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  • 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
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/82—Translation products from oncogenes
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • 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
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  • 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]

Definitions

• the present invention relates to methods and compositions for treating nucleic acid. and in particular, methods and compositions for detection and characterization of nucleic acid sequences and sequence changes.
• nucleic acid sequences and sequence changes have been utilized to detect the presence of viral or bacterial nucleic acid sequences indicative of an infection, the presence of variants or alleles of mammalian genes associated with disease and cancers, and the identification of the source of nucleic acids found in forensic samples, as well as in paternity determinations.
• Various methods are known in the art which may be used to detect and characterize specific nucleic acid sequences and sequence changes. Nonetheless, as nucleic acid sequence data of the human genome, as well as the genomes of pathogenic organisms accumulates, the demand for fast, reliable, cost-effective and user-friendly tests for specific sequences continues to grow.
• PCR Polymerase Chain Reaction
• PCR polymerase chain reaction
• PCR can be used to directly increase the concentration of the target to an easily detectable level.
• This process for amplifying the target sequence involves introducing a molar excess o two oligonucleotide primers which are complementary to their respective strands of the double-stranded target sequence to the DNA mixture containing the desired target sequence. The mixture is denatured and then allowed to hybridize. Following hybridization, the prime are extended with polymerase so as to form complementary strands. The steps of denaturation, hybridization, and polymerase extension can be repeated as often as needed, in order to obtain relatively high concentrations of a segment of the desired target sequence.
• the length of the segment of the desired target sequence is determined by the relativ positions of the primers with respect to each other, and. therefore, this length is a controllabl parameter. Because the desired segments of the target sequence become the dominant sequences (in terms of concentration) in the mixture, they are said to be "PCR-amplified.”
• LCR Ligase Chain Reaction
• LAR ligase chain reaction
• LCR LCR has also bee used in combination with PCR to achieve enhanced detection of single-base changes. Segev PCT Public. No. W09001069 Al (1990). However, because the four oligonucleotides used this assay can pair to form two short ligatable fragments, there is the potential lor the generation of target-independent background signal. The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
• an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5 ' end of the sequence of interest.
• a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase. RNase H. RNA polymerase and ribo-and deoxyribonucleoside triphosphates.
• the target sequence undergoes repeated rounds of transcription.
• cDNA synthesis and second-strand synthesis to amplify the area of interest.
• the use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g.. 200-300 base pairs).
• a probe which recognizes the sequence of interest is attached to the replicatable RNA template for Q ⁇ replicase.
• a previously identified major problem with false positives resulting from the replication of unhybridized probes has been addressed through use of a sequence-specific ligation step.
• available thermostable DNA ligases are not effective on this RNA substrate, so the ligation must be performed by T4 DNA ligase at low temperatures (37°C). This prevents the use of high temperature as a means of achieving specificity as in the LCR. the ligation event can be used to detect a mutation at the junction site, but not elsewhere.
• Table 1 lists some of the features desirable for systems useful in sensitive nucleic acid diagnostics, and summarizes the abilities of each of the major amplification methods (See also. Landgren. Trends in Genetics 9: 199 [1993]).
• a successful diagnostic method must be very specific.
• a straight-forward method of controlling the specificity of nucleic acid hybridization is by controlling the temperature of the reaction. While the 3SR/NASBA. and Q ⁇ systems are all able to generate a large quantity of signal, one or more of the enzymes involved in each cannot be used at high temperature (i.e.. >55°C). Therefore the reaction temperatures cannot be raised to prevent non-specific hybridization of the probes. If probes are shortened in order to make them melt more easily at low temperatures, the likelihood of having more than one perfect match in a complex genome increases. For these reasons. PCR and LCR currently dominate the research field in detection technologies.
• the basis of the amplification procedure in the PCR and LCR is the fact that the products of one cycle become usable templates in all subsequent cycles, consequently doubling the population with each cycle.
• "X" is the mean efficiency (percent copied in each cycle)
• "n” is the number of cycles
• "y” is the overall efficiency, or yield of the reaction (Mullis. PCR Methods Applic, 1 : 1 [1991 ]). If every copy of a target DNA is utilized as a template i every cycle of a polymerase chain reaction, then the mean efficiency is 100%. If 20 cycles o PCR are performed, then the yield will be 2 :o .
• nucleic acid detection technologies such as in studies of allelic variation, involve not only detection of a specific sequence in a complex background, but also the discrimination between sequences with few. or single, nucleotide differences.
• One method for the detection of allele-specific variants by PCR is based upon the fact that it is difficult for Tag polymerase to synthesize a DNA strand when there is a mismatch between the template strand and the 3 " end of the primer.
• An allele-specific variant may be detected by the use of a primer that is perfectly matched with only one of the possible alleles; the mismatch to the other allele acts to prevent the extension of the primer, thereby preventing the amplification of that sequence.
• the cycling probe reaction (CPR) (Duck et al.. BioTech., 9:142 [1990]). uses a long chimeric oligonucleotide in which a central portion is made of RNA while the two termini ar made of DNA. Hybridization of the probe to a target DNA and exposure to a thermostable RNase H causes the RNA portion to be digested. This destabilizes the remaining DNA portions of the duplex, releasing the remainder of the probe from the target DNA and allowing another probe molecule to repeat the process. The signal, in the form of cleaved probe molecules, accumulates at a linear rate.
• RNA portion of the oligonucleotide is vulnerable to RNases that may carried through sample preparation.
• Branched DNA branched DNA (bDNA). described by Urdea et al.. Gene 61 :253-264 (1987). involve oligonucleotides with branched structures that allow each individual oligonucleotide to carry 35 to 40 labels (e.g.. alkaline phosphatase enzymes). While this enhances the signal from a hybridization event, signal from non-specific binding is similarly increased.
• nucleic acid segments for mutations.
• One option is to determine the entire gene sequence of each test sample (e.g.. a bacterial isolate). For sequences under approximately 600 nucleotides. this may be accomplished using amplified material (e.g.. PCR reaction products). This avoids the time and expense associated with cloning the segment of interest.
• amplified material e.g.. PCR reaction products
• a given segment of nucleic acid may be characterized on several other levels.
• the size of the molecule can be determined by electrophoresis by comparison to a known standard run on the same gel. A more detailed picture of the molecule may be achieved by cleavage with combinations of restriction enzymes prior to electrophoresis. to allow construction of an ordered map. The presence of specific sequences within the fragment can be detected by hybridization of a labeled probe, or the precise nucleotide sequence can be determined by partial chemical degradation or by primer extension in the presence of chain-terminating nucleotide analogs.
• RFLPs Mutations are detected and localized by the presence and size of the RNA fragments generated by cleavage at the mismatches. Single nucleotide mismatches in DNA heteroduplexes are also recognized and cleaved by some chemicals, providing an alternative strategy to detect single base substitutions, generically named the "Mismatch Chemical Cleavage” (MCC) (Gogos et al.. Nucl. Acids Res.. 18:6807-6817 [ 1990]).
• MCC Mismatch Chemical Cleavage
• this method requires the use of osmium tetroxide and piperidine. Two highly noxious chemicals which are not suited for use in a clinical laboratory.
• RFLP analysis suffers from low sensitivity and requires a large amount of sample.
• ASOs can be designed to hybridize in proximity to the unknown nucleotide. such that a primer extension or ligation event can be used as the indicator of a match or a mis-match.
• Hybridization with radioactively labeled allelic specific oligonucleotides (ASO) also has been applied to the detection of specific point mutations (Conner et al . Proc. Natl. Acad. Sci.. 80:278-282 [1983]). The method is based on the differences in the melting temperature of short DNA fragments differing by a single nucleotide. Stringent hybridization and washing conditions can differentiate between mutant and wild-type alleles.
• the ASO method requires the use of many oligonucleotides to cover all possible oncogenic mutations.
• the precise location of the suspected mutation must be known in advance of the test. That is to say. they are inapplicable when one needs to detect the presence of a mutation of an unknown character and position within a gene or sequence of interest.
• Two other methods rely on detecting changes in electrophoretic mobility in response to minor sequence changes.
• DGGE Denaturing Gradient Gel Electrophoresis
• DGGE constant denaturant gel electrophoresis
• CDGE requires that gels be performed under different denaturant conditions in order to reach high efficiency for the detection of unknown mutations.
• An technique analogous to DGGE. termed temperature gradient gel electrophoresis (TGGE) uses a thermal gradient rather than a chemical denaturant gradient (Scholz. et al.. Hum. Mol. Genet. 2:2155 [1993]).
• TGGE requires the use of specialized equipment which can generate a temperature gradient perpendicularly oriented relative to the electrical field. TGGE can detect mutations in relatively small fragments of DNA therefore scanning of large gene segments requires the use of multiple PCR products prior to running the gel.
• SSCP Single-Strand Conformation Polymorphism
• the SSCP process involves denaturing a DNA segment (e.g. , a PCR product) that is labelled on both strands, followed by slow electrophoretic separation on a non-denaturing polyacrylamide gel. so that intra-molecular interactions can form and not be disturbed during the run.
• This technique is extremely sensitive to variations in gel composition and temperature.
• a serious limitation of this method is the relative difficulty encountered in comparing data generated in different laboratories, under apparently similar conditions.
• the dideoxy fingerprinting is another technique developed to scan genes for th presence of unknown mutations (Liu and Sommer, PCR Methods Appli.. 4:97 [1994]).
• the ddF technique combines components of Sanger dideoxy sequencing with SSCP.
• a dideoxy sequencing reaction is performed using one dideoxy terminator and then the reaction product are electrophoresised on nondenaturing polyacrylamide gels to detect alterations in mobility the termination segments as in SSCP analysis.
