WO1999042595A9 - Enzymes de clivage incompatibles provenant de microorganismes extremement thermophiles et leurs utilisations - Google Patents

Enzymes de clivage incompatibles provenant de microorganismes extremement thermophiles et leurs utilisations

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Publication number
WO1999042595A9
WO1999042595A9 PCT/US1999/003274 US9903274W WO9942595A9 WO 1999042595 A9 WO1999042595 A9 WO 1999042595A9 US 9903274 W US9903274 W US 9903274W WO 9942595 A9 WO9942595 A9 WO 9942595A9
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WIPO (PCT)
Prior art keywords
polynucleotide
enzyme
mismatch
probe
nucleotide sequence
Prior art date
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PCT/US1999/003274
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English (en)
Other versions
WO1999042595A1 (fr
Inventor
Jack G Chirikjian
Leonard S Bazar
Albert L George
Original Assignee
Trevigen Inc
Jack G Chirikjian
Leonard S Bazar
Albert L George
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Publication date
Application filed by Trevigen Inc, Jack G Chirikjian, Leonard S Bazar, Albert L George filed Critical Trevigen Inc
Priority to AU27664/99A priority Critical patent/AU2766499A/en
Publication of WO1999042595A1 publication Critical patent/WO1999042595A1/fr
Publication of WO1999042595A9 publication Critical patent/WO1999042595A9/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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • thermophilic mismatch cleavage enzyme While Horst et al. and WO 96/40902 report a thermophilic mismatch cleavage enzyme, there have been no reports of the isolation of an extreme thermophilic mismatch cleavage enzyme.
  • TIGR has reported the partial sequencing of the Thermotoga maritima genome, particularly disclosing sequences U71155 (designated "MutS”), U71053 (“MutL”),
  • an object of the present invention to provide an extreme thermophilic enzyme that cleaves at a mismatch formed between two polynucleotides in a duplex. It is a further object to provide a method of using the extreme thermophilic enzyme of the present invention to detect a mutation in a polynucleotide sequence or to detect the presence of a non-mutated sequence.
  • an extreme thermophilic enzyme that is contained in an enzyme composition obtained from an extreme thermophile.
  • an extreme thermophilic enzyme that is referred to herein as Thermotoga maritima Endo V (TM-Endo V).
  • TM-Endo V Thermotoga maritima Endo V
  • a method of detecting the presence of and determining the relative positions of at least two mutations in target polynucleotides comprising: (a) hybridizing single-stranded polynucleotide probes to target polynucleotides to form hybrid, double-stranded polynucleotides such that mismatches occur at the sites of the mutations, wherein the probes are complementary to a non-mutated sequence of the target polynucleotides and are labeled at one end but not both ends, and wherein the target polynucleotides are not labeled;
  • a method of detecting the presence or absence of a mismatch in a polynucleotide duplex comprising:
  • Figure 1 is an autoradiograph showing the cleavage products of an extreme thermophilic enzyme composition obtained from Thermotoga neopolitana as discussed in Example 2.
  • Figure 2 is a representation of a four-way junction used to assay resolvase activity.
  • Figure 3 shows a nucleotide and amino acid sequence for TM-Endo V.
  • Figures 4A and 4B are autoradiographs depicting cleavage products of TM- Endo V. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • extreme thermophile refers here to a microorganism with an optimal growth temperature of between approximately 70°C and approximately 85°C and preferably, approximately 85°C.
  • enzyme composition refers to one or more isolated enzymes. Exemplary methods of isolating the one or more enzymes are disclosed herein.
  • the term “extreme thermophilic” when used to describe the inventive "extreme thermophilic enzyme” or “extreme thermophilic enzyme composition” or “extreme thermophilic mismatch cleavage activity” connotes the ability to cleave a mismatch formed between two polynucleotides in a duplex at temperatures as high as approximately 85°C and preferably the ability to cleave such mismatches after the enzyme has been exposed to a temperature of approximately 85°C for a prolonged period.
  • the term “extreme thermophilic enzyme” connotes an enzyme that can withstand approximately 20 temperature cycles (approximately 95°C/30 sec, approximately 65°C/60 sec) while still maintaining the ability to cleave mismatches.
  • mismatch cleavage activity when used in the context of an enzyme, refers to the ability to cleave mismatches formed between two polynucleotides and when used in the context of a polynucleotide, refers to a polynucleotide that encodes an enzyme having that activity.
  • mismatch refers to the situation where one strand of a polynucleotide in a duplex does not or cannot pair through Watson-Crick base pairing to a nucleotide in the opposing complementary polynucleotide.
  • mismatches result from (i) a point mutation or (ii) an insertion or deletion mutation, which results in a bubble formation.
  • cleaves at a mismatch or cleaving “at the point of mismatch” includes (i) cleaving directly at the mismatch site or (ii) cleaving near the mismatch site of the probe polynucleotide.
  • Near the mismatch site includes a distance of approximately 4 or less nucleotides from the mismatch site, in either the 3' or 5' direction.
  • the description of the inventive enzyme as cleaving at a mismatch or at a point of mismatch contemplates that the inventive enzyme exhibits glycosylase activity, which results in an abasic sugar (an AP site) at the point of mismatch. Cleavage is then effectuated at the AP site.
