WO1992022650A1 - Analyse multiplex de fragments d'adn facilitee par systeme de recombinaison - Google Patents

Analyse multiplex de fragments d'adn facilitee par systeme de recombinaison Download PDF

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Publication number
WO1992022650A1
WO1992022650A1 PCT/US1992/004923 US9204923W WO9222650A1 WO 1992022650 A1 WO1992022650 A1 WO 1992022650A1 US 9204923 W US9204923 W US 9204923W WO 9222650 A1 WO9222650 A1 WO 9222650A1
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site
molecule
dna
recombinational
vector
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PCT/US1992/004923
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English (en)
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Robert L. Bebee
James L. Hartley
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Life Technologies, Inc.
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Publication of WO1992022650A1 publication Critical patent/WO1992022650A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the dideoxy-mediated method thus requires single- stranded templates, specific oligonucleotide primers, and high quality preparations of a DNA polymerase (typically the Klenow fragment of E. coli DNA polymerase I) . Initially, these requirements delayed the wide spread use of the method. However, with the ready availability of synthetic primers, and the availability of bacteriophage M13 and phagemid vectors (Maniatis, T. , et al. , Molecular Cloning, a Laboratory Manual. 2nd Edition. Cold Spring Harbor Press. Cold Spring Harbor, New York (1989) , herein incorporated by reference) , the dideoxy-mediated chain termination method is now extensively employed.
  • a DNA polymerase typically the Klenow fragment of E. coli DNA polymerase I
  • the sequence is obtained from the original DNA molecule, and not from an enzymatic copy.
  • the method can be used to sequence synthetic oligonucleotides, and to analyze DNA modifications such as methylation, etc. It can also be used to study both DNA secondary structure and protein-DNA interactions. Indeed, it has been readily employed in the identification of the binding sites of DNA binding proteins.
  • Both the above-described dideoxy-mediated method and the Maxam-Gilbert method of DNA sequencing require the prior isolation of the DNA molecule which is to be sequenced.
  • the sequence information is obtained by subjecting the reaction products to electrophoretic analysis (typically using polyacrylamide gels) .
  • electrophoretic analysis typically using polyacrylamide gels
  • a sample is applied to a lane of a gel, and the various species of nested fragments are separated from one another by their migration velocity through the gel.
  • the number of nested fragments which can be separated in a single lane is approximately 200-300 regardless of whether the Sanger or the Maxam-Gilbert method is used.
  • Those of great skill in the art can separate up to 600 fragments in a single lane.
  • the sequence of the entire molecule is obtained by orienting and ordering the sequence data obtained from each fragment.
  • Tags are in turn flanked by sites recognized by the NotI restriction endonuclease (which cuts only at infrequent sites) .
  • the vectors differ from each other only by their tag sequences, which are originally selected from a random collection of chemically synthesized oligonucleotides.
  • DNA is sonicated to produce fragments of 900-1500 base pairs.
  • Such DNA is rendered ligatable through treatment with Bal 31 exonuclease and then with T4 DNA polymerase and all four deoxynucleotide triphosphates.
  • the DNA fragments are then ligated separately into each of the vectors and the ligation mixtures are used to transform E. coli cells. This procedure thus results in a formation of 20 gene libraries, which can then be amplified by conventional means. After amplification, the vectors are treated with
  • the invention provides a method for analyzing a target DNA molecule, which comprises:
  • the invention also provides the embodiment of the above-described method for analyzing a target DNA molecule, wherein the analysis comprises ordering restriction endonuclease recognition sites in a target DNA molecule, and wherein, in step (B) , the analysis comprises
  • the newly formed linear molecule will contain an AttL and an AttR site at the termini • of the inserted molecule. Even in the presence of host factors, the ⁇ Int enzyme, by itself, is unable to catalyze the excision of the inserted molecule. Thus, the reaction is unidirectional. If a second ⁇ protein, the ⁇ Xis protein, is added to the reaction, the reverse reaction can proceed, and a site-specific recombinational event will occur between the AttR and AttL sites to regenerate the initial molecules.
  • the multiplex sequencing method of G.M. Church et al. requires the construction of a large number of vector libraries.
  • the present invention achieves the goal of multiplex sequencing without the need to construct multiple gene libraries.
  • DNA or cDNA from any desired source is obtained, and cloned into a cloning site of any of the well-known prokaryotic, eukaryotic, or shuttle vectors vectors, modified to contain a recombinational site. Examples of suitable vectors are provided by Maniatis, T. , et al. (In: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)). The sequence information is then obtained through the use of a novel method.
  • the loxP site is preferably located about 500 bases away from the target sequence (if a plasmid is employed) or 1000-2000 bases away from the target sequence
  • the vectors of the present invention contain at least one "recombinational site.”
  • the loxP site is the preferred recombinational site of the present invention.
  • the vector shall contain one loxP site.
  • the recombinational site will be incorporated into the vector at a location near the location of the cloning region.
  • the structure of the vector is thus depicted in Figure 6 (where the recombinational site is illustrated as a loxP site; the orientation of the loxP is always left to right relative to the other elements shown) .
  • a DNA molecule whose sequence is to be mapped by restriction endonuclease digestion is obtained from a suitable source.
  • target DNA has been isolated using a restriction endonuclease which is also capable of cleaving at a site within the cloning region of the above-described vectors.