• ddF is an improvement over SSCP in terms of increased sensitivity.
• ddF requires the use of expensive dideoxynucleotides and this technique is still limited to the analysis of fragments of the size suitable for SSCP (i.e.. fragments of 200-300 bases for optimal detection of mutations).
• the present invention relates to methods and compositions for treating nucleic acid. and in particular, methods and compositions for detection and characterization of nucleic acid sequences and sequence changes in human gene sequences and in microbial gene sequences.
• the present invention provides means for cleaving a nucleic acid cleavage structure in a site- specific manner.
• the means for cleaving is an enzyme capable of cleaving cleavage structures on a nucleic acid substrate, forming the basis of a novel method of detection of specific nucleic acid sequences.
• the present invention contemplates use of the novel detection method for. among other uses, clinical diagnostic purposes, including but not limited to detection and identification of 1) mutations in human gene sequences and 2) pathogenic organisms.
• the present invention contemplates a DNA sequence encoding a
• DNA polymerase altered in sequence i.e.. a "mutant" DNA polymerase
• DNA synthetic activity from that of the native (i.e.. "wild type") DNA polymerase.
• the enzymes of the invention are nucleases and are capable of cleaving nucleic acids in a structure-specific manner.
• the nucleases of the present invention are capable of cleaving cleavage structures to create discre cleavage products.
• the present invention contemplates nucleases from a variety of sources, including nucleases that are thermostable.
• Thermostable nucleases are contemplated as particularly useful, as they are capable of operating at temperatures where nucleic acid hybridization is extremely specific, allowing for allele-specific detection (including single-base mismatches).
• thermostable 5 ' nucleases are selected from the group consisting of altered polymerases derived from the native polymerases of various Thermits species, including, but not limited to Thermus aquaticus. Thermus flavus and Thermus thermophilus.
• the present invention is not limited to the use of thermostable nucleases.
• nucleases from mesophilic organisms may also be employed in the methods of the invention (e.g., E. coli Exo III. Saccharomyces cerevisiae Radi/Radl O complex).
• the present invention utilizes nucleases in methods for detection and characterization of nucleic acid sequences and sequence changes.
• the present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner. Nuclease activity is use to screen for known and unknown mutations, including single base changes, in nucleic acids.
• the present invention contemplates a method for treating nucleic acid, comprising: a) providing: i) a cleavage means and ii) nucleic acid substrate: b) treating the nucleic acid substrate under conditions such that the substrate forms one or more cleava structures; and c) reacting the cleavage means with the cleavage structures so that one or mo cleavage products are produced.
• the cleavage means is an enzyme.
• th cleavage means is a nuclease.
• the nuclease is select from the group consisting of the CleavaseTM BN enzyme. Thermus aquaticus DNA polymerase. Thermus thermophilus DNA polymerase. Escherichia coli Exo III. and the
• the nucleic acid substrate comprise a nucleotide analog, including but not limited to the group comprising 7-deaza-dATP. 7-deaza-dGTP and dUTP.
• the nucleic acid of step is substantially single-stranded. It is not intended that the nucleic acid substrate be limited to any particular form, indeed, it is contemplated that the nucleic acid substrate is single stranded or double stranded DNA or RNA.
• the treating step (b) comprises rendering the double-stranded nucleic acid substantially single- stranded and exposing the single-stranded nucleic acid to conditions such that the single- stranded nucleic acid assumes a secondary or characteristic folded structure.
• the double stranded nucleic acid is rendered substantially single-stranded by increased temperature.
• the method of the present invention further comprises the step of detecting said one or more cleavage products.
• the nucleic acid substrate comprises an oligonucleotide containing human p53 gene sequences. In an alternative embodiment, the nucleic acid substrate comprises an oligonucleotide containing microbial gene sequences.
• the present invention contemplates further a method for treating nucleic acid, comprising: a) providing: i) a cleavage means in a solution comprising manganese and ii) a nucleic acid substrate; b) treating the nucleic acid substrate with increased temperature; c) reducing the temperature under conditions such that the substrate forms one or more cleavage structures; d) reacting the cleavage means with the cleavage structures so that one or more cleavage products are produced: and e) detecting the cleavage products.
• the cleavage means may be an enzyme.
• the cleavage means may be a nuclease.
• the nuclease is selected from the group consisting of the CleavaseTM BN enzyme.
• Thermus aquaticus DNA polymerase Thermus thermophilus DNA polymerase, Escherichia coli Exo III. and the Saccharomyces cerevisiae Radl/Radl O complex.
• the nucleic acid substrate comprise a nucleotide analog, including, but not limited to, the group comprising 7-deaza-dATP, 7-deaza-dGTP and dUTP.
• the nucleic acid of step is substantially single-stranded. It is not intended that the nucleic acid substrate be limited to any particular form, indeed, it is contemplated that the nucleic acid substrate is single stranded or double stranded DNA or RNA.
• the nucleic acid substrate comprises an oligonucleotide containing human p53 gene sequences. In an alternative embodiment, the nucleic acid substrate comprises an oligonucleotide containing microbial gene sequences.
• the present invention contemplates further, a method for detecting mutation in the human p53 gene, comprising: a) providing: i) a cleavage means and ii) a nucleic acid substrate containing human p53 gene sequences; b) treating the nucleic acid substrate under conditions such that the substrate forms one or more cleavage structures; c) reacting the cleavage means with the cleavage structures so that one or more cleavage products are produced; and d) comparing said cleavage products to the cleavage products produced by cleavage of a reference p53 gene sequence.
• the cleavage products produced by cleavage of a reference p53 gene sequence are generated by the cleavage of a nucleic acid substrate containing the human p53 gene sequences selected from the group consisting of SEQ ID NOS:79-81. 84-89 and 94-97. Additional p53 mutant sequences are provided herein; SEQ ID NO:79 lists the sequence of the wild-type p53 cDNA. Table 2 below provides the identity and location of numerous known p53 mutations. Combination of the information in Table 2 with the sequence of the wild-type p53 cDNA in SEQ ID NO:79 allows the generation of the complet nucleotide sequence for cDNAs corresponding to the numerous p53 mutations described in Table 2.
• the method of the invention permits the screening of or "scanning" for heretofore uncharacterized mutations within human gene sequences, such as the human p53 gene.
• the present invention also contemplates a process for creating a record reference library of genetic fingerprints characteristic (i.e.. diagnostic) of one or more alleles of the human p53 gene comprising: a) providing: i) a cleavage means: and ii) nucleic acid substrate derived from human p53 gene sequences; b) contacting said nucleic acid substrate with a cleavage means under conditions such that said extracted nucleic acid forms one or more secondary structures and said cleavage means cleaves said secondary structures resulting in th generation of multiple cleavage products; c) separating said multiple cleavage products: and d maintaining a testable record reference of said separated cleavage products.
• genetic fingerprint it is meant that changes in the sequence of the nucleic acid (e.g.. a deletion, insertion or a single point substitution) alter the structures formed, thus changing the banding pattern (i.e., the "fingerprint” or "bar code”) to reflect the difference in the sequence, allowing rapid detection and identification of variants.
• the present invention also contemplates a process for creating a record reference library of genetic fingerprints characteristic (i.e.. diagnostic) of one or more alleles of one or more genes from a eukaryotic organism (e.g..).
• mammals comprising: a) providing: i) a cleavage means; and ii) nucleic acid substrate derived from one or more alleles of a gene derived from a eukaryotic organism; b) contacting said nucleic acid substrate with a cleavage means under conditions such that said extracted nucleic acid forms one or more secondary structures and said cleavage means cleaves said secondary structures resulting in the generation of multiple cleavage products; c) separating said multiple cleavage products: and d) maintaining a testable record reference of said separated cleavage products.
• the present invention also contemplates a method for identifying strains of microorganisms comprising: a) providing i) a cleavage means; and ii) a nucleic acid substrate containing sequences derived from one or more microorganism; b) treating said nucleic acid substrate under conditions such that said substrate forms one or more cleavage structures: and c) reacting said cleavage means with said cleavage structures so that one or more cleavage products are produced.
• the preferred cleavage means is an enzyme, such as a nuclease.
• enzymes that can be used with success with the method of the present invention include (but are not limited to) the CleavaseTM BN enzyme, Thermus aquaticus DNA polymerase. Thermus thermophilus DNA polymerase. Escherichia coli Exo III. and the Saccharomyces cerevisiae Radl /Radl O complex.
• the nucleic acid substrate comprise a nucleotide analog, including but not limited to the group comprising 7-deaza-dATP, 7-deaza-dGTP and dUTP.
• the nucleic acid of step is substantially single-stranded. It is not intended that the nucleic acid substrate be limited to any particular form, indeed, it is contemplated that the nucleic acid substrate is single stranded or double stranded DNA or RNA.
• the treating step (b) comprises rendering the double-stranded nucleic acid substantially single- stranded and exposing the single-stranded nucleic acid to conditions such that the single- stranded nucleic acid assumes a secondary or characteristic folded structure.
• the double stranded nucleic acid is rendered substantially single-stranded by increased temperature.
• the method of the present invention further comprises the step of detecting said one or more cleavage products.
• the microorganism(s) of the present invention be selected from a variety of microorganisms; it is not intended that the present invention be limited to am particular type of microorganism. Rather, it is intended that the present invention will be used with organisms including, but not limited to, bacteria, fungi, protozoa, ciliates. and viruses. It is not intended that the microorganisms be limited to a particular genus, species, strain, or serotype. Indeed, it is contemplated that the bacteria be selected from the group comprising, but not limited to members of the genera Campylobacter, Escherichia. Mycohacterium, Salmonella. Shigella,and Staphylococcus.