  • AP site cleavage is carried out by the inventive enzyme, itself.
  • AP site cleavage is carried out via conditions, such as increased temperature and increased pH, or the addition of an AP cleaving enzyme, such as an endonuclease or lyase. Such conditions for effectuating cleaving of an AP site are well known in the art.
  • mutation includes (i) single base pair point mutations, (ii) insertion mutations of one or more base pairs, and (iii) deletion mutations of one or more base pairs.
  • polynucleotide refers to two or more joined nucleotides, wherein the nucleotide is either (i) deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a derivative thereof as known to the skilled artisan, such as a peptide nucleic acid (PNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • PNA peptide nucleic acid
  • polynucleotides in a duplex or “hybrid double- stranded polynucleotide” includes, for example, either DNA/DNA duplexes or DNA/RNA duplexes.
  • the term “isolated,” when used to describe the inventive enzymes, refers to an enzyme that is essentially free of other polypeptides or other contaminants with which the enzyme is normally found in nature.
  • isolated when used to describe the inventive polynucleotides, refers to a polynucleotide that is essentially free of other polypeptides or other contaminants with which it is normally found in nature.
  • the present invention includes an extreme thermophilic enzyme composition that cleaves at a mismatch formed between two polynucleotides in a duplex.
  • the enzyme composition cleaves T/G, A/G, T/C, C/C, G/G, T/C, A/C, A/A, and T/T mismatches, and also cleaves at a bubble formation created by an insertion or deletion mutation.
  • the inventive enzyme composition is obtained by isolating an enzyme composition having mismatch cleavage properties from an extreme thermophile.
  • the enzyme composition may be isolated by lysis of extreme thermophile cells, for example by repeated passage through a French press, followed by centrifugation and subjecting the supernatant to column chromatography, such as ion exchange chromatography.
  • column chromatography such as ion exchange chromatography.
  • a phosphocellulose column is preferred as it is inexpensive and binds to charged proteins which are typical of DNA binding proteins. The resulting fractions may then be tested for mismatch cleavage activity.
  • extreme thermophiles are Thermotoga neopolitana, Thermotoga maritima, Thermus aquaticus, or Methanococcus jannaschii.
  • Other extreme thermophiles may be isolated by methods known to the skilled artisan from high temperature environments such as geysers, volcanoes, and underwater thermal vents. See, generally, Genetic Engineering News (February 1, 1998) at 16.
  • the present invention also is directed to an extreme thermophilic enzyme composition which does not require divalent cations to effectuate mismatch cleavage.
  • mismatch cleavage may be carried out in a buffer solution that does not contain magnesium.
  • a GATC nucleotide sequence is not necessary in order to effectuate mismatch cleavage.
  • the present invention is also directed to an extreme thermophilic enzyme composition that does not exhibit resolvase activity.
  • the lack of resolvase activity is shown by a failure to cleave the four-way junction shown in Figure 2.
  • B. TM-Endo V Thermotoga maritima Endo V, abbreviated herein as TM-Endo V refers to an enzyme comprising the amino acid sequence shown in Figure 3.
  • TM-Endo V further comprehends an enzyme that (1) exhibits extreme thermophilic mismatch cleavage activity, (2) does not exhibit resolvase activity, (3) does not require a GATC sequence to effectuate mismatch cleavage, and (4) does not require a divalent cation to effectuate cleavage.
  • TM-Endo V cleaves A/G, C/C, G/G, T/C, A/C, A/A, and T/T mismatches, but does not cleave T/G mismatches or a bubble formation caused by an insertion or deletion mutation.
  • TM-Endo V when used in conjunction with an enzyme specificity altering agent, such as dimethyl sulfoxide (DMSO), does cleave at a bubble formation caused by an insertion or deletion mutation and cleaves T/G mismatches.
  • TM-Endo V also refers to an enzyme encoded by a nucleotide sequence that hybridizes under high stringency or stringent conditions to the complement of the nucleotide sequence of Figure 3 or the complement of a nucleotide sequence which encodes the polypeptide of Figure 3.
  • high stringency conditions refers to 5 X SSC at 65 °C, followed by washing in 0.1 X SSC at 65 °C for thirty minutes.
  • Stringent conditions refers to 5 X SSC at 65 °C, followed by washing in 1 X SSC at 65 °C for thirty minutes.
  • a general discussion of hybridization techniques is found in Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL (Cold Spring Harbor Press, 1989) at ⁇ 11.1-11.61, incorporated by reference herein.
  • TM-Endo V further includes an enzyme encoded by a nucleotide sequence having, (i) 60% or greater sequence identity, (ii) 80% or greater sequence identity, or preferably, (iii) 90% or greater, or (iv) 95% or greater sequence identity to the nucleotide sequence of Figure 3 or to a nucleotide sequence that encodes the polypeptide of Figure 3.
  • percent identity is calculated by FastDB based upon the following parameters: Mismatch Penalty 1.00; Gap Penalty 1.00; Gap Size Penalty 0.33; Joining Penalty 30.0.