  • conventional methods can be used to adapt the ends of the target such that they are now capable of being ligated into a restriction site of the cloning region. This can, for example, be accomplished with any target by treating overhanging ends to produce a blunt ended target molecule.
  • the target molecule is then introduced into the vector, and the vector is recircularized through the action of a DNA ligase. Procedures for accomplishing these steps are disclosed by Maniatis, T., et al. (In: Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)).
  • these nested fragments contain the probe/primer region of the original molecule, a probe having a sequence substantially complementary to that of the probe/primer region will be able to hybridize to the fragments. By labelling such a probe, it is thus possible to visualize the nested fragments which hybridize to the probe/primer region.
  • the position of the restriction sites can be readily determined by measuring the sizes of the "bands," as shown in Figure 13. It is possible to prepare multiple sets of nested fragments using different restriction endonucleases, and loxP-containing oligonucleotides having different probe/primer regions.
  • Each set of nested fragments can be visualized by incubating the total set of fragments with a probe substantially complementary to the probe/primer region of the respective set of nested fragments.
  • the present invention permits one to sequentially analyze all of these nested sets of fragments. Indeed, if different labels are employed on different probes, it is possible to simultaneously analyze different sets of nested fragments.
  • the present invention greatly facilitates the process of restriction mapping.
  • the sequence of a cloned region can be determined in a multiplex analysis.
  • the method utilizes two types of DNA molecules.
  • the first molecule is a cloning vector which contains a loxP site. Any of the well-known prokaryotic, eukaryotic, or shuttle vectors vectors may be modified to permit their use in the present invention.
  • the vector shall contain at least one recombinational site, preferably loxP, which precedes and is adjacent to and, most preferably. immediately adjacent to a cloning region.
  • the structure of the vector is as shown in Figure 6 or Figure 7.
  • a DNA molecule whose sequence is to be determined i.e. a "target" sequence
  • a suitable source i.e. a "target" sequence
  • target DNA has been isolated using a restriction endonuclease which is also capable of cleaving at a site within the cloning region of the above-described vector.
  • conventional methods can be used to adapt the ends of the target such that they are now capable of being ligated into a restriction site of the cloning region. This can, for example, be accomplished with any target by treating overhanging ends to produce a blunt ended target molecule.
  • the target molecule is then introduced into the vector, and the vector is recircularized through the action of a DNA ligase.
  • Procedures for accomplishing these steps are disclosed by Maniatis, T. , et al. (In: Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)). The construction is as shown in Figure 8 and Figure 9.
  • the target DNA Once the target DNA has been inserted into any of the above-described vectors, it can then be amplified, by propagating the vector in a suitable host. Individual members of the library (either as transformed cells, or isolated DNA) can then be isolated.
  • This feature of the present invention permits a multiplex analysis to be performed.
  • members of the unfractionated vector library are separately permitted to recombine with one of a plurality of linear molecules each of which differs from the other in the sequence of its probe/primer sequence.
  • the result of such recombination may be depicted asshown in Figure 15.
  • oligonucleotides of class II are employed, a single primer would be used in the sequencing reactions, and different probes (1, 2, etc.) would be used. This latter embodiment is preferred, except that target sequence would not be reached until about 77 nucleotides from the 5' end of the primer (i.e. 20 nucleotides of the primer, 20 nucleotides of the probe, 34 nucleotides of the loxP site, and 3 nucleotides from the remainder of the cloning site (e.g. Smal) .
  • the priming sites would be completely single stranded, even without denaturation, since the recombination oligonucleotide would be single stranded in the primer domain.
  • the present invention facilitates the sequencing of cos id molecules.
  • a cosmid is constructed so as to contain a loxP site ( Figure 19) .
  • the molecule is incubated in the presence of Cre and a loxP-containing oligonucleotide, preferably, the oligonucleotide is single-stranded, and will possess a sequence which causes it to snap back upon itself ( Figure 20) .
  • a linear molecule will be produced having the structure shown in Figure 21.
  • an array of partial-digestion products such as those shown in Figure 22 are obtained.
  • the effect of the reaction has been to produce a series of oligonucleotides which contain at most, only one loxP site.
  • This mixture of oligonucleotides is then incubated with a DNA ligase in the presence of a second loxP-containing oligonucleotide, which will preferably be single-stranded, and possess a sequence which causes it to snap back upon itself, such as shown in Figure 20.
  • a DNA ligase in the presence of a second loxP-containing oligonucleotide, which will preferably be single-stranded, and possess a sequence which causes it to snap back upon itself, such as shown in Figure 20.
  • three general classes of molecules will be present in the reaction:
  • Cre/loxP mediated site-specific recombination as a method to facilitate multiplex mapping was demonstrated by the following procedure.
  • the target molecules were pLox, a 2.9 kb plasmid with a loxP site cloned into a polylinker region, and pSPORT-lox, a 4.1 kb plasmid with a loxP site inserted into its multiple cloning site (MCS) .
  • Recombinant molecules to be eventually used as substrates for multiplex mapping were generated as follows.
  • the reaction contained 1 pmol of plasmid, 4 pmol of oligonucleotides and 5 units of Cre (NEN) in buffer composed of 50 mM Tris-HCl (pH 7.5), 33 mM NaCl, 5 mM spermidine, 0.5 g/ml bovine serum albumin (BSA);- incubations were at 37 ⁇ C for 15 minutes.
  • BSA bovine serum albumin