• the microorganism(s) comprise strains of multi-drug resistant Mycohacterium tuberculosis. It is also contemplated that the present invention be used with viruses, including but not limited to hepatitis C virus and simian immunodeficiency virus.
• Another embodiment of the present invention contemplates a method for detecting and identifying strains of microorganisms, comprising the steps of extracting nucleic acid from a sample suspected of containing one or more microorganisms and contacting the extracted nucleic acid with a cleavage means under conditions such that the extracted nucleic acid forms one or more secondary structures, and the cleavage means cleaves the secondary structures to produce one or more cleavage products.
• the method further comprises the step of separating said cleavage products. In yet another embodiment, the method further comprises the step of detecting said cleavage products.
• the present invention further comprises comparing said detected cleavage products generated from cleavage of the extracted nucleic acid isolated fro the sample with separated cleavage products generated by cleavage of nucleic acids derived from one or more reference microorganisms.
• the sequence of the nucleic acid from one or more reference microorganisms may be related but different (e.g.. a wild-type control for a mutant sequence or a known or previously characterized mutant sequence).
• the present invention further comprises the step of isolating a polymorphic locus from said extracted nucleic acid after the extraction ste so as to generate a nucleic acid substrate, wherein the substrate is contacted with the cleavag means.
• the isolation of a polymorphic locus is accomplished by nucleic acid amplification.
• the invention is limited by the method of nucleic acid amplification employed.
• One method of achieving nucleic acid amplification is the polymerase chain reaction.
• the nucleic acid amplification is conducted in the presence of a nucleotide analog, including but not limited to the group comprising 7-deaza- dATP. 7-deaza-dGTP and dUTP.
• the nucleic acid amplification (e.g.. PCR) will employ oligonucleotide primers which either 1 ) match consensus gene sequences derived from the polymo ⁇ hic locus (i.e.. the primers comprise the same sequence found on a strand of nucleic acid derived from the polymo ⁇ hic locus) or 2) are complementary to consensus gene sequences derived from said polymo ⁇ hic locus (i.e.. they are the complement to a strand of nucleic acid derived from the polymo ⁇ hic locus).
• the polymo ⁇ hic locus comprises a ribosomal RNA gene.
• the ribosomal RNA gene is a 16S ribosomal RNA gene.
• the cleavage means is an enzyme, such as a nuclease.
• the nuclease is selected from the group including, but not limited to CleavaseTM BN, Thermus aquaticus DNA polymerase. Thermus thermophilus DNA polymerase, Escherichia coli Exo III. and the Saccharomyces cerevisiae Radl /Radl O complex.
• the enzyme may have a portion of its amino acid sequence that is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase derived from a eubacterial thermophile. the latter being selected from the group consisting of Thermus aquaticus. Thermus flavus and Thermus thermophilus.
• nucleic acid substrate is single stranded or double-stranded
• the treating step of the method may comprise rendering double-stranded nucleic acid substantially single- stranded, and exposing the single-stranded nucleic acid to conditions such that the single- stranded nucleic acid has secondary structure.
• double-stranded nucleic acid is rendered substantially single-stranded by increased temperature.
• the microorganism(s) of the present invention be selected from a variety of microorganisms; it is not intended that the present invention be limited to any particular type of microorganism. Rather, it is intended that the present invention will be used with organisms including, but not limited to. bacteria, fungi, protozoa, ciliates. and viruses. It is not intended that the microorganisms be limited to a particular genus, species. strain, or serotype. Indeed, it is contemplated that the bacteria be selected from the group comprising, but not limited to members of the genera Campylohacter, Escherichia, Mycohacterium. Salmonella. Shigella.and St ⁇ phylococcus.
• the microorganism(s) comprise strains of multi-drug resistant Mycohacterium tuberculosis. It is also contemplated that the present invention be used with viruses, including but not limited t hepatitis C virus and simian immunodeficiency virus.
• the present invention also contemplates a process for creating a record reference library of genetic fmge ⁇ rints characteristic (i.e. , diagnostic) of one or more alleles of the various microorganisms, comprising the steps of providing a cleavage means and nucleic acid substrate derived from microbial gene sequences; contacting the nucleic acid substrate with a cleavage means under conditions such that the extracted nucleic acid forms one or more secondary structures and the cleavage means cleaves the secondary structures, resulting in the generation of multiple cleavage products; separating the multiple cleavage products: and maintaining a testable record reference of the separated cleavage products.
• the detection and identification is application to all organisms, including viruses and bacteria.
• the present invention also contemplates a process for creating a record reference (e.g. library) of genetic fingerprints characteristic (i.e.. diagnostic) of pathogenic microorganisms comprising: a) providing: i) a cleavage means; and ii) a nucleic acid substrate characteristic o (e.g..
• the present invention also contemplates a nucleic acid treatment kit.
• a nucleic acid treatment kit comprising: a) a enzyme capable of reacting with cleavage structures so as to generate cleavage products, and b) a solution comprising manganese.
• the enzyme of the kit may be a nuclease.
• the nuclease is elected from the group including, but not limited to CleavaseTM BN.
• Thermus aquaticus DNA polymerase Thermus thermophilus DNA polymerase. Escherichia coli Exo III. and the Saccharomyces cerevisiae Radl/RadlO comple
• the present invention contemplates other reagents useful for the treatment of nucleic acid.
• the kit may include reagents for detecting said cleavage products.
• the kit may include reagents for the cleavage reaction including salt solutions ⁇ e.g.. KG and NaCl solutions), manganese chloride solutions, buffer solutions and solutions which terminate the cleavage reaction.
• the methods of the present invention allow for simultaneous analysis of both strands (e.g.. the sense and antisense strands) and are ideal for high-level multiplexing.
• the products produced are amenable to qualitative, quantitative and positional analysis.
• the methods may be automated and may be practiced in solution or in the solid phase (e.g.. on a solid support).
• the methods are powerful in that they allow for analysis of longer fragments of nucleic acid than current methodologies.
• Figure 1 is a comparison of the nucleotide structure of the DNAP genes isolated from Thermus aquaticus (SEQ ID NO:l), Thermus flavus (SEQ ID NO:2) and Thermus thermophilus (SEQ ID NO:3); the consensus sequence (SEQ ID NO:7) is shown at the top of each row.
• Figure 2 is a comparison of the amino acid sequence of the DNAP isolated from Thermus aquaticus (SEQ ID NO:l), Thermus flavus (SEQ ID NO:2) and Thermus thermophilus (SEQ ID NO:3); the consensus sequence (SEQ ID NO:7) is shown at the top of each row.
• Figure 2 is a comparison of the amino acid sequence of the DNAP isolated from
• Thermus aquaticus (SEQ ID NO:4).
• Thermus flavus (SEQ ID NO:5).
• Thermus thermophilus (SEQ ID NO:6); the consensus sequence (SEQ ID NO: 8) is shown at the top of each row.
• Figure 3 is a schematic showing the CFLPTM method of generating a characteristic fingerprint from a nucleic acid substrate.
• Figures 4 depicts the organization of the human p53 gene; exons are represented by the solid black boxes and are labelled 1-11. Five hot spot regions are shown as a blow-up of the region spanning exons 5-8; the hot spot regions are labelled A. A'. B. C. and D.
• Figure 5 provides a schematic showing the use of a first 2-step PCR technique for the generation DNA fragments containing p53 mutations.
• Figure 6 provides a schematic showing the use of a second 2-step PCR technique for the generation DNA fragments containing p53 mutations.
• Figure 7 depicts a structure which cannot be amplified using ONAPTaq.
• Figure 8 is a ethidium bromide-stained gel demonstrating attempts to amplify a bifurcated duplex using either OUAPTaq or DNAPStf (Stoffel).
• Figure 9 is an autoradiogram of a gel analyzing the cleavage of a bifurcated duplex by DNAPTaq and lack of cleavage by DNAPStf.
• Figures 10 A-B are a set of autoradiograms of gels analyzing cleavage or lack of cleavage upon addition of different reaction components and change of incubation temperatur during attempts to cleave a bifurcated duplex with DNAPTaq.
• Figures 1 1 A-B are an autoradiogram displaying timed cleavage reactions, with and without primer.
• Figures 12 A-B are a set of autoradiograms of gels demonstrating attempts to cleave a bifurcated duplex (with and without primer) with various DNAPs.
• Figures 13A shows the substrates and oligonucleotides used to test the specific cleavage of substrate DNAs targeted by pilot oligonucleotides.
• Figure 13B shows an autoradiogram of a gel showing the results of cleavage reactions using the substrates and oligonucleotides shown Fig. 13 A.
• Figure 14A shows the substrate and oligonucleotide used to test the specific cleavage of a substrate RNA targeted by a pilot oligonucleotide.
• Figure 14B shows an autoradiogram of a gel showing the results of a cleavage reactio using the substrate and oligonucleotide shown in Fig. 14A.
• Figure 15 is a diagram of vector pTTQ18.
• Figure 16A-G are a set of diagrams of wild-type and synthesis-deficient DNAPTaq genes.
• Figure 17 is a diagram of vector pET-3c Figure 18A depicts the wild-type Thermus flavus polymerase gene.
• Figure 18B depicts a synthesis-deficient Thermus flavus polymerase gene.
• Figures 19A-E depict a set of molecules which are suitable substrates for cleavage by the 5 " nuclease activity of DNAPs.
• Figure 20 is an autoradiogram of a gel showing the results of a cleavage reaction run with synthesis-deficient DNAPs.
• Figure 21 is an autoradiogram of a PEI chromatogram resolving the products of an assay for synthetic activity in synthesis-deficient ONAPTaq clones.
• Figure 22A depicts the substrate molecule used to test the ability of synthesis-deficien DNAPs to cleave short hai ⁇ in structures.