  • TM-Endo V further includes an enzyme encoded by the nucleotide sequence of Figure 3 and fragments thereof that have mismatch cleavage activity or a nucleotide sequence that encodes the polypeptide of Figure 3 and fragments thereof that have mismatch cleavage activity.
  • TM-Endo V further includes conservative variations of the amino acid sequence of Figure 3.
  • conservative variation denotes the replacement of an amino acid residue by another, biologically active similar residue. Examples of conservative variations include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • TM-Endo V also includes insertion or deletional variants of the amino acid sequence of Figure 3. Also included within the present invention are polynucleotides that encode
  • TM-Endo V the polynucleotide encoding TM-Endo V is shown in Figure 3.
  • Polynucleotides encoding TM-Endo V also include those that hybridize under high stringency or stringent conditions, as defined above, to the nucleotide sequence of Figure 3 or a nucleotide sequence which encodes the polypeptide of Figure 3 as well as those having the percent sequence identity, as defined above, to the nucleotide sequence of Figure 3 or to a nucleotide sequence which encodes the polypeptide of Figure 3.
  • Polynucleotides encoding TM-Endo V further include fragments of the nucleotide sequence of Figure 3 that have mismatch cleavage activity.
  • the present invention also comprehends that a number of different polynucleotide sequences will encode the amino acid sequence of Figure 3 due to the degeneracy of the genetic code. There are 20 natural amino acids, most of which are specified by more that one codon (a three base sequence). Therefore, all degenerate nucleotide sequences that encode the amino acid sequence of Figure 3 are included in the present invention.
  • the present invention further includes allelic variations (naturally-occurring base changes in the species population which may or may not result in an amino acid change) of the polynucleotide sequence of Figure 3.
  • TM-Endo V The amino acid sequence of TM-Endo V can be synthesized by such commonly used methods as t-BOC or FMOC protection of alpha-amino groups. Both methods involve step-wise syntheses whereby a single amino acid is added at each step starting from the C-terminus of the peptide (Coligan et al, Current Protocols in Immunology, Wiley Interscience, Unit 9, 1991).
  • TM-Endo V can be synthesized by solid phase synthesis methods (Merrifield, J. Am. Chem. Soc. 85:2149, 1962; Steward and Young, Solid Phase Peptide Synthesis, Freeman, San Francisco pp.
  • copolyol styrene-divinylbenzene
  • polypeptides can be deprotected and cleaved from the polymer by treatment with liquid HF 10% anisole for about 15-60 min at 0 °C. After evaporation of the reagents, the peptides are extracted from the polymer with 1% acetic acid solution, which is then lyophilized to yield crude material. This can normally be purified by such techniques as gel filtration of Sephadex G-15 using 5% acetic acid as a solvent.
  • Lyophilization of appropriate fractions of the column will yield a homogeneous polypeptide or polypeptide derivatives, which are characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility and quantitated by solid phase Edman degradation.
  • Polynucleotide sequences encoding TM-Endo V include DNA, RNA and cDNA sequences. Polynucleotides encoding TM-Endo V can be obtained by several methods. For example, the DNA can be isolated using hybridization procedures which are known in the art. Such hybridization procedures include, for example, hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences, antibody screening of expression libraries to detect common antigenic epitopes or shared structural features and synthesis by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Hybridization procedures are useful for screening recombinant clones by using labeled mixed synthetic oligonucleotides probes, wherein each probe is potentially the complete complement of a specific DNA sequence in a hybridization sample which includes a heterogeneous mixture of denatured double-stranded DNA.
  • hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA.
  • Hybridization is particularly useful for detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present.
  • TM-Endo V variants and related enzymes can be identified by screening a cDNA expression library, such as lambda gtl 1, using antibodies specific for the amino acid sequence of Figure 3. Such antibodies can be either polyclonal or monoclonal, derived from the entire sequence of Figure 3 or fragments thereof. It is further understood that given knowledge of the sequence of TM-Endo V as described herein, one of skill in the art can generate TM-Endo V polynucleotides via conventional chemical synthesis methodologies. 3. Expressing TM-Endo V Polynucleotides
  • TM-Endo V polynucleotides can be accomplished by insertion of a polynucleotide encoding the enzyme into an appropriate recombinant expression vector and then expressing this vector in an appropriate recombinant expression system.
  • recombinant expression vector refers to a plasmid, virus or other vehicle that has been manipulated by insertion or incorporation of genetic sequences. Such expression vectors contain a promoter sequence which facilitates efficient transcription of TM- Endo V in a host.
  • a polynucleotide encoding TM-Endo V can be inserted into the expression vector by standard cloning techniques.
  • a suitable recombinant expression vector for use in the present invention is one with a bacterial origin of replication, a bacterial promoter, a ribosome binding site for expression in bacteria, and one or more genes conferring a trait, such as antibiotic resistance, which allows for phenotypic selection of transformed cells.
  • the bacterial promoter may also be an inducible promoter.
  • the polynucleotide encoding TM-Endo V may also be ligated to a nucleotide sequence encoding an amino acid sequence which can be used to purify the expressed
  • TM-Endo V Such a polynucleotide construct encodes what is known as a fusion protein. Examples of suitable fusion proteins are known in the art.
  • TM-Endo V is an E. coli expression system.
