WO2007117857A2 - Nouvelle adn polymérase issue de caldicellulosiruptor kristjanssonii - Google Patents

Nouvelle adn polymérase issue de caldicellulosiruptor kristjanssonii Download PDF

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Publication number
WO2007117857A2
WO2007117857A2 PCT/US2007/064125 US2007064125W WO2007117857A2 WO 2007117857 A2 WO2007117857 A2 WO 2007117857A2 US 2007064125 W US2007064125 W US 2007064125W WO 2007117857 A2 WO2007117857 A2 WO 2007117857A2
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WIPO (PCT)
Prior art keywords
seq
dna
dna polymerase
polymerase
amino acid
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PCT/US2007/064125
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English (en)
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WO2007117857A3 (fr
Inventor
Elena Garnova
Cuong Lam
Scott Hamilton
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Ge Healthcare Bio-Sciences Corp.
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Application filed by Ge Healthcare Bio-Sciences Corp. filed Critical Ge Healthcare Bio-Sciences Corp.
Publication of WO2007117857A2 publication Critical patent/WO2007117857A2/fr
Publication of WO2007117857A3 publication Critical patent/WO2007117857A3/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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the present invention relates to novel DNA polymerases obtainable from the thermophilic organism Caldicellulosiruptor kristjanssonii, to certain deletions and mutants of this enzyme, to genes and vectors encoding the wild type and mutant polymerases and their use in strand displacement activity, polymerase chain reaction, and DNA sequencing.
  • DNA polymerases are a family of enzymes involved in DNA repair and replication.
  • DNA polymerases have been isolated from E. coli (e.g. E. coli DNA polymerase I and the Klenow fragment thereof) and bacteriophageT4 DNA polymerase and more recently thermostable DNA polymerases have been isolated (e.g. from T. aquaticus, US Patent 4,889,818, and from T. litoralis).
  • Thermostable DNA polymerases have been suggested for use in amplifying existing nucleic acid sequences in amounts that are large compared to that originally present (US Patent 4,683,195).
  • the polymerase chain reaction (PCR, US Patent 4,683,202) and strand displacement amplification (SDA) are two methods of amplifying nucleic acid sequences.
  • DNA polymerases e.g. Sequenase, Klenow, Taq, etc, have also been extensively used in DNA sequencing, see for example "Molecular Cloning: A Laboratory Manual” (Sambrook, Fritsch, and Maniatis, 2nd edition, Cold Spring Harbor Laboratory Press, 1989).
  • Figure 2 is a contiguous open reading frame capable of encoding the full length polymerase from Caldicellulosiruptor kristjanssonii (SEQ ID NO:2). Translation is of the open reading frame spanning SEQ ID NO:1 as shown in Figure 1, encoding native polymerase.
  • Figure 8 is a multi-sequence alignment of several DNA polymerase protein sequences. Included in the alignment are DNA polymerase sequence from Ckristjanssonii (SEQ ID NO: 19), Anaerocellum thertnophilum (SEQ ID NO: 20), Caldicellulosiruptor saccharolyticus (SEQ ID NO: 21), Thermoanaerobacter ethanolicus ATCC 33223 (SEQ ID NO: 22), Thermoanaerobacter tengcongensis MB4 (SEQ ID NO: 23), Clostridium perfringens str. 13 (SEQ ID NO: 24), and Dictyoglomus thermophilum (SEQ ID NO: 25) Detailed Description of the Invention
  • the present invention provides a purified DNA polymerase or fragment thereof having the DNA polymerase activity of Caldicellulosiruptor kristjanssonii and having at least 90% amino acid homology, preferably at least 95% homology, more preferably at least 98% amino acid homology, to at least a contiguous 40 amino acid sequence shown in Figure 2 (SEQ ID NO:2).
  • Figure 2 represents the translation of the open reading frame of DNA sequence encoding a DNA polymerase from Caldicellulosiruptor kristjanssonii ( Figure 1; SEQ ID NO:1) encoding the native polymerase.
  • amino acid homology means amino acid identity or conservative amino acid changes thereto.
  • identity is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical or homologous residues by the total number of residues and multiplying the product by 100. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.
  • Buffers around neutral pH (5-9) such as 5-100 mM TrisHCl, HEPES or MES are suitable for use in the current invention.
  • the present invention also provides a gene encoding a polymerase of the present invention.
  • Fig. 1 represents nucleotides of the cloned gene encoding the polymerase of the present invention (SEQ ID NO: 1).
  • the entire amino acid sequence of the polymerase is not required for enzymatic activity.
  • the exonuclease domain of the enzyme has been deleted to give an enzyme which retains enzyme activity.
  • This exonuclease-free enzyme is analogous to the Klenow fragment of E. coli DNA polymerase I.