• Figure 22B shows an autoradiogram of a gel resolving the products of a cleavage reaction run using the substrate shown in Fig. 22 A.
• Figure 23 provides the complete 206-mer duplex sequence employed as a substrate fo the 5 " nucleases of the present invention
• Figures 24A and B show the cleavage of linear nucleic acid substrates (based on the 206-mer of Figure 23) by wild type DNAPs and 5 " nucleases isolated from Thermus aquaticus and Thermus flavus.
• Figure 25A shows the "nibbling" phenomenon detected with the DNAPs of the present invention.
• Figure 25B shows that the "nibbling" of Figure 25A is 5' nucleolytic cleavage and not phosphatase cleavage.
• Figure 26 demonstrates that the "nibbling" phenomenon is duplex dependent.
• Figure 27 shows an autoradiograph of a gel resolving the products of cleavage reactions run in the presence of either MgCL or MnCL.
• Figure 28 shows an autoradiograph of a gel resolving the products of cleavage reactions run on four similarly sized DNA substrates.
• Figure 29 shows an autoradiograph of a gel resolving the products of cleavage reactions run using a wild-type and two mutant tyrosinase gene substrates.
• Figure 30 shows an autoradiograph of a gel resolving the products of cleavage reactions run using either a wild-type or mutant tyrosinase substrate varying in length from 157 nucleotides to 1.587 kb.
• Figure 31 shows an autoradiograph of a gel resolving the products of cleavage reactions run in various concentrations of MnCl 2 .
• Figure 32 shows an autoradiograph of a gel resolving the products of cleavage reactions run in various concentrations of KG.
• Figure 33 shows an autoradiograph of a gel resolving the products of cleavage reactions run for different lengths of time.
• Figure 34 shows an autoradiograph of a gel resolving the products of cleavage reactions run at different temperatures.
• Figure 35 shows an autoradiograph of a gel resolving the products of cleavage reactions run using different amounts of the enzyme CleavaseTM BN.
• Figure 36 shows an autoradiograph of a gel resolving the products of cleavage reactions run using four different preparations of the DNA substrate.
• Figure 37 shows an autoradiograph of a gel resolving the products of cleavage reactions run on either the sense or antisense strand of four different tyrosinase gene substrates.
• Figure 38 shows an autoradiograph of a gel resolving the products of cleavage reactions run on a wild-type ⁇ -globin substrate in two different concentrations of KC1 and at four different temperatures.
• Figure 39 shows an autoradiograph of a gel resolving the products of cleavage reactions run on two different mutant ⁇ -globin substrates in five different concentrations of
• Figure 40 shows an autoradiograph of a gel resolving the products of cleavage reactions run on a wild-type and three mutant ⁇ -globin substrates.
• Figure 41 shows an autoradiograph of a gel resolving the products of cleavage reactions run on an RNA substrate.
• Figure 42 shows an autoradiograph of a gel resolving the products of cleavage reactions run using either the enzyme CleavaseTM BN or Taq DNA polymerase as the 5 " nuclease.
• Figure 43 shows an autoradiograph of a gel resolving the products of cleavage reactions run on a double-stranded DNA substrate to demonstrate multiplexing of the cleavag reaction.
• Figure 44 shows an autoradiograph of a gel resolving the products of cleavage reactions run on double-stranded DNA substrates consisting of the 41 and 422 mutant allele derived from exon 4 of the human tyrosinase gene in the presence of various concentrations of MnCl,.
• Figure 45 displays two traces representing two channel signals (JOE and FAM fluorescent dyes) for cleavage fragments derived from a cleavage reaction containing two differently labelled substrates (the wild-type and 422 mutant substrates derived from exon 4 the tyrosinase gene).
• the thin lines represent the JOE-labelled wild-type substrate and the thick lines represent the FAM-labelled 422 mutant substrate.
• Above the tracing is an autoradiograph of a gel resolving the products of cleavage reactions run on double-stranded DNA substrates consisting of the wild-type and 422 mutant alleles derived from exon 4 of th tyrosinase gene.
• Figure 46 depicts the nucleotide sequence of six SIV LTR clones corresponding to SEQ ID NOS:63-68.
• Figure 47 shows an autoradiograph of a gel resolving the products of cleavage reactions run on six different double-stranded SIV LTR substrates which contained a biotin label on the 5 " end of the (-) strand.
• Figure 48 shows an autoradiograph of a gel resolving the products of cleavage reactions run on six different double-stranded SIV LTR substrates which contained a biotin label on the 5 " end of the (+) strand.
• Figure 49 shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run in various concentrations of NaCl.
• Figure 50 shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run in various concentrations of (NH 4 ) 2 S0 4 .
• Figure 51 shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run in increasing concentrations of KC1.
• Figure 52 shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run in two concentrations of KC1 for various periods of time.
• Figure 53 shows an autoradiograph of a gel resolving the products of cleavage reactions run on either the single-stranded or double-stranded form of the same substrate.
• Figure 54 shows an autoradiograph of a gel resolving the products of double-stranded cleavage reactions run in various concentrations of KG.
• Figure 55 shows an autoradiograph of a gel resolving the products of double-stranded cleavage reactions run in various concentrations of NaCl.
• Figure 56 shows an autoradiograph of a gel resolving the products of double-stranded cleavage reactions run in various concentrations of (NH 4 ) 2 S0 4 .
• Figure 57 shows an autoradiograph of a gel resolving the products of double-stranded cleavage reactions run for various lengths of time.
• Figure 58 shows an autoradiograph of a gel resolving the products of double-stranded cleavage reactions run using various amounts of GeavaseTM BN enzyme for either 5 seconds or 1 minute.
• Figure 59 shows an autoradiograph of a gel resolving the products of double-stranded cleavage reactions run at various temperatures.
• Figure 60 shows an autoradiograph of a gel resolving the products of double-stranded cleavage reactions run using various amounts of GeavaseTM BN enzyme.
• Figure 61 A shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run in buffers having various pHs.
• Figure 61 B shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run in buffers having a pH of either 7.5 or 7.8.
• Figure 62A shows an autoradiograph of a gel resolving the products of double- stranded cleavage reactions run in buffers having a pH of either 8.2 or 7.2.
• Figure 62B shows an autoradiograph of a gel resolving the products of double-strand cleavage reactions run in buffers having a pH of either 7.5 or 7.8.
• Figure 63 shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run in the presence of various amounts of human genomic DNA.
• Figure 64 shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run using the Tfl DNA polymerase in two different concentrations of KG.
• Figure 65 shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run using the Tth DNA polymerase in two different concentrations of KG
• Figure 66 shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run using the E. coli Exo III enzyme in two different concentrations of KG.
• Figure 67 shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run on three different tyrosinase gene substrates (SEQ ID NOS:34. 41 an
• Figure 68 is a schematic drawing depicting the location of the 5 * and 3 " cleavage site on a cleavage structure.
• Figure 69 shows an autoradiograph of a gel resolving the products of single-stranded cleavage reactions run on three different tyrosinase gene substrates (SEQ ID NOS:34. 41 an
• Figure 70 shows an autoradiograph of a gel resolving the products of double-stranded cleavage reactions run on a wild-type and two mutant ⁇ -globin substrates.
• Figure 71 A shows an autoradiograph of a gel resolving the products of single-strande cleavage reactions run on a wild-type and three mutant ⁇ -globin substrates.
• Figure 71 B shows an autoradiograph of a gel resolving the products of single-strande cleavage reactions run on five mutant ⁇ -globin substrates.
• Figure 72 shows an autoradiograph of a gel resolving the products of double-stranded cleavage reactions which varied the order of addition of the reaction components.
• Figure 73 shows an autoradiograph of a gel resolving the products of cleavage reactions run on a wild-type and two mutant p53 substrates.
• Figure 74 shows an autoradiograph of a gel resolving the products of cleavage reactions run on a wild-type and three mutant p53 substrates.
• Figure 75 shows an autoradiograph of a gel resolving the products of cleavage reactions run on a wild-type and a mutant p53 substrate where the mutant and wild-type substrates are present in various concentrations relative to one another.
• Figure 76 provides an alignment of HCV clones 1.1 (SEQ ID NO: 108). HCV2.1 (SEQ ID NO:109), HCV3.1 (SEQ ID NO:1 10). HCV4.2 (SEQ ID NO:l 1 1 ). HCV6.1 (SEQ ID NO: 108).
• Figure 77 shows a fluoroimager scan of a gel resolving the products of cleavage reactions run on six double-stranded HCV substrates labeled on either the sense or anti-sense strand.
• Figure 78 shows an autoradiogram of a gel resolving the products of cleavage reactions run on a wild-type and two mutant M. tuberculosis rpoB substrates.
• Figure 79A shows a fluoroimager scan of a gel resolving the products of cleavage reactions run on a wild-type and two mutant M. tuberculosis rpoB substrates prepared using either dTTP or dUTP.
• Figure 79B shows a fluoroimager scan of the gel shown in Figure 85A following a longer period of electrophoresis.
• Figure 80 shows an autoradiogram of a gel resolving the products of cleavage reactions run on a wild-type and three mutant M. tuberculosis katG substrates labeled on the sense strand.
• Figure 81 shows a fluoroimager scan of a gel resolving the products of cleavage reactions run on a wild-type and three mutant M. tuberculosis katG substrates labeled on the anti-sense strand.
• Figure 82 shows the location of primers along the sequence of the E. coli rrsE gene (SEQ ID NO: 145).
• Figure 83 provides an alignment of the E. coli rrsE (SEQ ID NO: 145).
• Figure 84 shows a fluoroimager scan of a gel resolving the products of cleavage reactions run on four bacterial 16S rRNA substrates.
