WO1997048791A1 - Clonage direct de fragments d'adn - Google Patents

Clonage direct de fragments d'adn Download PDF

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Publication number
WO1997048791A1
WO1997048791A1 PCT/US1997/009696 US9709696W WO9748791A1 WO 1997048791 A1 WO1997048791 A1 WO 1997048791A1 US 9709696 W US9709696 W US 9709696W WO 9748791 A1 WO9748791 A1 WO 9748791A1
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WO
WIPO (PCT)
Prior art keywords
vector
nucleotide
dna
residue
overhang
Prior art date
Application number
PCT/US1997/009696
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English (en)
Inventor
Robert C. Mierendorf
Robert E. Novy
Kristin M. Kolb
David O'reilly
Original Assignee
Novagen, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9612713.9A external-priority patent/GB9612713D0/en
Priority claimed from GBGB9614765.7A external-priority patent/GB9614765D0/en
Application filed by Novagen, Inc. filed Critical Novagen, Inc.
Priority to AU35681/97A priority Critical patent/AU3568197A/en
Publication of WO1997048791A1 publication Critical patent/WO1997048791A1/fr

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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
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • T-vectors are formed from two closely spaced sequences that are recognized by a specific restriction enzyme that cleaves the plasmid DNA to leave a single 3' dTMP overhang on each end of the linearized vector following cleavage. The use and construction of such a vector is described in US Patent Number 5,487,993.
  • the T- vector is intended to take advantage of a phenomenon associated with some of the DNA polymerases that are used in the PCR process.
  • a popularly used DNA polymerase Taq DNA polymerase
  • Taq DNA polymerase often leaves a single unpaired dAMP residue at each end of the DNA molecules which are the product of the PCR reaction.
  • the propensity of Taq DNA polymerase to add single 3' dAMP extensions varies with the composition of the 3' ends of the PCR products, the PCR conditions, and the conditions under which the completed reactions are stored (Hu, DNA Cell Biol. 12, 763 (1993); Magnuson et al. , BioTechniques 21, 700, (1996)) .
  • dAMP residues at the 3' end of a duplex are inhibitory to dAMP addition (presumably leaving a blunt end) , and other bases such as dCMP, dGMP or dTMP can be preferentially added to ends having specific sequences. Therefore, under many conditions the fraction of PCR products that possess single 3' dAMP extensions on both ends can be relatively low. Therefore, vectors which have only a single 3' dTMP overhang cannot efficiently ligate with these other fragments.
  • T vectors There are other methods for preparing T vectors including the addition of single dideoxy TMP residues at each end of a blunt and linearized plasmid using terminal transferase Holton & Graham, Nucleic Acids Res. 19:1156 (1991), or the addition of linkers containing a single unpaired dTMP residue to a linearized plasmid. However, no method has been described for the preparation of vectors having bases other than dTMP as the single 3' overhang.
  • a vector for use in the direct cloning of DNA fragments contains at at least one end thereof an overhang of a single nucleotide, the nucleotide being a nucleotide which does not normally occur in DNA, such as a uracil or an inosine residue.
  • the resulting single overhang vector can conveniently be used for the direct cloning of the products of PCR reactions in a convenient and useful manner. It is another object of the present invention to describe a method for cloning the direct products of PCR reaction using linearized vectors having a single overhang of non-natively occurring nucleotides, including uracil and inosine.
  • the cloning vectors created with single overhangs including non-naturally occurring DNA nucleotides, have the inherent characteristic of binding to a wider range of PCR reaction products single nucleotide overhangs than previously available vectors having only a single T overhang.
  • a cloning vector in which there is a single nucleotide overhang at the 3' end of each end of the vector.
  • the single nucleotide overhang is of a nucleotide which does not occur normally in DNA, the preferable nucleotides being uracil (U) and inosine (I) .
  • Uracil and inosine are both naturally occurring nucleotides, uracil being incorporated in both mRNA and tRNA and inosine also being present in tRNA.
  • Uracil forms hydrogen bonds with adenine (A) while inosine preferentially binds to cytosine (C) .
  • This cloning plasmid can be readily used for the direct cloning of the products of polymerase chain reaction
  • PCR PCR protocols which normally contain a complementary single nucleotide overhang on those products.
  • the vectors described in this patent application are intended to be an extension of, and an improvement upon, the vectors having a single T overhang as described in US Patent No. 5,487,993, the specification of which is hereby incorporated by reference.
  • the vectors of the present invention extend the capability of in vi tro methods of cloning PCR reaction products, and vector preparation, to incorporate nucleotide residues that do not naturally occur in native DNA. These vectors improve the cloning of DNA fragments tailed with single dAMP overhangs.
  • the use of dUMP in place of dTMP as the single end overhang at each end of the vector allows for the potential of more efficient cloning of dTMP-containing inserts since at least for RNA to DNA interactions, the hydrogen bond formed between the nucleotides A and U is stronger than that between the nucleotides T and A.