  • a suitable fusion protein system is the pBAD/His C (Invitrogen). Fusion protein systems are also available which allow for the excision of the fusion partner from the TM-Endo V protein.
  • the extreme thermophilic enzymes described herein can be used to detect a mutation in a target polynucleotide.
  • This detection entails hybridizing a single-stranded polynucleotide probe to a target polynucleotide to form a hybrid, double- stranded polynucleotide.
  • this hybridization occurs under stringent conditions.
  • Stringent conditions designates those conditions under which only nucleotide sequences which have a high frequency of complementary base sequences will hybridize with each other. Stringent conditions are established by a number of factors well known in the art, such as the size and nature of the probe, temperature and salt conditions. For example, see Sambrook et al.
  • the probe is complementary to a non-mutated sequence of target polynucleotide, there will be a mismatch between the non-mutated probe and the mutated target polynucleotide at the site of the mutation.
  • the probe is then cleaved at the site of the mismatch by the inventive extreme thermophilic enzyme resulting in disassociation of the cleaved fragments at a predetermined temperature.
  • the temperature of disassociation can be determined based on a calculation of the melting temperature (Tm) of the probe-target hybrid. Methods for calculating melting temperatures are well known in the art. See Sambrook et al , supra, at ⁇ 11.46-47.
  • hybridization of the probe polynucleotide to the target polynucleotide and cleavage by the extreme thermophilic enzyme is effected at . a temperature from approximately 65°C to approximately 85°C. This elevated temperature is advantageous because it allows for (i) the stringent hybridization of relatively large probes to target and (ii) the spontaneous disassociation of relatively large cleaved fragments.
  • the amount of cleaved polynucleotide probe can be determined by techniques known to the skilled artisan and discussed below.
  • the amount of cleaved probe can be quantified to indicate the amount of target polynucleotide in a given sample that contains a mutation and the size of the cleaved probe fragment indicates the site of the mutation in the target sample.
  • the present invention also includes detecting a known sequence in a target polynucleotide in a biological sample.
  • This aspect of the invention includes hybridizing a single-stranded polynucleotide probe to a target polynucleotide to form a hybrid double-stranded polynucleotide.
  • the probe is designed such that it includes a mutation, when compared to the target polynucleotide, such that the probe-target hybrid will form a mismatch.
  • the probe strand of the duplex is then cleaved at the point of mismatch.
  • the cleaved strands are then detected and it is determined based on the number and size of cleaved fragments whether the target polynucleotide contains the known sequence of interest.
  • hybridization of the probe polynucleotide to the target polynucleotide and cleavage by the extreme thermophilic enzyme is effected at a temperature from approximately 65°C to approximately 85°C. Also, as discussed above hybridization preferably occurs under stringent conditions.
  • General methodologies for detecting known sequences with a mismatch cleavage enzymes are described in U.S. Patent No. 5,656,430 and PCT Publication . WO 96/40902, supra.
  • the present invention also includes an oscillation reaction whereby the extreme thermophilic enzymes cleave the polynucleotide probe strand of the probe- target hybrid and the shortened cleaved probe fragments dissociate from the target polynucleotide at a predetermined temperature. That is, the probe is designed so that, at the predetermined temperature the probe fragments dissociate from the target polynucleotide after cleavage by the extreme thermophilic enzyme. A cycle or oscillation reaction then occurs because the target polynucleotide hybridizes to another probe and the cleavage process is repeated.
  • the oscillation reaction may be performed at an isothermal temperature, t ' .e. , the temperature of the dissociation is the same as the temperature at which mismatch cleavage occurs.
  • the oscillation reaction includes temperature cycling such that the mismatch cleavage is carried out at certain temperature and then the temperature is raised to enhance the dissociation of cleaved fragments from the target polynucleotide. The temperature is then lowered to allow hybridization of another probe and the process is repeated.
  • hybridization and mismatch cleavage are carried out from approximately 65°C to approximately 85°C, after which the temperature is raised to approximately 93°C-95°C to enhance dissociation. The temperature is then returned to approximately 65°C to approximately 85°C and the cycle is repeated one or more times and preferably ten times or more.
  • hybridization and mismatch cleavage are carried out from approximately 45°C to approximately 55°C, after which the temperature is raised to approximately 93°C-95°C to enhance dissociation. The temperature is then returned to approximately 45°C to approximately 55°C and the cycle is repeated one or more times and preferably ten times or more.
  • General methodologies for the above described oscillation reaction are described in U.S. Patent No. 5,656,430 and PCT Publication WO 96/40902, supra. .
  • the present invention also includes detecting two or more mutations in target polynucleotides and determining the relative position of each mutation.
  • This aspect of the present invention entails hybridizing single- stranded polynucleotide probes to target polynucleotides to form hybrid, double-stranded polynucleotides. The hybridization preferably occurs under stringent conditions as described above.
  • the polynucleotide probe also is labeled. Probe labeling allows for the detection of cleaved probe fragments and may be accomplished by a number of art recognized methods as discussed below. In the present invention, the probe is labeled at one end but not both. Additionally, the target polynucleotide is not labeled.
  • the probe is complementary to a non-mutated sequence of the target polynucleotide, there will be mismatches between non-mutated probe and mutated target polynucleotides at each site of the mutation.
