homologous first and second adapter sequences can be at least 25 or 30 bp. The homologous first and second adapter sequences can be at least 40 bp. Also, the homologous first and second adapter sequences can be at least 50 bp. In preferred embodiments the homologous first and second adapter sequences are at least 60 bp. In more preferred embodiments the homologous first and second adapter sequences are at greater than 60 bp.
  • the nucleic acid fragment 10 can also include a coding region 26 for a sequence or gene of interest.
  • coding region refers generically to a region of the nucleic acid fragment 10 that can encode, for example, an operably linked promoter, a termination sequence, an operon, a fusion tag, a signal peptide for intracellular or intercellular trafficking, a peptide, a protein, an antisense sequence, a ribozyme, a protein binding site, and the like.
  • the coding region 26 can encode a protein.
  • the protein can include enzymes receptors, transcription factors, lymphokines, hormones, antigens.
  • the coding region 26 can include a gene encoding a product which is absent or present at reduced levels in an organism.
  • Nonlimiting examples of these gene products are the cystic fibrosis transmembrane regulator (CFTR), insulin, dystrophin, interleukin-2, interleukin-12, erythropoietin, gamma interferon, and granulocyte macrophage colony stimulating factor (GM-CSF).
  • CFTR cystic fibrosis transmembrane regulator
  • GM-CSF granulocyte macrophage colony stimulating factor
  • the nucleic acid fragment 10 having first and second adapter sequences 12 , 14 can be generated by methods well known to those of skill in the art. Referring to the embodiment of the invention depicted in FIG. 2, a gene of interest 26 with known 5′ and 3′ sequences undergoes PCR along with overlapping 5′ and 3′ priming oligonucleotides 30 , 32 .
  • the priming oligonucleotides can be obtained by methods known in the art, including manufacture by commercial suppliers.
  • a primary fragment 34 with adapter sequences is generated.
  • the adapter sequences flanking the gene of interest can be homologous to sequences on a vector or to sequences from other 5′ or 3′ fragments to be used in a subsequent PCR, as will be discussed more fully below.
  • the method depicted in FIG. 2 is more fully described in U.S. patent application Ser. No. 09/535,262, “Methods for Generating Transcriptionally Active DNA Fragments,” which is hereby incorporated by reference in
  • the nucleic acid fragment depicted in FIG. 1 can also include a functional element.
  • the first and second adapter sequences 12 , 14 can include the functional element.
  • FIG. 2 illustrates one method for generating a nucleic acid fragment with functional elements.
  • the primary fragment generated as discussed above, has flanking sequences homologous to sequences on a 5′ fragment 36 and a 3′ fragment 40 , respectively.
  • the 5′ and 3′ fragments 36 , 40 include functional elements 46 and 50 , as well as a first and a second adapter sequence homologous to sequences on the primary fragment 34 .
  • a 5′ primer 42 and 3′ primer 44 for PCR can also be included. All undergo PCR.
  • the resulting fragment 52 has a new 5′ element 54 and a new 3′ element 56 that include a functional element and terminal flanking sequences homologous to sequences on a vector.
  • the method is more fully described in U.S. patent application Ser. No. 09/535,262, “Methods for Generating Transcriptionally Active DNA Fragments,” Liang, et al, which is hereby incorporated by reference in its entirety.
  • “transcriptionally active PCR fragment” or “transcriptionally active DNA fragment” refers to a nucleic acid fragment having a promoter and terminator sequence included therewith such that the fragment can be transcribed within a host cell.
  • the resulting vectors are useful for a variety of different applications.
  • the functional element can be a promoter, a terminator, a nucleic acid fragment encoding a selection marker gene, a nucleic acid fragment encoding a known protein, such as a fusion tag, a nucleic acid fragment encoding a portion of a selection marker gene, a nucleic acid fragment encoding a growth promoting protein, a nucleic acid fragment encoding a transcription factor, a nucleic acid fragment encoding an autofluorescent protein (e.g. GFP), and the like.
  • GFP autofluorescent protein
  • Nucleic acid fragments flanked by adapter sequences suitable for the purposes of the present invention can be generated using the TAP ExpressTM system (Gene Therapy Systems, San Diego, Calif.).
  • the TAP ExpressTM uses nested PCR to append adapter sequences, which can include additional sequences such as a promoter and a terminator sequence, onto PCR fragments so that they become transcriptionally active and can be used directly in in vitro and in vivo transfection experiments.
  • the TAP ExpressTM system can be used to generate a large numbers of genes that can be conveniently amplified and introduced into functional assays in a single day, a task that is impractical or impossible using conventional cloning methodology.
  • promoter is a DNA sequence which extends upstream from the transcription initiation site and is involved in binding of RNA polymerase.