• Figure 85A shows a fluoroimager scan of a gel resolving the products of cleavage reactions run on five bacterial 16S rRNA substrates.
• Figure 85B shows bacterial a fluoroimager scan of a gel resolving the products of cleavage reactions run on five bacterial 16S rRNA substrates.
• Figure 86 shows bacterial a fluoroimager scan of a gel resolving the products of cleavage reactions run on various bacterial 16S rRNA substrates.
• Figure 87 shows bacterial a fluoroimager scan of a gel resolving the products of cleavage reactions run on eight bacterial 16S rRNA substrates.
• Figure 88 shows an autoradiogram of a gel resolving the products of cleavage reactions run on a wild-type and mutant tyrosinase gene substrates prepared using naturally occurring deoxynucleotides or deoxynucleotide analogs.
• the term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor.
• the polypeptide can b encoded by a full length coding sequence or by any portion of the coding sequence so long a the desired enzymatic activity is retained.
• wild-type refers to a gene or gene product which has the characteristics o that gene or gene product when isolated from a naturally occurring source. A wild-type gen is that which is most frequently observed in a population and is thus arbitrarily designed the "normal” or “wild-type” form of the gene.
• the term “modified” or “mutant” refer to a gene or gene product which displays modifications in sequence and or functional properties (i.e.. altered characteristics) when compared to the wild-type gene or gene product.
• mutants can be isolated; these are identified by the fact th they have altered characteristics when compared to the wild-type gene or gene product.
• recombinant DNA vector refers to DNA sequences containing a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
• DNA sequences necessary for expression in procaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences.
• Eukaryotic cells are known to utilize promoters, polyadenlyation signals and enhancers.
• LTR refers to the long terminal repeat found at each end o a provirus (i.e.. the integrated form of a retrovirus).
• the LTR contains numerous regulator) signals including transcriptional control elements, polyadenylation signals and sequences needed for replication and integration of the viral genome.
• the viral LTR is divided into three regions called U3. R and U5.
• the U3 region contains the enhancer and promoter elements.
• the U5 region contains the polyadenylation signals.
• the R (repeat) region separates the U3 and U5 regions and transcribed sequences of the R region appear at both the 5 * and 3 " ends of the viral RNA.
• oligonucleotide as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides. preferably more than three, and usually more than ten. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide.
• the oligonucleotide may be generated in any manner, including chemical synthesis. DNA replication, reverse transcription, or a combination thereof.
• an end of an oligonucleotide is referred to as the "5 " end” if its 5' phosphate is not linked to the 3 * oxygen of a mononucleotide pentose ring and as the "3 " end” if its 3 " oxygen is not linked to a 5 * phosphate of a subsequent mononucleotide pentose ring.
• a nucleic acid sequence even if internal to a larger oligonucleotide. also may be said to have 5 ' and 3 " ends.
• the former When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3 " end of one oligonucleotide points towards the 5 " end of the other, the former may be called the "upstream” oligonucleotide and the latter the "downstream” oligonucleotide.
• primer refers to an oligonucleotide which is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated.
• An oligonucleotide “primer” may occur naturally, as in a purified restriction digest or may be produced synthetically.
• a primer is selected to be “substantially” complementary to a strand of specific sequence of the template.
• a primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur.
• a primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5 * end of the primer, with the remainder of the primer sequence being substantially complementary to the strand.
• Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.
• “Hybridization” methods involve the annealing of a complementary sequence to the target nucleic acid (the sequence to be detected). The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the "hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 ( 1960) and Doty et al .
• the target sequence must be made accessible to the probe via rearrangements of higher-order structure.
• These higher-order structural rearrangements may concern either the secondary structure or tertiary structure of the molecule.
• Secondary structure is determined by intramolecular bonding. In the case of DNA or RNA targets this consists of hybridization within a single, continuous strand of bases (as opposed to hybridization between two different strands). Depending on the extent and position of intramolecular bonding, the probe can be displaced from the target sequence preventing hybridization.
• complementarity it is important for some diagnostic applications to determine whether the hybridization represents complete or partial complementarity. For example, where it is desired to detect simply the presence or absence of pathogen DNA (such as from a virus, bacterium, fungi, mycoplasma, protozoan) it is only important that the hybridization method ensures hybridization when the relevant sequence is present; conditions can be selected where both partially complementary probes and completely complementary probes will hybridize. Other diagnostic applications, however, may require that the hybridization method distinguish between partial and complete complementarity. It may be of interest to detect genetic polymorphisms. For example, human hemoglobin is composed, in part, of four polypeptide chains.
• Two of these chains are identical chains of 141 amino acids (alpha chains) and two of these chains are identical chains of 146 amino acids (beta chains).
• the gene encoding the beta chain is known to exhibit polymorphism.
• the normal allele encodes a beta chain having glutamic acid at the sixth position.
• the mutant allele encodes a beta chain having valine at the sixth position.
• This difference in amino acids has a profound (most profound when the individual is homozygous for the mutant allele) physiological impact known clinically as sickle cell anemia. It is well known that the genetic basis of the amino acid change involves a single base difference between the normal allele DNA sequence and the mutant allele DNA sequence.
• the probe will hybridize to both the normal and variant target sequence.
• Hybridization regardless of the method used. requires some degree of complementarity between the sequence being assayed (the target sequence) and the fragment of DNA used to perform the test (the probe). (Of course, one ca obtain binding without any complementarity but this binding is nonspecific and to be avoided.)
• nucleic acid sequence refers to an oligonucleotid which, when aligned with the nucleic acid sequence such that the 5 ' end of one sequence is paired with the 3 ' end of the other, is in "antiparallel association.”
• Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases.
• Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide. base composition and sequence of the oligonucleotide. ionic strength and incidence of mismatched base pairs.
• T m melting temperature
• probe refers to a labeled oligonucleotide which forms a duplex structure with a sequence in another nucleic acid, due to complementarity of at least one sequence in the probe with a sequence in the other nucleic acid.
• label refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry. gravimetry. X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
• cleavage structure refers to a region of a single-stranded nucleic acid substrate containing secondary structure, said region being cleavable by a cleavage means, including but not limited to an enzyme.
• the cleavage structure is a substra for specific cleavage by said cleavage means in contrast to a nucleic acid molecule which is substrate for non-specific cleavage by agents such as phosphodiesterases which cleave nucleic acid molecules without regard to secondary structure (i.e.. no folding of the substrate is required).
• cleavage means refers to any means which is capable of cleaving a cleavage structure, including but not limited to enzymes.
• the cleavage means may include native DNAPs having 5 * nuclease activity (e.g., Taq DNA polymerase. E. coli DNA polymerase I) and, more specifically, modified DNAPs having 5 ' nuclease but lacking synthetic activity.
• native DNAPs having 5 * nuclease activity e.g., Taq DNA polymerase. E. coli DNA polymerase I
• modified DNAPs having 5 ' nuclease but lacking synthetic activity.
• structure-specific cleavage is useful to detect internal sequence differences in nucleic acids without prior knowledge of the specific sequence of the nucleic acid. In this manner, they are structure-specific enzymes.
• Structure-specific enzymes are enzymes which recognize specific secondary structures in a nucleic molecule and cleave these structures.
• the site of cleavage may be on either the 5' or 3' side of the cleavage structure: alternatively the site of cleavage may be between the 5 ' and 3 * side (i.e.. within or internal to) of the cleavage structure.
• the cleavage means of the invention cleave a nucleic acid molecule in response to the formation of cleavage structures; it is not necessary that the cleavage means cleave the cleavage structure at any particular location within the cleavage structure.
• the cleavage means is not restricted to enzymes having 5 " nuclease activity.
• the cleavage means may include nuclease activity provided from a variety of sources including the enzyme GeavaseTM. Taq DNA polymerase, E. coli DNA polymerase I and eukaryotic structure-specific endonucleases. murine FEN-1 endonucleases [Harrington and Liener, ( 1994) Genes and Develop. 8:1344] and calf thymus 5' to 3 ' exonuclease [Murante. R.S.. et al. ( 1994) J. Biol. Chem. 269:1 191]).
• enzymes having 3 " nuclease activity such as members of the family of DNA repair endonucleases (e.g.. the Rrpl enzyme from Drosophila melanogaster. the yeast RAD1/RAD10 complex and E. coli Exo III), are also suitable cleavage means for the practice of the methods of the invention.
• cleavage products refers to products generated by the reaction of a cleavage means with a cleavage structure (i.e.. the treatment of a cleavage structure with a cleavage means).
• the nucleic acid substrate may comprise single- or double- stranded DNA or RNA.
• substantially single-stranded when used in reference to a nucleic acid substrate means that the substrate molecule exists primarily as a single strand of nucleic acid in contrast to a double-stranded substrate which exists as two strands of nucleic acid which are held together by inter-strand base pairing interactions.
• Nucleic acids form secondary structures which depend on base-pairing for stability. When single strands of nucleic acids (single-stranded DNA. denatured double-stranded DNA or RNA) with different sequences, even closely related ones, are allowed to fold on themselves, they assume characteristic secondary structures. At “elevated temperatures” the duplex regions of the structures are brought to the brink of instability, so that the effects of small changes in sequence are maximized, and revealed as alterations in the cleavage pattern In other words, "an elevated temperature” is a temperature at which a given duplex region o the folded substrate molecule is near the temperature at which that duplex melts.
• transient formation of these structures allows recognition and cleavage of these structures by said cleavage mean
• the formation or disruption of these structures in response to small sequence changes results in changes in the patterns of cleavage.
• Temperatures in the range of 40-85°C. with the rang of 55-85°C being particularly preferred, are suitable elevated temperatures for the practice o the method of the invention.