  • the target polynucleotide will have at least two mismatch sites.
  • the probe strands of the hybrid probe-target polynucleotides then are partially digested with the extreme thermophilic enzyme of the instant invention such that probe fragments of differing lengths are generated.
  • the probe fragments are then separated by size in a medium suitable for visualizing the separated fragments.
  • the separated fragments are then visualized, sized, and the presence and relative position of the mutations are determined.
  • Methods for measuring the size of nucleotide bands in visualizing medium are known to those of skill in the art. For example, a size marker can be used to determine the size of a nucleotide band in an electrophoresis gel.
  • the polynucleotide probe is designed not to have self complementary regions or palindromic regions.
  • General parameters for probe design can be found, for example, in Lowe et al. , Nucl. Acids Res. 18: 1757-61 (1990).
  • the probe may be as large as desired, limited to the extent that it should not be so large that it may not be detected or manipulated by detection methods available to the skilled artisan.
  • the polynucleotide probe is from approximately 8 bases to approximately lkb in length.
  • the probe may be a synthetic polynucleotide, or may be derived from genomic DNA, cDNA, plasmid DNA, or DNA from pathogens as well as RNA from RNA pathogens such as HIV which may be converted to DNA by reverse transcriptase.
  • polynucleotide probes of the instant invention may be labeled by methods known to the skilled artisan.
  • the probes may be labeled with 32 P via ⁇ - 3 P-ATP and polynucleotide kinase, or with biotin, digoxigenin, horse radish peroxidase, alkaline phosphatase, or any fluorophore by procedures familiar to those experienced in the state of the art.
  • Resulting radiolabeled or fluorophore-labeled cleavage products may be detected by denaturing polyacrylamide gel electrophoresis and autoradiography or by capillary electrophoresis followed by laser induced fluorescence, respectively.
  • the probe can be labeled using fluorescence resonance energy transfer (FRET).
  • FRET refers to the transfer of electronic excitation energy from a fluorescence donor group to an unexcited acceptor group, through dipole-dipole interactions. The efficiency of this interaction depends on the orientation of the donor and acceptor, the distance between donor and acceptor, and the spectral properties of the donor and acceptor. In FRET, the energy transfer causes a decrease in the emission intensity of the donor and an increase in the intensity of the acceptor.
  • the polynucleotide probe is conjugated with two fluorophores, one on either side of the mismatch cleavage site.
  • the two fluorophores are placed close enough together so as to interact with each other through FRET, but are placed so as not to sterically prevent the cleavage activity of mismatch cleavage enzyme employed.
  • the cleavage products After cleavage by an enzyme of the present invention, the cleavage products have a melting temperature which is lower than the operating temperature of the reaction and therefore fall off the target DNA. The presence or absence of cleavage products can then be assayed by measuring a change in emission levels resulting from either the proximity (no cleavage) or distance (cleavage) of the donor and acceptor.
  • the polynucleotide probe is biotinylated.
  • the polynucleotide probe is biotinylated on its 5' end and conjugated with a fluorophore on its 3' end.
  • the probe is hybridized to a target polynucleotide and is then cleaved at a point of mismatch with an enzyme of the present invention.
  • the cleavage products have a melting temperature which is lower than the operating temperature of the reaction and therefore fall off the target.
  • polystyrene beads for example, coated with streptavidin, are added.
  • the biotinylated probe and biotinylated probe fragments bind to the beads.
  • the beads are analyzed by flow cytometry. The decrease in fluorescence of the beads as compared to a standard (e.g., a sample where no mismatch cleavage enzyme has been added or where no mismatch occurs between the probe and the target) indicates the presence of mismatch between the probe and target.
  • the probe polynucleotide is biotinylated on its 5' end and has a dideoxynucleotide on its 3' end.
  • the sequence of the probe is such that it creates a mismatch when hybridized to the mutated target polynucleotide, but not to wild-type polynucleotide.
  • the probe is mixed with target polynucleotide.
  • the polynucleotide is denatured by heating at, preferably, approximately 95 °C for 3 min, and allowed to reanneal by slow cooling to, preferably, approximately 65 °C.
  • An extreme thermophilic enzyme of the present invention is added, along with a mixture of deoxynucleotide triphosphates, one of which is conjugated with a fluorophore.
  • the mismatch cleavage enzyme cleaves the probe at the site of mismatch.
  • the newly created fragment containing the 5' biotin now has a 3' -OH which acts as a primer.
  • a polymerase for example Taq polymerase, can then be used to extend the primer using the target strand as template, incorporating a number of fluorophore molecules during the extension process.
  • the DNA is denatured, for example, by alkali and/or heat, streptavidin coated microbeads, for example, are added.
  • the biotinylated probe binds to the beads.
  • the beads are injected into a flow cytometer for analysis. An increase in the observed fluorescence on the beads indicates the probe has been cleaved. If no mismatch is present, then the probe is not cleaved and no primer extension can occur (because the 3' nucleotide on the uncut probe is a dideoxynucleotide). It is further understood that the beads can be added at the beginning of the reaction.
  • FRET fluorophores are used in conjunction with biotinylation.