  • the present invention also provides fragments of the polymerase which retain the DNA polymerase activity of Caldicellulosiruptor kristjanssonii but have one or more amino acids deleted, preferably from the amino-terminus, while still having at least 80% amino acid homology to at least a 40 contiguous amino acid sequence shown in Figure 2 (SEQ ID NO:2) .
  • the present invention provides a DNA polymerase which corresponds to the DNA polymerase from Caldicellulosiruptor kristjanssonii in which up to one third of the amino acid sequence at the amino- terminus has been deleted.
  • a DNA polymerase which corresponds to the DNA polymerase from Caldicellulosiruptor kristjanssonii in which up to one third of the amino acid sequence at the amino- terminus has been deleted.
  • fragments of Caldicellulosiruptor kristjanssonii having N-terminal 270 - 272 amino acid deletions have been generated ( Figures 5, 6 and 7; SEQ ID NO:5, 6, and 7).
  • the DNA polymerases of the present invention can be constructed using standard techniques familiar to those who practice the art.
  • mutagenic PCR primers can be designed to incorporate the desired Phe to Tyr amino acid change (FY mutation in one primer). Deletion of the exonuclease function is carried out by PCR to remove the amino terminus, or standard techniques of site directed mutagenesis to generate point mutations.
  • the present invention provides a host cell comprising a vector containing the gene encoding the DNA polymerase activity of the present invention, e.g., encoding an amino acid sequence corresponding to native
  • the DNA polymerases of the present invention are suitably used in SDA, preferably in combination with a thermostable restriction enzyme. Accordingly, the present invention provides a composition which comprises a DNA polymerase of the present invention in combination with a thermostable restriction enzyme, for example BsoBI from Bacillus stearothermophilus.
  • a thermostable restriction enzyme for example BsoBI from Bacillus stearothermophilus.
  • the invention also features a kit or solution for SDA comprising a DNA polymerase of the present invention in combination and a thermostable restriction enzyme.
  • the polymerases of the present invention are also useful in methods for generating and amplifying a nucleic acid fragment via a strand displacement amplification (SDA) mechanism.
  • SDA strand displacement amplification
  • the method generally comprises: a) specifically hybridizing a first primer 5' to a target nucleic acid sequence, the first primer containing a restriction enzyme recognition sequence 5' to a target binding region; b) extending the 3' ends of the hybridized material using a DNA polymerase of the present invention, preferably one in which the exonuclease activity has been removed, in the presence of three dNTPs and one dNTP ⁇ S; c) nicking at the hemiphosphorothioate recognition site with a restriction enzyme, preferably; d) extending the 3' end at the nick using a DNA polymerase of the present invention, displacing the downstream complement of the target strand; and e) repeating steps (c) and (d).
  • This SDA method proceeds at a linear amplification rate if one primer is used as above. However, if two primers are used which hybridize to each strand of a double-stranded DNA fragment, then the method proceeds exponentially (Walker, G. T., Little, M.C., Nadeau, J.G. and Shank D.D. (1992) Proc. Natl. Acad. ScL USA 89:392-396).
  • the present invention also provides a method for determining the nucleotide base sequence of a DNA molecule.
  • the method includes providing a DNA molecule, annealing with a primer molecule able to hybridize to the DNA molecule; and incubating the annealed molecules in a vessel containing at least one, and preferably four deoxynucleotide triphosphate, and a DNA polymerase of the present invention preferably one containing the phenylalanine to tyrosine mutation.
  • at least one DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base.
  • the method further includes separating the DNA products of the incubating reaction according to size, whereby at least a part of the nucleotide base sequence of the DNA molecule can be determined.
  • the DNA polymerase has less than 1000, 250, 100, 50, 10 or even 2 units of exonuclease activity per mg of polymerase (measured by standard procedure, see below) and is able to utilize primers having only 4, 6 or 10 bases; and the concentration of all four deoxynucleoside triphosphates at the start of the incubating step is sufficient to allow DNA synthesis to continue until terminated by the agent, e.g. a ddNTP.
  • the agent e.g. a ddNTP.
  • more than 2, 5, 10 or even 100 fold excess of a dNTP is provided to the corresponding ddNTP.
  • the method also features providing a second chain terminating agent in the hybridized mixture at a concentration different from the first chain terminating agent, wherein the DNA polymerase causes production of a second series of second DNA products differing in length of the elongated primer, with each second DNA product having the second chain terminating agent at its elongated end.