• sequence variation refers to differences in nucleic acid sequence between two nucleic acid templates.
• a wild-type structural gene and mutant form of this wild-type structural gene may vary in sequence by the presence of singl base substitutions and/or deletions or insertions of one or more nucleotides. These two for of the structural gene are said to vary in sequence from one another.
• a second mutant form of the structural gene may exits. This second mutant form is said to vary in sequence from both the wild-type gene and the first mutant form of the gene. It is noted, however, that the invention does not require that a comparison be made between one or more forms of a gene to detect sequence variations.
• a characteristic "finge ⁇ rint" may be obtained from any nucleic substrate without reference to a wild-type or other control.
• the invention contemplates the use of the method for both "fingerprinting" nucleic acids without reference to a control and identification of mutant forms of a substrate nucleic acid by comparison of the mutant form of the substrate with a wild-type or known mutant control.
• the term “liberating” as used herein refers to the release of a nucleic acid fragment from a larger nucleic acid fragment, such as an oligonucleotide, by the action of a 5 " nuclease such that the released fragment is no longer covalently attached to the remainder of the oligonucleotide.
• substrate strand as used herein, means that strand of nucleic acid in a cleavage structure in which the cleavage mediated by the 5 " nuclease activity occurs.
• template strand means that strand of nucleic acid in a cleavage structure which is at least partially complementary to the substrate strand and which anneals to the substrate strand to form the cleavage structure.
• K m refers to the Michaelis-Menten constant for an enzyme and is defined as the concentration of the specific substrate at which a given enzyme yields one-half its maximum velocity in an enzyme catalyzed reaction.
• nucleotide analog refers to modified or non-naturally occurring nucleotides such as 7-deaza purines (i.e., 7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogs and comprise modified forms of deoxyribonucleotides as well as ribonucleotides. As used herein the term “nucleotide analog” when used in reference to substrates present in a nucleic acid amplification mixture (e.g.. a PCR mixture) refers to the use of nucleotides other than dATP, dGTP.
• dUTP a naturally occurring dNTP
• dTTP a nucleotide analog in the PCR.
• a PCR product generated using dUTP. 7-deaza-dATP. 7- deaza-dGTP or any other nucleotide analog in the reaction mixture is said to contain nucleotide analogs.
• Oligonucleotide primers matching or complementary to a gene sequence refers to oligonucleotide primers capable of facilitating the template-dependent synthesis of single or double-stranded nucleic acids. Oligonucleotide primers matching or complementary to a gen sequence may be used in PCRs, RT-PCRs and the like.
• a "consensus gene sequence” refers to a gene sequence which is derived by comparison of two or more gene sequences and which describes the nucleotides most often present in a given segment of the genes; the consensus sequence is the canonical sequence.
• polymo ⁇ hic locus is a locus present in a population which shows variatio between members of the population (i.e., the most common allele has a frequency of less tha 0.95).
• a "monomo ⁇ hic locus” is a genetic locus at little or no variations seen between members of the population (generally taken to be a locus at which the most commo allele exceeds a frequency of 0.95 in the gene pool of the population).
• microorganism as used herein means an organism too small to be observe with the unaided eye and includes, but is not limited to bacteria, virus, protozoans, fungi, an ciliates.
• microbial gene sequences refers to gene sequences derived from a microorganism.
• sequences derived from one or more microorganisms refers to nucleic aci sequences extracted from one or a mixture of more than one microorganism.
• the extracted sequences may be subjected to further treatment, such as nucleic acid amplification (e.g.. polymerase chain reaction) prior to treatment to form and subsequently cleave cleavage structures comprising the microbial nucleic acid sequences.
• nucleic acid amplification e.g.. polymerase chain reaction
• bacteria refers to any bacterial species including eubacterial and archaebacterial species.
• virus refers to obligate, ultramicroscopic. intracellular parasites incapable of autonomous replication (i.e., replication requires the use of the host celfs machinery).
• multi-drug resistant or multiple-drug resistant refers to a microorganism which is resistant to more than one of the antibiotics or antimicrobial agents used in the treatment of said microorganism.
• CFLPTM GelavaseTM Fragment Length Polymo ⁇ hism
• nucleic acid substrates which differ in sequence from a control or reference nucleic acid substrate will, when analyzed by this method, show an altered representation of the fragments within the pool of cleavage products, indicating the presence of said differences in sequence between the substrates.
• the present invention relates to methods and compositions for treating nucleic acid. and in particular, methods and compositions for detection and characterization of nucleic acid sequences and sequence changes.
• the present invention relates to means for cleaving a nucleic acid cleavage structure in a site-specific manner.
• the present invention relates to a cleaving enzyme having 5 " nuclease activity without interfering nucleic acid synthetic ability.
• This invention provides 5 " nucleases derived from thermostable DNA polymerases which exhibit altered DNA synthetic activity from that of native thermostable DNA polymerases. The 5" nuclease activity of the polymerase is retained while the synthetic activity is reduced or absent.
• Such 5 ' nucleases are capable of catalyzing the structure- specific cleavage of nucleic acids in the absence of interfering synthetic activity. The lack of synthetic activity during a cleavage reaction results in nucleic acid cleavage products of uniform size.
• novel properties of the polymerases of the invention form the basis of a method of detecting specific nucleic acid sequences. This method relies upon the amplification of the detection molecule rather than upon the amplification of the target sequence itself as do existing methods of detecting specific target sequences.
• DNA polymerases such as those isolated from E. coli or from thermophilic bacteria of the genus Thermus, are enzymes that synthesize new DNA strands.
• DNAPs include associated nuclease activities in addition to the synthetic activity of the enzyme.
• Some DNAPs are known to remove nucleotides from the 5 * and 3 * ends of DNA chains [Kornberg. DNA Replication, W.H. Freeman and Co.. San Francisco, pp. 127-139 ( 1980)].
• These nuclease activities are usually referred to as 5 " exonuclease and 3 " exonuclease activities, respectively.
• DNAPs participates in the removal of RNA primers during lagging strand synthesis during DNA replication and the removal of damaged nucleotides during repair.
• Some DNAPs such as the £ coli DNA polymerase (DNAPEc l ). also e a 3' exonuclease activity responsible for proof-reading during DNA synthesis (Kornberg. supra)
• DNAPTaq A DNAP isolated from Thermus aquaticus, termed Taq DNA polymerase (DNAPTaq has a 5 ' exonuclease activity, but lacks a functional 3 * exonucleolytic domain [Tindall and Kunkell. Biochem 27 6008 (1988)] Derivatives of DNAPEcl and DNAP7 ⁇ /. respectively called the Klenow and Stoffel fragments, lack 5 " exonuclease domains as a result of enzymatic or genetic manipulations [Brutlag et al . Biochem Biophvs Res C o mun 37 982
• nucleases which clea ⁇ e the nucleic acid molecule at internal rather than terminal sites
• the nuclease activity associated with some thermostable DNA polymerases cleaves endonucleolyticalh but this cleavage requires contact with the 5 " end of the molecule being cleaved Therefore these nucleases are referred to as 5 " nucleases
• the Klenow or large proteolytic cleavage fragment of DNAPEc 1 contains the polymerase and 3' exonuclease activity but lacks the 5 * nuclease activity.
• the Stoffel fragment of DNAP7 ⁇ g (DNAPStf) lacks the 5' nuclease activity due to a genetic manipulation which deleted the N-terminal 289 amino acids of the polymerase molecule [Erlich el al.. Science 252: 1643 (1991)].
• WO 92/06200 describes a thermostable DNAP with an altered level of 5' to 3' exonuclease.
• U.S. Patent No. 5.108.892 describes a Thermus aquaticus DNAP without a 5' to 3 ' exonuclease.
• the art of molecular biology lacks a thermostable DNA polymerase with a lessened amount of synthetic activity.
• the present invention provides 5 ' nucleases derived from thermostable Type A DNA polymerases that retain 5 * nuclease activity but have reduced or absent synthetic activit .
• the ability to uncouple the synthetic activity of the enzyme from the 5 * nuclease activity proves that the 5 " nuclease activity does not require concurrent DNA synthesis as was previously reported (Gelfand. PCR Technology, supra).
• the description of the invention is divided into: I. Generation of 5 * Nucleases Derived From Thermostable DNA Polymerases; II. GeavaseTM Fragment Length Polymo ⁇ hism for the Detection of Secondary Structure; III. Detection of Mutations in the p53 Tumor Suppressor Gene Using the CFLPTM Method and IV. Detection and Identification of Pathogens Using the CFLPTM Method.
• the methods of the present invention employ 5 " nucleases for the detection of specific nucleic acid sequences.
• the 5 " nuclease may be derived from a thermostable DNA polymerase: however, the methods of the invention are not limited to the use of a 5 ' nuclease. Any cleavage agent capable of generating a unique (i.e.. characteristic) pattern of cleavage products from a substrate nucleic acid may be employed.
• the 5 * nuclease may be derived from a thermostable DNA polymerase as describe below.
• thermostable polymerases include those isolated from Thermus aquaticus, Thermus flavus. and Thermus thermophilus. Howeve other thermostable Type A polymerases which have 5 " nuclease activity are also suitable.
• Figures 1 and 2 compare the nucleotide and amino acid sequences of the three above mentioned polymerases. In Figures 1 and 2, the consensus or majority sequence derived fro a comparison of the nucleotide (Fig. 1) or amino acid (Fig. 2) sequence of the three thermostable DNA polymerases is shown on the top line.
• SEQ ID NOS: l-3 display the nucleotide sequences and SEQ ID NOS:4
• SEQ ID NO: 6 corresponds to the amino acid sequences of the three wild-type polymerases.