  • dIMP single nucleotide overhanging additions other than dTMP, such as dIMP
  • dIMP can in theory base pair with multiple residues, but practical experience to date suggests dIMP has a practical preference for annealing with dCMP.
  • Virtually any nucleotide that can be synthesized and incorporated into a DNA chain can serve as the 3' extended base allowing for improvements in cloning efficiency and versatility as other nucleotide modifications are developed. It thus becomes possible to clone a variety of species found in a PCR reaction product mix with greater efficiency and selectability than it was heretofore possible.
  • the cloning vectors of the present invention will be created by making a blunt ended cloning vector and then adding to each end of the blunt end a single 3' nucleotide overhang.
  • Circular DNA vectors are commonly blunt-ended by digestion with restriction enzymes which have a cutting site leaving a blunt end, such as EcoRV or Smal .
  • the blunt ended vector can be incubated in a reaction mixture with only the nucleotide sought to be added (e.g., dUTP or dITP) , in the presence of an enzyme, such as Taq or Tth DNA polymerases, each of which will add a single nucleotide overhang to a blunt ended double stranded DNA segment.
  • An alternative strategy for the products of some PCR reactions is to construct a vector having one blunt end and a single U or I overhang at the other end.
  • a vector of this variation is particularly useful for cloning of PCR products which have a single A overhang only at one of their ends. Since the addition of the dAMP overhang to a PCR product by Taq DNA polymerase, for instance, is dependent to some degree on the DNA sequence of the PCR product, it is not uncommon for a significant fraction of such products to have an overhang only at one end. To make such a single end single overhang vector, one could begin with a vector which is linearized with a restriction enzyme, such as ECORV, which produces blunt ends.
  • the single uracil or inosine residue can be added to both ends of the linear vector.
  • digestion with another blunt-ending endonuclease, such as Smal could cleave the vector closely adjacent to only one end to leave one blunt end and one single overhand end.
  • This variation has the advantage of directionality, i.e., the PCR product will ligate into the vector in only one orientation.
  • the cloning vector constructed in accordance with the present invention may also have any of the many other components commonly carried on cloning and expression vectors . Such components may include a selectible marker such as for antibiotic resistance.
  • the vector should also have an origin of replication to permit high copy number replication in commonly used host cells.
  • Such cloning vectors also usually include a plurality of restriction sites forming a polylinker or multiple cloning site, located adjacent to each side of the single nucleotide overhang to allow easy subcloning of the fragments into other vectors or for the addition of promoters for expression.
  • the vector may also include a bacteriophage origin of replication to allow production of single stranded copies of the recombinant vector for use in, among other things, sequencing procedures.
  • the vector may also include a gene spanning the cloning site which creates an easily detectible phenotype to allow for selection of inserts, such as for example the LacZ ⁇ gene which is used to span a cloning site to allow for convenient blue-white color screening for recombinant vectors which include an insert at the site within the LacZ ⁇ gene.
  • a gene spanning the cloning site which creates an easily detectible phenotype to allow for selection of inserts, such as for example the LacZ ⁇ gene which is used to span a cloning site to allow for convenient blue-white color screening for recombinant vectors which include an insert at the site within the LacZ ⁇ gene.
  • Another feature which can be included in such a vector is a positive selection sequence which permits survival of recombinants which disrupt a lethal gene.
  • the vector in accordance with the present invention can be sold separately or as part of a cloning kit .
  • the vector would typically be a linearized piece of DNA with the single U or I nucleotide overhang at each 3' end thereof.
  • the vector can be sold with competent host cells with a suitable genotype and which are competent for transformation.
  • the vector may also be sold with an aliquot of a DNA ligase enzyme and buffers which may be conveniently used to ligate PCR amplification products into the vector.
  • a kit including the vector might also include nuclease free water or control inserts with mononucleotide overhangs to check procedures for using the vector.
  • the kit may also include a supercoiled plasmid used as a transformation control.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Vecteur de clonage direct des produits d'une réaction de polymérase en chaîne (PCR) incorporant des saillies constituées par des mononucléotides à une ou aux deux extrémités d'un segment d'ADN rendu linéaire. Ces saillies de mononucléotides sont des résidus d'uracil ou d'inosine permettant, selon ce qu'on désire, de faciliter le clonage des produits souhaités de PCR.
PCT/US1997/009696 1996-06-18 1997-06-18 Clonage direct de fragments d'adn WO1997048791A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU35681/97A AU3568197A (en) 1996-06-18 1997-06-18 Direct cloning of dna fragments