  • the promoter may contain several short ( ⁇ 10 base pair) sequence elements that bind transcription factors, generally dispersed over >200 base pairs.
  • a promoter that contains only elements recognized by general and upstream factors is usually transcribed in any cell type.
  • Such promoters may be responsible for expression of cellular genes that are constitutively expressed (sometimes called housekeeping genes).
  • tissue-specific promoters limited to particular cell types, such as the human metallothionein (MT) promoter that is upregulated by heavy metal ions and glucocorticoids.
  • MT human metallothionein
  • the promoter can be selected based upon consideration of the desired use for the nucleic acid fragment.
  • One skilled in the art easily can select an appropriate promoter according the uses of the nucleic acid fragment.
  • a promoter capable of promoting transcription in mammalian cells can be selected.
  • Other examples of a promoter includes a promoter from a plant or a plant pathogen, such as cauliflower mosaic virus, and the like.
  • the promoter can be from a mammal or a mammalian pathogen such as CMV, SV40, MMV, HIV, and the like.
  • the promoter can be from a fungus such as a yeast (Gal 4 promoter), and the like, while in other examples it can be from bacteria or bacterial phage, for example lambda, T3, T7, SP6, and the like.
  • terminator is a DNA sequence represented at the end of the transcript that causes RNA polymerase to terminate transcription. This occurs at a discrete site downstream of the mature 3′ end, which is generated by cleavage and polyadenylation.
  • the terminator sequence can be derived from a plant, a procaryotyic or a eukaryotic source, such as SV40, bovine growth hormone, rabbit beta-globin i, and the like.
  • operon is a controllable unit of transcription consisting of a number of structural genes transcribed together.
  • An operon can contain at least two distinct regions, the operator and the promoter. Examples of operons include the lac operon, Tet/on operon, Tet/off operon, trp operon, and the like.
  • fusion tag is used herein to refer generally to a nucleic acid sequence encoding a molecule used to quantify, capture, purify, visualize, etc., the expressed protein to which the fusion tag is fused or attached.
  • fusion tags include 6 ⁇ or 8 ⁇ his-tag, GST tag, fluorescent protein tag, Flag tag, HA tag, and the like.
  • the nucleic acid fragment 10 and the vector 16 are introduced together into a host cell 24 .
  • the nucleic acid fragment 10 incorporates into the vector 16 by in vivo homologous recombination.
  • the homologous sequence between the nucleic acid fragment and the vector can be recognized by the DNA recombination and repair mechanism (e.g., in E. coli ) and joined together.
  • the vector 16 can be a plasmid, a cosmid, a bacterial artificial chromasome (BAC), or the like.
  • a plasmid include CoE1, PR100, R2, pACYC, and the like.
  • FIG. 3 depicts one example of a plasmid that can be used in the present invention.
  • the plasmid can include a functional or intact selection marker for growth.
  • FIG. 3 illustrates a plasmid vector 60 that includes an intact selection marker 62 for growth.
  • Examples of a functional selection marker include a resistance gene for kanamycin, kenamycin, ampicillin, blasticidin, carbonicillin, tetracycline, chloramphenicol, and the like.
  • the vector can be maintained in the host cell under the selection condition selecting for the functional selection marker.
  • embodiments of the present invention include methods for selecting for successful transformation of a vector by a nucleic acid fragment.
  • the vector can include a dysfunctional selection marker that lacks a critical element.
  • the lacked critical element is supplied to the vector by the nucleic acid fragment.
  • the homologous recombination and repair mechanism is not yet well characterized, it is of a relatively low frequency, making the identification of the intended recombined vector difficult.
  • the inclusion of a critical element necessary for the viability of the host cell will facilitate selection of the intended vector, because only the correctly recombined vector can survive with the host, while a host only carrying either the insert or the vector alone cannot survive.
  • a successfully transformed host can also be selecting for by a negative selection method.
  • the vector can include a negative selection element.
  • a negative selection element can be a sequence that encodes a molecule that is detrimental to growth of the host cell, such as, for example, the mouse GATA-1 gene product which is toxic to some cells.
  • the toxic gene can be interrupted or replaced by a nucleic acid sequence that is correctly incorporated into the vector by homologous recombination. Only cells with a successfully incorporated “interrupting” sequence survive, because only those cells lack the toxic gene product.
  • the negative selection element can also encode for molecules that block cell metabolism or prevent efficient transcription, and the like. One of skill in the art can easily select other elements that will work with the present invention.
  • the host cell can be a bacterium.
  • the bacterium is capable of in vivo recombination.
  • Examples of a bacterium include JC8679, TB1, DH5 ⁇ , DH5, HB101, JM101, JM109, LE392, and the like.