• SEQ ID NO: l corresponds to the nucleic acid sequence of the wild type Thermus aquaticus DNA polymerase gene isolated from the YT-1 strain [Lawyer et al, J. Biol Chem. 264:6427 ( 1989)].
• SEQ ID NO:2 corresponds to the nucleic acid sequence of the wild type Thermus flavus DNA polymerase gene [Akhmetzjanov and Vakhitov, Nucl Acids Res. 20:5839 (1992)
• SEQ ID NO:3 corresponds to the nucleic acid sequence of the wild type Thermus thermophilus DNA polymerase gene [Gelfand et al. WO 91/09950 (1991 )].
• SEQ ID NOS:7 8 depict the consensus nucleotide and amino acid sequences, respectively for the above three DNAPs (also shown on the top row in Figs. 1 and 2).
• the 5 " nucleases of the invention derived from thermostable polymerases have reduc synthetic ability, but retain substantially the same 5 ' exonuclease activity as the native DNA polymerase.
• substantially the same 5 * nuclease activity means tha the 5 " nuclease activity of the modified enzyme retains the ability to function as a structure- dependent single-stranded endonuclease but not necessarily at the same rate of cleavage as compared to the unmodified enzyme.
• Type A DNA polymerases may also be modified so a to produce an enzyme which has increases 5 " nuclease activity while having a reduced level of synthetic activity. Modified enzymes having reduced synthetic activity and increased 5 ' nuclease activity are also envisioned by the present invention.
• reduced synthetic activity as used herein it is meant that the modified enzyme has less than the level of synthetic activity found in the unmodified or “native" enzyme.
• the modified enzyme may have no synthetic activity remaining or may have that level of synthetic activity that will not interfere with the use of the modified enzyme in the detection assay described below.
• the 5 ' nucleases of the present invention are advantageous in situations where the cleavage activity of the polymerase is desired, but the synthetic abilit is not (such as in the detection assay of the invention).
• the invention contemplates a variety of methods, including but not limited to: 1 ) proteolysis; 2) recombinant constructs (including mutants); and 3) physical and/or chemical modification and/or inhibition.
• Thermostable DNA polymerases having a reduced level of synthetic activity are produced by physically cleaving the unmodified enzyme with proteolytic enzymes to produce fragments of the enzyme that are deficient in synthetic activity but retain 5 " nuclease activity . Following proteolytic digestion, the resulting fragments are separated by standard chromatographic techniques and assayed for the ability to synthesize DNA and to act as a 5 " nuclease. The assays to determine synthetic activity and 5 ' nuclease activity are described below.
• thermostable DNA polymerase is cloned by isolating genomic DNA using molecular biological methods from a bacteria containing a thermostable Type A DNA polymerase. This genomic DNA is exposed to primers which are capable of amplifying the polymerase gene by
• This amplified polymerase sequence is then subjected to standard deletion processes t delete the polymerase portion of the gene. Suitable deletion processes are described below i the examples.
• Deletion of amino acids from the protein can be done either by deletion of the encoding genetic material, or by introduction of a translational stop codon by mutation or frame shift.
• proteolytic treatment of the protein molecule can be performed to remove segments of the protein.
• specific alterations of the Taq gene were: a deletion between nucleotides 1601 and 2502 (the end of the coding region), a 4 nucleotide insertion at positio 2043. and deletions between nucleotides 1614 and 1848 and between nucleotides 875 and 1778 (numbering is as in SEQ ID NO: l ).
• SEQ ID NO: 9-12 are examples of the examples and at SEQ ID NOS:9-12.
• deletions are also suitable to create the 5 " nucleases of the present invention. I is preferable that the deletion decrease the polymerase activity of the 5 " nucleases to a level which synthetic activity will not interfere with the use of the 5 ' nuclease in the detection assay of the invention. Most preferably, the synthetic ability is absent. Modified polymeras are tested for the presence of synthetic and 5 ' nuclease activity as in assays described below . Thoughtful consideration of these assays allows for the screening of candidate enzymes who structure is heretofore as yet unknown. In other words, construct "X" can be evaluated according to the protocol described below to determine whether it is a member of the genus of 5 " nucleases of the present invention as defined functionally, rather than structurally .
• the PCR product of the amplified Thermits aquaticus genomic DNA did not have the identical nucleotide structure of the native genomic DNA and did not have the same synthetic ability of the original clone.
• Base pair changes which result due to the infidelity of DNAPTaq during PCR amplification of a polymerase gene are also a metho by which the synthetic ability of a polymerase gene may be inactivated.
• the examples belo and Figs. 4A and 5A indicate regions in the native Thermus aquaticus and flavus DNA polymerases likely to be important for synthetic ability. There are other base pair changes and substitutions that will likely also inactivate the polymerase.
• the present invention contemplates that the nucleic acid construct of the present invention be capable of expression in a suitable host.
• Those in the art know methods for attaching various promoters and 3 * sequences to a gene structure to achieve efficient expression.
• the examples below disclose two suitable vectors and six suitable vector constructs. Of course, there are other promoter/vector combinations that would be suitable.
• a host organism be used for the expression of the nucleic acid constructs of the invention.
• expression of the protein encoded by a nucleic acid construct may be achieved through the use of a cell-free in vitro transcription/translation system.
• An example of such a cell-free system is the commercially available TnTTM Coupled Reticulocyte Lysate System (Promega Co ⁇ oration, Madison, WI).
• nuclease may be produced from the construct.
• the examples below and standard molecular biological teachings enable one to manipulate the construct by different suitable methods.
• the polymerase is tested for both synthetic and nuclease activity as described below.
• thermostable DNA polymerase may be reduced by chemical and/or physical means.
• the cleavage reaction catalyzed by the 5 " nuclease activity of the polymerase is run under conditions which preferentially inhibit the synthetic activity of the polymerase.
• the level of synthetic activity need only be reduced to that level of activity which does not interfere with cleavage reactions requiring no significant synthetic activity.
• concentrations of Mg " greater than 5 mM inhibit the polymerization activity of the native DNAPT ⁇ cy.
• the ability of the 5' nuclease to function under conditions where synthetic activity is inhibited is tested by running the assays for synthetic and 5 " nuclease activity, described below, in the presence of a range of Mg * concentrations (5 to 10 mM).
• the effect of a given concentration of Mg " is determined by quantitation of the amount of synthesis and cleavage in the test reaction as compared to the standard reaction for each assay.
• nucleic acid binding chemicals such as. actinomycin D. ethidium bromide and psoralens, are tested b their addition to the standard reaction buffers for the synthesis and 5 ' nuclease assays. Thos compounds having a preferential inhibitory effect on the synthetic activity of a thermostable polymerase are then used to create reaction conditions under which 5' nuclease activit
• thermostable polymerases are destroyed by exposure of the polymerase to extreme heat (typically 96 to 100°C) for extended periods of time (greater than or equal to 20 minutes). While these are minor differences with respect t the specific heat tolerance for each of the enzymes, these are readily determined. Polymeras are treated with heat for various periods of time and the effect of the heat treatment upon th synthetic and 5 " nuclease activities is determined.
• extreme heat typically 96 to 100°C
• extended periods of time greater than or equal to 20 minutes
• Nucleic acids assume secondary structures which depend on base-pairing for stability . When single strands of nucleic acids (single-stranded DNA. denatured DNA or RNA) with different sequences, even closely related ones, are allowed to fold on themselves, they assum characteristic secondary structures. These differences in structures account for the ability of single strand conformation polymorphism (SSCP) analysis to distinguish between DNA fragments having closely related sequences.
• SSCP single strand conformation polymorphism
• the 5 " nuclease domains of certain DNA polymerases are specific endonucleases that recognize and cleave nucleic acids at specific structures rather than in a sequence-specific manner (as do restriction endonucleases).
• the isolated nuclease domain of DNAPTaq described herein (termed the enzyme GeavaseTM) recognizes the end of a duplex that has non- base paired strands at the ends. The strand with the 5 " end is cleaved at the junction between the single strand and the duplex.
• Figure 3 depicts a wild-type substrate and a mutant substrate wherein the mutant substrate differs from the wild-type by a single base change (A to G as indicated).
• substrate structures form when nucleic acids are denatured and allowed to fold on themselves (See Figure 3. steps 1 and 2).
• the step of denaturation may be achieved by treating the nucleic acid with heat, low ( ⁇ 3) or high pH (>10). the use of low salt concentrations, the absence of cations, chemicals (e.g.. urea, formamide) or proteins (e.g., helicases). Folding or renaturation of the nucleic acid is achieved by lowering of the temperature, addition of salt, neutralization of the pH. withdrawal of the chemicals or proteins.
• the manner in which the substrate folds is dependent upon the sequence of the substrate.
• the 5 ' nucleases of the invention cleave the structures (See Figure 3. step 3).
• the end points of the resulting fragments reflect the locations of the cleavage sites.
• the cleavage itself is dependent upon the formation of a particular structure, not upon a particular sequence at the cleavage site.
• nucleic acid substrate When the 5 ' nucleases of the invention cleave a nucleic acid substrate, a collection of cleavage products or fragments is generated. These fragments constitute a characteristic fingerprint of the nucleic acid which can be detected [e.g. , by electrophoresis on a gel (see step 4)]. Changes in the sequence of a nucleic acid (e.g., single point mutation between a wild-type and mutant gene) alter the pattern of cleavage structures formed. When the 5 " nucleases of the invention cleave the structures formed by a wild-type and an altered or mutant form of the substrate, the distribution of the cleavage fragments generated will differ between the two substrates reflecting the difference in the sequence of the two substrates (See Figure 3. step 5).
• the GeavaseTM enzyme generates a unique pattern of cleavage products for a substrate nucleic acid. Digestion with the GeavaseTM enzyme can be used to detect single base changes in DNA molecules of great length (e.g.. 1.6 kb in length) to produce a characteristic pattern of cleavage products.