  • the polynucleotide probe is biotinylated on its 5' end, a donor fluorophore is conjugated 5' of the mismatch and an acceptor fluorophore is conjugated 3' of the mismatch. Accordingly, after mismatch cleavage, the emission of the probe bound to beads is measured and an increase in the observed fluorescence indicates that the probe has been cleaved.
  • the above biotinylation embodiments allow for the use of two or more probes in a single reaction container. For example, a specific bead size is joined to a specific probe in the container, and then prior to assaying for fluorescence the beads are separated by size.
  • the probe can be covalently linked to a microbead by methods known to the skilled artisan. It is understood that such linkage is appropriately spaced from the mismatch site so that it does not sterically interfere with mismatch cleavage.
  • Target Polynucleotide is used here to denote a nucleotide sequence which may be as large as desired, limited to the extent that it should not be so large that it may not be detected or manipulated by detection methods available to the skilled artisan.
  • the target polynucleotide is from approximately 8 bases to approximately lkb in length.
  • target polynucleotides may be genomic DNA, cDNA, plasmid DNA, or DNA from pathogens as well as RNA from pathogens such as HIV which may be converted to DNA by reverse transcriptase.
  • polynucleotide targets include the genes or portions of genes which have been isolated from genomic or cDNA libraries by methods known to the skilled artisan, such as PCR amplification.
  • genes of interest would be those known to exhibit point mutations eliciting disease states. Examples include sickle cell anemia hypoxanthine phosphotransferase and p53, a tumor suppressor gene, as well as several oncogenes and cancer genes.
  • Other illustrations of polynucleotide targets include cDNAs generated by reverse transcriptase, and a specific deoxyoligonucleotide primer encompassing a short sequence immediately downstream of a hotspot for mutations within a gene such as BRCA1. J. Detecting the Presence or Absence of a Mismatch in a Polynucleotide Duplex
  • the present invention includes using the extreme thermophilic enzymes described herein to detect the presence or absence of a mismatch in a polynucleotide duplex.
  • This detection entails providing a first and second polynucleotide both of which are single-stranded, hybridizing the first and second polynucleotides to form a duplex, and contacting the duplex with the inventive extreme thermophilic enzyme. Cleavage will result if there is a mismatch in the duplex. Such cleavage or lack of cleavage indicates the presence or absence of a mismatch in the duplex.
  • the first and second single-stranded polynucleotides may comprise any two nucleotides for which one wishes to determine if one or more mismatches is created in a duplex formed between the two nucleotides.
  • a determination that one or more mismatches exists in a duplex formed between the first and second polynucleotides will inform a skilled artisan as to the relative sequences of the first and second polynucleotides based on what is already known concerning the two sequences.
  • the first and second polynucleotides can be obtained from any appropriate two sources of DNA for which a comparison is sought, for example, from genomic DNA, cDNA, plasmid DNA, or DNA from pathogens as well as RNA from RNA pathogens such as HIV which may be converted to DNA by reverse transcriptase.
  • the size of the first and second nucleotides may be as large as desired, limited to the extent that it should not be so large that it may not be detected or manipulated by detection methods available to the skilled artisan.
  • the first and second nucleotides are from approximately 8 bases to approximately lkb in length.
  • the first and second sequences may be, respectively, a test DNA sequence and a control DNA sequence.
  • the objective here would be to see if the test sequence contains a mutation relative to the control sequence.
  • the test sequence may be derived from a patient's DNA and the control sequence from a source of DNA that is believed to represent the wild-type, i.e. , non- mutated sequences for a gene of interest.
  • the hybridization of the first and second nucleotides occurs under stringent conditions as described above.
  • the temperature at which cleavage occurs is from approximately 65°C to approximately 85°C.
  • Labeling and detection methods for detecting the presence or absence of cleavage products are known to the skilled artisan. For instance, the labeling and detection methods described above may be employed. Such labeling and detection methods will indicate the presence or absence of a mismatch in the duplex based on an analysis of the cleavage products.
  • the present invention includes using the extreme thermophilic enzymes described herein to cleave mismatches that are created during amplification of DNA sequences by Polymerase Chain Reaction (PCR) using Taq polymerase or other extreme thermophilic DNA polymerases.
  • PCR Polymerase Chain Reaction
  • the term "Polymerase Chain Reaction” is used here to include any amplification methodologies that employs annealing of primers to target nucleotides and then thermocycling and reannealing. This would include conventional PCR as well as modifications to conventional PCR.
  • Taq DNA Polymerase incorporates approximately one incorrect base per 500 bases, an unacceptably high error rate if long stretches of DNA (greater than 500 bp) are to be amplified. If the incorrect base is incorporated in the early cycles, the majority of the amplicons will display this erroneous base.
  • the addition of the extreme thermophilic enzymes described herein can be used to correct the error as the incorrect base is incorporated.
  • the extreme thermophilic enzyme will cleave either the template strand or the growing daughter strand; these cleavage products are then no longer capable of exponential amplification in subsequent PCR cycles.