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GBGB9612713.9A GB9612713D0 (en) 1996-06-18 1996-06-18 U-vector cloning
GB9612713.9 1996-06-18
GBGB9614765.7A GB9614765D0 (en) 1996-07-13 1996-07-13 Direct cloning of mononucleotide-tailed DNA fragments
GB9614765.7 1996-07-13

Publications (1)

Publication Number Publication Date
WO1997048791A1 true WO1997048791A1 (fr) 1997-12-24

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/009696 WO1997048791A1 (fr) 1996-06-18 1997-06-18 Clonage direct de fragments d'adn

Country Status (2)

Country Link
AU (1) AU3568197A (fr)
WO (1) WO1997048791A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1396540A2 (fr) * 1990-09-27 2004-03-10 Invitrogen Corporation Clonage direct d'acides nucléiques amplifiés par réaction en chaine de lapolymérase

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5487993A (en) * 1990-09-27 1996-01-30 Invitrogen Corporation Direct cloning of PCR amplified nucleic acids

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5487993A (en) * 1990-09-27 1996-01-30 Invitrogen Corporation Direct cloning of PCR amplified nucleic acids

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NUCLEIC ACIDS RESEARCH, 1991, Vol. 19, No. 5, HOLTON et al., "A Simple and Efficient Method for Direct Cloning of PCR Products Using ddT-Tailed Vectors", page 1156. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1396540A2 (fr) * 1990-09-27 2004-03-10 Invitrogen Corporation Clonage direct d'acides nucléiques amplifiés par réaction en chaine de lapolymérase
EP1396540A3 (fr) * 1990-09-27 2004-06-09 Invitrogen Corporation Clonage direct d'acides nucléiques amplifiés par réaction en chaine de lapolymérase
EP1790723A2 (fr) * 1990-09-27 2007-05-30 Invitrogen Corporation Clonage direct d'acides nucléiques amplifiers par réaction en chaîne de la polymérase PCR
EP1790723A3 (fr) * 1990-09-27 2008-10-22 Invitrogen Corporation Clonage direct d'acides nucléiques amplifiers par réaction en chaîne de la polymérase PCR

Also Published As

Publication number Publication date
AU3568197A (en) 1998-01-07

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