  • the nucleic acid fragment and the vector can be introduced together into the host cell.
  • the vector first can be introduced into the cell followed by a later introduction of the nucleic acid fragment or simply the nucleic acid fragment can be introduced into the host cell in order to transform the cell.
  • the host cell can include a vector that replicates with the cell, thus obviating the need to introduce a vector into the host cell.
  • the nucleic acid fragment and/or vector can be introduced by electroporation, chemical transformation, and the like.
  • the nucleic acid fragment and the vector are introduced into an E. Coli cell by high efficiency electroporation.
  • the present invention also includes high efficiency electroporation-competent cells. These cells significantly facilitate the introduction of insert and Vector into the host cells, thus improving the efficiency of recombination, which is a bi-molecule reaction, that is exponentially dependent on the amount of substrate (the fragment and vector). Examples of these cells include JC8679, TB1, HB 101, and the like.
  • a cloning vector will include a selection marker, such as a resistance gene, so that only a host cell having a properly incorporated DNA insert and vector will grow in a selective medium.
  • the host cell may incorporate a vector having the resistance gene without the desired insert or with only a portion of the insert.
  • host cell colonies will have to be screened, potentially at a significant time, material and labor cost, in order to identify a colony having the proper vector and insert.
  • Embodiments of the present invention relate to methods for selecting for the successful transformation of a vector by a nucleic acid insert.
  • the vector 16 is prepared with a dysfunctional selection marker that lacks a critical element.
  • the nucleic acid fragment 10 can include the critical element.
  • critical element can refer to any sequence on the nucleic acid fragment 10 that, upon incorporation with the vector 16 , restores functionality to a selection marker.
  • the vector can have a dysfunctional antibiotic resistance gene.
  • the vector can be prepared having an interrupted antibiotic resistance gene.
  • the nucleic acid fragment is engineered to restore the functional antibiotic resistance gene upon incorporation into the vector by homologous recombination.
  • the host cell having the “restored” vector can then be plated in a selective growth media. Any host cell lacking the “restored” vector will be unable to grow in the selective media.
  • the vector can have a negative selection element that is detrimental to cell growth.
  • the negative selection element can be sequence that encodes a molecule that is toxic to the cell, a molecule that stops or prevents transcription, a molecule that is otherwise detrimental to growth of the host cell, and the like.
  • the negative selection element is disabled. Disabling the negative selection element allows the host cell to grow, thus only cells with proper insertion of the nucleic acid fragment into the vector will survive and be selected.
  • the negative selection element can be inducible.
  • the vector can have a functional suicide gene or other negative selection element.
  • the suicide gene can be replaced or disabled upon incorporation of the nucleic acid fragment into the vector by homologous recombination.
  • a vector 76 can be prepared having a negative selection element 80 , in this embodiment a mouse GATA-1 transcription factor gene.
  • the negative selection element can be inserted between the first adapter sequence 82 and the second adapter sequence 84 .
  • the first and second adapter sequences 82 , 84 have regions homologous to the ends of a nucleic acid fragment 68 that is to be cloned.
  • the nucleic acid fragment 68 can be generated by PCR or any other suitable method, as discussed herein.
  • the nucleic acid fragment 68 can encode some gene of interest 70 as discussed above.
  • the fragment 68 includes a first adapter sequence 72 and a second adapter sequence 74 , which are homologous to the first and second adapter sequences 82 , 84 on the vector 76 .
  • the first and second adapter sequences 72 , 74 as discussed above can also include additional elements, such as sequences encoding a promoter, a terminator, an operon, a fusion tag, and the like.
  • the negative selection element 80 in this case the GATA-1 gene, is under the control of TAC promoter inducible by IPTG, and its product is able to bind to the bacterial origin of replication, therefore resulting in a rapid arrest of cell growth.
  • the nucleic acid fragment 68 upon incorporation into the vector 76 by homologous recombination will replace the negative selection element 80 , thus enabling the host cell to grow in a selective media. Any host cells lacking the recombined vector will be unable to grow in the selective media.
  • the negative selection methods and systems can be combined with the other systems, methods, and kits, including for example, forced cloning.
  • the nucleic acid insert can encode a critical element, as described above, that restores function to a disabled selection marker, while at the same time disabling a negative selection element, such as a suicide gene.
  • the forced cloning methods, systems and kits can be used independently, in conjunction with the negative selection methods, systems and kits.
  • two nucleic acid fragments may be introduced into the host cell.
  • Another embodiment of the present invention relates to a system for cloning a nucleic acid fragment into a vector without restriction enzyme, ligase, gyrase, single stranded DNA binding protein, or other DNA modifying enzymes.
  • the system can include a nucleic acid fragment flanked by first and second adapter sequences, and a vector having sequences homologous to the first and second adapter sequences.