  • the present invention further includes a chimera of an extreme thermophilic
  • TM-Endo V can be covalently linked to Thermus aquaticus DNA polymerase, either by the use of a cross-linking reagent, or by incorporating a recombinant TM-Endo V DNA sequence upstream or downstream of a Taq DNA polymerase DNA sequence in an
  • Sample Thermotoga neopolitana was obtained from Dr. Robert Kelly at North Carolina State University. Two grams of Thermotoga neopolitana were suspended in 35 ml of 50 mM Tris-Cl, pH 7.8. 1 mM dithiothreitol, 1 mM EDTA, and 0.1 mM PMSF (Buffer A) and lysed by repeated passage through a French press.
  • the homogenate was clarified by centrifugation and the supernatant was applied to a 30 ml column of phosphocellulose (BioRad Pl l), previously equilibrated with Buffer A.
  • the column was developed as follows: (1) Buffer A, 200 ml; (2) a
  • Example 2 Fractions as obtained in Example 1 were screened for mismatch repair activity using 32p-labeled oligonucleotide pairs in a buffer consisting of 50 mM Tris-Cl, pH 7.8, 100 mM KC1, 10 mM EDTA, a temperature of 65°C for 1 hr, and resolving the cleavage products by 20% urea denaturing polyacrylamide gel electrophoresis and autoradiography. Activity was present in the fractions containing protein that did not adhere to the phosphocellulose column.
  • Mismatch activity was assayed as follows. Oligonucleotides of 27 or 37 bases were radiolabeled on 5' ends with ⁇ - 32 P-ATP and polynucleotide kinase. Appropriate oligonucleotides were paired in order to generate the mismatches and three base bubble indicated in Figure 1 (as well as an A/G mismatch). The three base bubble and the T/G mismatch of this Example and those that follow was created using the following oligonucleotides:
  • RO620 3 ' -TGTCTACCCTGTGATTCTAAAAGAC-5 ' Mutant RO618 was used to hybridize to RO619 or to RO620 to generate a three base bubble or a T/G mismatch, respectively.
  • the reactions (20 ⁇ l) contained 1 pmole of 32 P-oligonucleotide duplex, and l ⁇ l of lysis buffer or T. Neopolitana flow-through fraction from the phosphocellulose column.
  • the tubes were incubated at 65°C for 1 hr in a buffer containing 50 mM Tris-Cl, pH 7.8, lOOmM KCl, 10 mM EDTA.
  • the reactions were terminated by the addition of 6 ⁇ l of a 4 X loading buffer (95% formamide, 100 mM NaOH, 0.1 % bromophenol blue) and the DNA was denatured by heating at 95°C for 3 min.
  • the samples were loaded onto a 20% denaturing polyacrylamide gel, electrophoresed at 250 volts, and subjected to autoradiography. Cleavage products were evident in all the mismatches tested A/G, T/G, C/C, G/G, T/C, A/C, A/A, T/T, and a 3 base bubble. No cleavage products were observed with oligonucleotide duplexes which lack base mismatches or a bubble.
  • T. maritima was obtained from Dr. Robert Kelly at North Carolina State University. Five grams of T. maritima were suspended in 35 ml of 25 mM Tris-Cl, pH 7.8, ImM dithiothreitol, ImM MgCh, and 0.1 mM PMSF (Buffer A). Lysis of the T. maritima was achieved by passage through a French press.
  • Endogenous DNA was cleaved to small fragments by the addition of Benzonase to a final concentration of 1 unit/ml and incubation at 25°C for 1 hr.
  • the homogenate was clarified by centrifugation and applied to a 30 ml column of Q Sepharose fast flow (Pharmacia), previously equilibrated with 25mM Tris-CL, pH 7.8, ImM dithiothreitol, ImM EDTA and O. lmM PMSF (Buffer B).
  • the column was developed as follows: (1) Buffer B, 200 ml; (2) a 300 ml linear gradient from Buffer B to Buffer B + 1.0 M KCl Fractions of 6 ml were collected and assayed according to the methods of Example 2 and similar results were obtained, i.e. , the composition ⁇ cleaved A/G, T/G, C/C, G/G, T/C, A/C, A/A, T/T, and a 3 base bubble, but no cleavage products were observed with oligonucleotide duplexes which lacked base mismatches or a bubble.
  • composition from Thermotoga maritima Composition from Thermotoga maritima
  • Oligonucleotides 1-4 as designated in Figure 2 were end labeled with 32 P-ATP and polynucleotide kinase.
  • Labeled oligonucleotide 1 was mixed with equal amounts of unlabeled oligonucleotides 2, 3, and 4.
  • Labeled oligonucleotide 2 was mixed with equal amounts of unlabeled oligonucleotides 1, 3, and 4.
  • Labeled oligonucleotide 3 was mixed with unlabeled oligonucleotides 1, 2, and 4.
  • Labeled oligonucleotide 4 was mixed with unlabeled oligonucleotides 1, 2, and 3.
  • the oligonucleotide mixtures were heated at 95°C for five minutes and slowly cooled to room temperature to create four of the four-way junctions shown in Figure 2, each labeled on one of the four oligonucleotide strands.
  • the T. maritima enzyme composition isolated by the methods of Example 3 was then added and the reaction proceeded at 65°C for 1 hr in a buffer consisting of 50 mM Tris-Cl, pH 7.8, 100 mM KCl, 10 mM EDTA.