  • the number of molecules of each second DNA product is approximately the same for substantially all second DNA products differing in length from each other by from 1 to 20 bases, and is distinctly different from the number of molecules of all the first DNA products having a length differing by no more than 20 bases from that of said second DNA products.
  • three or four such chain terminating agents can be used to make different products and the sequence reaction is provided with a magnesium ion, or even a manganese or iron ion (e. g. at a concentration between 0.05 and 100 mM, preferably between 1 and 1OmM); and the DNA products are separated according to molecular weight in four or less lanes of a gel.
  • the invention features a method for sequencing a nucleic acid by combining an oligonucleotide primer, a nucleic acid to be sequenced, between one and four deoxyribonucleoside triphosphates, a DNA polymerase of the present invention, and at least two chain terminating agents in different amounts, under conditions favoring extension of the oligonucleotide primer to form nucleic acid fragments complementary to the nucleic acid to be sequenced.
  • the method further includes separating the nucleic acid fragments by size and determining the nucleic acid sequence.
  • the agents are differentiated from each other by intensity of a label in the primer extension products.
  • the invention features a method for polymerase chain reaction in the presence of a polymerase stabilizing agent, utilizing an enzymatically active DNA polymerase having at least 80% identity in its amino acid sequence to the DNA polymerase of Caldicellulosiruptor kristjanssonii and an exonuclease activity removed.
  • the polymerase stabilizing agents include, but are not limited to, glycerol (10 to 50% final concentration), trimethylamine-N-oxide (TMANO; up to 4 M final concentration), and N-methylmorpholine-N-oxide (MMO; up to 3 M final concentration).
  • TMANO trimethylamine-N-oxide
  • MMO N-methylmorpholine-N-oxide
  • glycerol is used at a final concentration of 30%.
  • polymerase stabilizing agent an agent which allows the use of the polymerase in PCR and RT/PCR. These agents reduce the denaturing temperature of the template and stabilize the polymerase. By stabilize is meant make temperature stable. By final concentration is meant the final concentration of the agent in the PCR or RT/PCR solution.
  • the invention also features kits, with polymerase stabilizing agents, for polymerase chain reaction having an enzymatically active DNA polymerase with at least 80% identity in its amino acid sequence to the DNA polymerase of Caldicellulosiruptor kristjanssonii and an exonuclease activity removed.
  • glycerol is used at a final concentration of 30%.
  • the exonuclease activity is removed by an N-terminal deletion, by deleting up to one third of the amino acid sequence at the N-terminal, or by substitutition of an amino acid in the amino terminal one third of the protein, and the glycerol concentration is 30%.
  • the primers used for the final cloning of the complete DNA polymerase gene were designed using the known gene sequence, with the introduction of restriction enzyme sites.
  • the primer at N-terminus (with SacII site) is 5 '- TCC CCG CGG ATG AAA CTG GTT ATA TTC GAT GGA - 3' (SEQ ID NO: 8).
  • the primer at the C-terminus (with HindIII site) is 5' - CCC AAG CTT CTA TTT TGT CTC ATA CCA GTT CAG TCC - 3' (SEQ ID NO:9).
  • the PCR product was generated using Pfu High-Fidelity DNA polymerase and cloned into PKK vector.
  • CAC GTC TTG CAA GAC CAT-3' (SEQ ID NO:13); (3) YC, forward: 5'- AGC GAA TAC CAA GAA TGC AAA GCT AAC AGA -3' (SEQ ID NO: 14), and reverse: 5'- TCT GTT AGC TTT GCA TTC TTG GTA TTC GCT -3' (SEQ ID NO:15).
  • Pfu High-Fidelity polymerase and standard PCR conditions were used.
  • the N-terminal deletion versions of the FY/DR mutant were generated.
  • the truncated clones start at amino acid sequence 271, 272, and 273 of SEQ ID NO:2, repectively .
  • the reverse primer used is the primer of SEQ ID NO:9, while the forward primer for truncated clone start at amino acid sequence position 271, is 5'- TCC CCG CGG ATG ATT TTG GTC CAG TTA GAG -3' (SEQ ID NO: 16); for truncated clone start at amino acid sequence position 272, is 5'- TCC CCG CGG ATG TTG GTC CAG TTA GAG -3' (SEQ ID NO: 17); for truncated clone start at amino acid sequence position 273, is 5 '- TCC CCG CGG ATG GTC CAG TTA GAG TTC AAA AGT ATA -3 ' (SEQ ID NO: 18). Amplified products were cloned into PKK vector.
  • the supernatant was loaded onto a 5ml HiTrap Q.
  • the column was washed with 10 column volumes with Buffer A (5OmM Tris pH 8.0; ImM EDTA; ImM DTT). Elution was done by gradient from 0 to 100% Buffer B (5OmM Tris pH 8.0; ImM EDTA; ImM DTT, IM NaCl) in 20 fraction volumes. Elution was continued by stepping up to 100% buffer B for 5 column volumes. 5ml fractions were collected.