  • the nucleic acid fragment is adapted to incorporate into the vector by homologous recombination.
  • the nucleic acid fragment flanked by the first and the second adapter sequences can be generated by PCR without the use of a restriction enzyme, a ligase, a gyrase, a single stranded DNA binding protein, or any other DNA modifying enzyme as discussed above or according to any other method known in the art.
  • the nucleic acid fragment flanked by the first and the second adapter sequences can be a transcriptionally active PCR fragment.
  • One embodiment of the present invention relates to a system for cloning a nucleic acid fragment into a bacterium bearing a vector, without the use of a restriction enzyme, a ligase, a gyrase, a single stranded DNA binding protein, or any other DNA modifying enzyme.
  • the system can include a nucleic acid fragment flanked by first and second adapter sequences and a bacterium bearing a vector having sequences homologous to the first and second adapter sequences.
  • the nucleic acid fragment is adapted to incorporate into the vector within the bacterium by homologous recombination.
  • a further embodiment relates to a kit for cloning a nucleic acid fragment into a vector.
  • the kit can include reagents for amplification of the nucleic acid fragment.
  • Suitable reagents may include, for example, TAQ polymerase and/or PCR reagents such as adapter sequences capable of acting as primers for nested PCR and including regions of homology to a nucleic fragment of interest and regions added onto the ends of the nucleic acid fragment of interest upon successful amplification steps, as explained in more detail in U.S. application Ser. No. 09/535,262, discussed above.
  • the reagents upon amplification can provide for a nucleic acid fragment flanked by first and second adapter sequences, a vector, a competent cell, or a competent cell bearing the vector.
  • the competent cell can form a part of the kit and can be ready to be transformed by electroporation, chemical transformation, or any like method known in the art.
  • the competent cell or the competent cell bearing the vector is bacteria.
  • the bacteria can be capable of in vivo recombination.
  • PCR polymerase chain reaction
  • DNA template containing CAT coding region 200 ⁇ M dNTP and 1 unit Taq DNA polymerase.
  • PCR was performed as follows: denaturation at 94° C. for 30 seconds, annealing for 45 seconds at 55° C. and extension for 2 minutes at 72° C. for 25 cycles.
  • the PCR product was analyzed by electrophoresis in 1% agarose gel and purified using a commercial PCR cleaning kit.
  • a second PCR reaction was carried out using the product from the first PCR as template.
  • the reaction mix also contained 5 ng of DNA fragment (800 bp) comprising a modified promoter sequence from human cytomegalovirus (Gene Thearpy Systems, San Diego, Calif.), 5 ng of DNA fragment (200 bp) SV40 transcription terminator region, and 400 ng of primers CMV154 and SV40-2.
  • the PCR was performed under similar conditions as above except the annealing temperature was raised to 60° C. and the extension time was extended to 3 minutes.
  • the resulting PCR product was transcriptionally active and was used directly for transfection of cells in vitro or tisuues in vivo.
  • a plasmid is constructed in such that the toxic gene GATA-1 is under the control of tac (IPTG-inducible) promoter.
  • the GATA-1-expressing unit is then flanked by TAP promoter (modified CMV IE promoter/intron, 800 bp) and TAP terminator (SV40 transcription terminator, 200 bp) sequences.
  • TAP promoter modified CMV IE promoter/intron, 800 bp
  • TAP terminator SV40 transcription terminator, 200 bp
  • a Transcriptionally active PCR fragment encoding CAT gene is generated using the same promoter and terminator elements. 2 ⁇ g of such TAP fragments is transformed into competent bacteria cells that contains the GATA-1/TAC plasmid and prepared in the absence of IPTG. After transformation, bacteria are plated on a LB plate containing 10 ng/ml IPTG. Only the cells bearing the plasmid in which the TAC/ATA-1 is replaced by the TAP fragment encoding gene of interest are able to grow

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US09/836,436 US20020165175A1 (en) 2001-04-17 2001-04-17 Fast and enzymeless cloning of nucleic acid fragments
PCT/US2002/012334 WO2002083871A2 (fr) 2001-04-17 2002-04-16 Clonage rapide et sans enzyme de fragments d'acide nucleique
US10/125,789 US6936470B2 (en) 2001-04-17 2002-04-16 Rapid and enzymeless cloning of nucleic acid fragments
AU2002256280A AU2002256280A1 (en) 2001-04-17 2002-04-16 Rapid and enzymeless cloning of nucleic acid fragments
US11/045,686 US20050233422A1 (en) 2001-04-17 2005-01-28 Rapid and enzymeless cloning of nucleic acid fragments

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US20030044820A1 (en) 2003-03-06

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