  • the T. maritima enzyme composition isolated by the methods of Example 3 was tested for extreme thermophilic activity in a cycling reaction as follows.
  • a series of tubes were set up containing 2 ⁇ l of 10 X Buffer (500 mM Tris-Cl, pH 7.8, 1000 mM KCl, 100 mM EDTA), 15 ⁇ l deionized water, 1 ⁇ l enzyme composition.
  • the tubes were heated at 95 °C for 30 sec, and at 65 °C for 60 sec (one cycle) for 0, 1, 2, 5, 10, 20 cycles.
  • the following was then added to the tubes: 1 ⁇ l of 200 .
  • These oligonucleotides were designed to form a C/C mismatch in the middle of the sequence.
  • the tubes were incubated at 65 °C for 1 hr. The reactions were terminated with 10 ⁇ l of 95% formamide, 100 mM NaOH, and 0.2% bromophenol blue. The tubes were heated at 95 °C for 3 min, cooled to room temp. , and loaded onto a 20% denaturing polyacrylamide gel. The electrophoresis was done at 400 volts for 2 hr. The gel was autoradiographed for analysis. The enzyme composition cleaved the labeled oligonucleotide even after 20 temperature cycles without any loss in activity.
  • TM Thermotoga maritima
  • the PCR product was cut with Pstl and Hindlll, gel purified on 1.2% agarose gels, and cloned in frame into the Pstl-Hindlll site of pBAD/His C (Invitrogen), yielding the plasmid pBAD/HISC/tm-endoV.
  • the LMG104 strain of E. coli was transformed with pBAD/HISC/tm-endoV. Plasmid minipreps of several clones of pBAD/HISC/tm-endoV were analyzed by restriction with Pstl and Hind III to confirm the presence of the insert.
  • One clone was selected for protein expression and purification.
  • coli was harvested and lysed with 80 ml of 50 mM KPI, pH 7.2, 500 mM NaCI, 0.5% Triton X-100 (Buffer A) containing 0.2 mM PMSF using a laboratory press. The homogenate was centrifuged at 27,000 x g for 30 min at 4°C. To the supernatant was added 30 ml of Pro Bond Nickle Resin (Invitrogen), previously equilibrated with Buffer A. Batch absorption to the beads continued with stirring for 30 min. The resin was poured into a column and washed with Buffer A (200 ml) and Buffer E + 5 mM Imidazole (Buffer B, 100 ml).
  • Buffer A 200 ml
  • the assay was performed by hybridizing 32 P -labeled oligonucleotide probes (100 fmoles) to complementary oligonucleotide targets (lpmole) to generate base mismatches A/A, T/T, T/G, C/C, G/G, T/C, A/C, A/G, no mismatch, and 3 base bubble. Reactions were carried out for 1 hour at 60°C with 10 ng of TM-Endo V in a volume of 20 ⁇ l in 50nm Tris-Cl, pH 7.8, 100 mM KCL, 10 mM EDTA. Cleavage products were analyzed by 20% denaturing PAGE and autoradiography and are shown in Figures 4A and 4B.
  • TM-Endo V cleaved C/C, G/G, T/C, A/C, A/A, and T/T mismatch.
  • the enzyme did not cleave a perfectly complementary oligonucleotide pair (no mismatch), a T/G mismatch or a three base bubble.
  • TM-Endo V cleaves a A/G mismatch according to the above protocol and in a further experiment it was found that TM- Endo V cleaved a 3 base bubble as well a T/G mismatch, when used in conjunction with dimethyl sulfoxide (DMSO) at 10% volume to volume.
  • DMSO dimethyl sulfoxide
  • TM-Endo V Resolvase activity of TM-Endo V was assayed following the protocol of Example 4.
  • Oligonucleotides 1-4 as designated in Figure 2 were end labeled with 32 P-ATP and polynucleotide kinase.
  • Labeled oligonucleotide 1 (100 fmoles) was mixed with (500 fmoles each of) unlabeled oligonucleotides 2, 3, and 4.
  • Labeled oligonucleotide 2 was mixed with unlabeled oligonucleotides 1, 3, and 4.
  • Labeled oligonucleotide 3 was mixed with unlabeled oligonucleotides 1, 2, and 4.
  • Labeled oligonucleotide 4 was mixed with unlabeled oligonucleotides 1, 2, and 3. Reactions were carried out in volumes of 20 ⁇ l at 65 °C for 1 hr in the presence and absence of l ⁇ l of recombinant TM-Endo V in a 1 X buffer consisting of 50 mM Tris-Cl, pH 7.8, 100 mM KCl, 10 mM EDTA. To test that the branched structure had indeed formed, the DNA was treated in those tubes which had either radiolabeled oligonucleotide 1 or oligonucleotide 4 with EcoRI at 37°C for 1 hr. The cleavage products were analyzed by 20% denaturing PAGE and autoradiography.

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Abstract

La présente invention concerne des enzymes de clivage incompatibles extrêmement thermophiles, ainsi que leurs utilisations.
PCT/US1999/003274 1998-02-19 1999-02-19 Enzymes de clivage incompatibles provenant de microorganismes extremement thermophiles et leurs utilisations WO1999042595A1 (fr)

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