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Abstract

La présente invention concerne une ADN polymérase issue de Caldicellulosiruptor kristjanssonii. Cette enzyme est utile pour des procédures qui requièrent une synthèse d'ADN impliquant le déplacement de brins, telle que l'amplification par déplacement de brin (SDA), pour un séquençage de l'ADN, et la réaction en chaîne par polymérase (PCR). La présente invention inclut également dans son champ de réalisation divers mutants (délétion et substitution) qui conservent la capacité de répliquer l'ADN sous la forme de la polymérase native de Caldicellulosiruptor kristjanssonii.
PCT/US2007/064125 2006-03-31 2007-03-16 Nouvelle adn polymérase issue de caldicellulosiruptor kristjanssonii WO2007117857A2 (fr)

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US74404506P 2006-03-31 2006-03-31
US60/744,045 2006-03-31

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WO2007117857A3 WO2007117857A3 (fr) 2008-02-21

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020102577A1 (en) * 2000-07-31 2002-08-01 Raillard Sun Ai Nucleotide incorporating enzymes
US20050019789A1 (en) * 1996-10-03 2005-01-27 Roche Diagnostics Gmbh Thermostable DNA polymerase from anaerocellum thermophilum

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050019789A1 (en) * 1996-10-03 2005-01-27 Roche Diagnostics Gmbh Thermostable DNA polymerase from anaerocellum thermophilum
US20020102577A1 (en) * 2000-07-31 2002-08-01 Raillard Sun Ai Nucleotide incorporating enzymes

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