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Abstract

Cette invention concerne l'utilisation de la recombinaison généralisée, ou plus préférablement, de la recombinaison à spécificité de site pour faciliter l'analyse de la séquence ou des fragments de molécules d'ADN. On emploie plus préférablement le système de recombinaison à spécificité de site du bactériophage P1 pour faciliter une analyse de la séquence de ce type.
PCT/US1992/004923 1991-06-17 1992-06-12 Analyse multiplex de fragments d'adn facilitee par systeme de recombinaison WO1992022650A1 (fr)

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US71668391A 1991-06-17 1991-06-17
US716,683 1991-06-17

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024654A1 (fr) * 1992-06-02 1993-12-09 Boehringer Mannheim Gmbh Sequencage simultanee d'acides nucleiques
US6720140B1 (en) 1995-06-07 2004-04-13 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6828093B1 (en) 1997-02-28 2004-12-07 Baylor College Of Medicine Rapid subcloning using site-specific recombination
US8304189B2 (en) * 2003-12-01 2012-11-06 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same
US8883988B2 (en) 1999-03-02 2014-11-11 Life Technologies Corporation Compositions for use in recombinational cloning of nucleic acids
US8945884B2 (en) 2000-12-11 2015-02-03 Life Technologies Corporation Methods and compositions for synthesis of nucleic acid molecules using multiplerecognition sites

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4942124A (en) * 1987-08-11 1990-07-17 President And Fellows Of Harvard College Multiplex sequencing
US4959317A (en) * 1985-10-07 1990-09-25 E. I. Du Pont De Nemours And Company Site-specific recombination of DNA in eukaryotic cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4959317A (en) * 1985-10-07 1990-09-25 E. I. Du Pont De Nemours And Company Site-specific recombination of DNA in eukaryotic cells
US4942124A (en) * 1987-08-11 1990-07-17 President And Fellows Of Harvard College Multiplex sequencing

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
NUCLEIC ACIDS RESEARCH, Volume 14, No. 5, issued 1986, R.H. HOESS et al., "The Role of the loxP Spacer Region in P1 Site-Specific recombination", pages 2287-2300. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024654A1 (fr) * 1992-06-02 1993-12-09 Boehringer Mannheim Gmbh Sequencage simultanee d'acides nucleiques
US5714318A (en) * 1992-06-02 1998-02-03 Boehringer Mannheim Gmbh Simultaneous sequencing of nucleic acids
US6720140B1 (en) 1995-06-07 2004-04-13 Invitrogen Corporation Recombinational cloning using engineered recombination sites
US6828093B1 (en) 1997-02-28 2004-12-07 Baylor College Of Medicine Rapid subcloning using site-specific recombination
US8883988B2 (en) 1999-03-02 2014-11-11 Life Technologies Corporation Compositions for use in recombinational cloning of nucleic acids
US9309520B2 (en) 2000-08-21 2016-04-12 Life Technologies Corporation Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites
US8945884B2 (en) 2000-12-11 2015-02-03 Life Technologies Corporation Methods and compositions for synthesis of nucleic acid molecules using multiplerecognition sites
US8304189B2 (en) * 2003-12-01 2012-11-06 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same
US20130316350A1 (en) * 2003-12-01 2013-11-28 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same
US9534252B2 (en) * 2003-12-01 2017-01-03 Life Technologies Corporation Nucleic acid molecules containing recombination sites and methods of using the same

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