• the method of the invention is termed "GeavaseTM Fragment Length Polymo ⁇ hism" (CFLPTM).
• CFLPTM Fragment Length Polymo ⁇ hism
• suitable enzymatic cleavage activity may be provided fro a variety of sources including the GeavaseTM enzyme, Taq DNA polymerase.
• coli DNA polymerase 1 and eukaryotic structure- specific endonucleases (e.g.. the yeast RAD2 protein and RAD1/RAD10 complex [Harrington. J.J. and Liener (1994) Genes and Develop. 8: 1344].
• murine FEN-1 endonucleases (Harrington and Liener, supra) and calf thymus 5 " to 3' exonuclease [Murante, R.S., et al. (1994) J. Biol. Chem. 269: 1 191 ]).
• human experimental data is provided herein which demonstrates that numerous enzymes may be use to generate a unique pattern of cleavage products for a substrate nucleic acid.
• Enzymes whic are shown herein to be suitable for use in the CFLPTM method include the GeavaseTM BN enzyme. Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase. E. coli Exo III. and the yeast Radl/RadlO complex.
• the invention demonstrates that numerous enzymes may be suitable for use in the CFLPTM method including enzymes which have been characterized in the literature a being 3 exonucleases.
• an enzyme is suitable for use as a cleavage means in the CFLPTM method (i.e.. capable of generating a unique pattern of cleavage products for a substrate nucleic acid)
• the following steps are taken. Careful consideration of the steps described below allows the evaluation of any enzyme (“enzyme X”) for use in the CFLPTM method.
• An initial CFLPTM reaction is prepared using a previously characterized substrate nucleic acid [for example the 157 nucleotide fragment of exon 4 of the human tyrosinase ge (SEQ ID NO:34)].
• the substrate nucleic acid (approximately 100 fmoles; the nucleic acid template may contain a 5' end or other label to permit easy detection of the cleavage products) is placed into a thin wall microcentrifuge tube in a solution which comprises reaction conditions reported to be optimal for the characterized activity of the enzyme (i.e.. enzyme X).
• enzyme X is a DNA polymerase
• the initial reaction conditions would utilize a buffer which has been reported to be optimal for the polymerizati activity of the polymerase.
• enzyme X is not a polymerase. or if no specific components a reported to be needed for activity, the initial reaction may be assembled by placing the substrate nucleic acid in a solution comprising IX CFLPTM buffer ( 10 M MOPS. 0.05%
• the substrate nucleic acid is denatured by heating the sample tube to 95°C for 5 seconds and then the reaction is cooled to a temperature suitable for the enzyme being tested (e.g.. if a thermostable polymerase is being tested the cleavage reaction may proceed at elevated temperatures such as 72°C: if a mesophilic enzyme is being tested the tube is cooled to 37°C for the cleavage reaction).
• a temperature suitable for the enzyme being tested e.g. if a thermostable polymerase is being tested the cleavage reaction may proceed at elevated temperatures such as 72°C: if a mesophilic enzyme is being tested the tube is cooled to 37°C for the cleavage reaction.
• the cleavage reaction is initiated by the addition of a solution comprising 1 to 200 units of the enzyme to be tested (i.e.. enzyme X; the enzyme may be diluted into IX
• the cleavage reaction is allowed to proceed at the target temperature for 2 to 5 minutes.
• the cleavage reaction is then terminated [this may be accomplished by the addition of a stop solution (95% formamide. 10 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol)] and the cleavage products are resolved and detected using any suitable method (e.g.. electrophoresis on a denaturing polyacrylamide gel followed by transfer to a solid support and nonisotopic detection).
• a stop solution 95% formamide. 10 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol
• the enzyme to be evaluated is used in at least two separate cleavage reactions run on different occasions using the same reaction conditions. If substantially the same cleavage pattern is obtained on both occasions, the enzyme is capable of reproducibly generating a cleavage pattern and is therefore suitable for use in the CFLPTM method.
• enzymes derived from mesophilic organisms When enzymes derived from mesophilic organisms are to be tested in the CFLPTM reaction they may be initially tested at 37°C. However it may be desirable to use theses enzymes at higher temperatures in the cleavage reaction. The ability to cleave nucleic acid substrates over a range of temperatures is desirable when the cleavage reaction is being used to detect sequence variation (i.e.. mutation) between different substrates. Strong secondary structures that may dominate the cleavage pattern are less likely to be destabilized by single- base changes and may therefore interfere with mutation detection. Elevated temperatures can then be used to bring these persistent structures to the brink of instability, so that the effects of small changes in sequence are maximized and revealed as alterations in the cleavage pattern.
• Mesophilic enzymes may be used at temperatures greater than 37°C under certain conditions known to the art. These conditions include the use of high (i.e.. 10-30%) concentrations of glycerol in the reaction conditions. Furthermore, it is noted that while an enzyme may be isolated from a mesophilic organism this fact alone does not mean that the enzyme may not demonstrate thermostability; therefore when testing the suitability of a mesophilic enzyme in the CFLPTM reaction, the reaction should be run at 37°C and at higher temperatures. Alternatively, mild denaturants can be used to destabilize the nucleic acid substrate at a lower temperature (e.g., 1-10% formamide. 1-10% DMSO and 1 -10% glycerol have been used in enzymatic reactions to mimic thermal destabilization).
• mild denaturants can be used to destabilize the nucleic acid substrate at a lower temperature (e.g., 1-10% formamide. 1-10% DMSO and 1 -10% glycerol have been used in enzymatic
• Nucleic acid substrates that may be analyzed using a cleavage means include many types of both RNA and DNA. Such nucleic acid substrates may all be obtained using standard molecular biological techniques. For example, substrates may be isolated from a tissue sample, tissue culture cells, bacteria or viruses, may be transcribed in vitro from a DNA template, or may be chemically synthesized. Furthermore, substrates may be isolated from an organism, either as genomic material or as a plasmid or similar extrachromosomal DNA, or it may be a fragment of such material generated by treatment with a restriction endonuclease or other cleavage agents or it may be synthetic.
• Substrates may also be produced by amplification using the PCR.
• the substrate may be produced using the PCR with preferential amplification of one strand (asymmetric PCR).
• Single-stranded substrates may also be conveniently generated in other ways.
• a double-stranded molecul containing a biotin label at the end of one of the two strands may be bound to a solid suppo (e.g.. a magnetic bead) linked to a streptavidin moiety.
• the biotin-labeled strand is selectively captured by binding to the streptavidin-bead complex.
• the subsequent cleavage reaction may be performed using substrate attached to the solid support. as the enzyme GeavaseTM can cleave the substrate while it is bound to the bead.
• a single- stranded substrate may also be produced from a double-stranded molecule by digestion of o strand with exonuclease.
• the nucleic acids of interest may contain a label to aid in their detection following t cleavage reaction.
• the label may be a radioisotope (e.g.. a j2 P or S-labeled nucleotide) placed at either the 5 ' or 3 * end of the nucleic acid or alternatively the label may be distributed throughout the nucleic acid (i.e.. an internally labeled substrate).
• the label may a nonisotopic detectable moiety, such as a fluorophore which can be detected directly, or a reactive group which permits specific recognition by a secondary agent.
• biotinylated nucleic acids may be detected by probing with a streptavidin molecule which is coupled to an indicator (e.g...
• alkaline phosphatase or a fluorophore alkaline phosphatase or a fluorophore
• a hapten such as digoxigenin
• unlabeled nucleic acid may be cleaved and visualized by staining (e.g.. ethidium bromide staining or silver staining) or by hybridization using a labeled probe.
• the substrate nucleic acid is labeled at the 5 * end with a biotin molecule and is detected using avidin or streptavidin coupled to alkaline phosphatase.
• the substrate nucleic acid is labeled at the 5 " end with a fluorescein molecule and is detected using an anti -fluorescein antibody-alkaline phosphatase conjugate.
• the cleavage patterns are essentially partial digests of the substrate in the reaction.
• the substrate When the substrate is labelled at one end (e.g., with biotin). all detectable fragments share a common end. Many of the structures recognized as active cleavage sites are likely to be onl a few base-pairs long and would appear to be unstable at the elevated temperatures used in the GeavaseTM reaction. The formation or disruption of these structures in response to small sequence changes results in changes in the patterns of cleavage.
• the products of the cleavage reaction are a collection of fragments generated by structure specific cleavage of the input nucleic acid.
• Nucleic acids which differ in size may be analyzed and resolved by a number of methods including electrophoresis, chromatography. fluorescence polarization, mass spectrometry and chip hybridization.
• the invention is illustrated using electrophoretic separation. However, it is noted that the resolution of the cleavage products is not limited to electrophoresis. Electrophoresis is chosen to illustrate the method of the invention because electrophoresis is widely practiced in the art and is easily accessible to the average practitioner.
• the CFLPTM reaction is useful to rapidly screen for differences between similar nucleic acid molecules. To optimize the CFLPTM reaction for any desired nucleic acid system
• CFLPTM a single substrate from the test system (for example, the wild-type substrate) to determine the best CFLPTM reaction conditions.
• a sin e suitable condition is chosen for doing the comparison CFLPTM reactions on the other molecules of interest.
• a cleavage reaction may be optimized for a wild-type sequence and mutant sequences may subsequently be cleaved under the same conditions for comparison with the wild-type pattern.
• the objective of the CFLPTM optimization test is the identification of a s of conditions which allow the test molecule to form an assortment (i.e..
• a panel of reaction conditions with varying salt concentration and temperature is firs performed to identify an optimal set of conditions for the single-stranded CFLPTM.
• Optima CFLPTM is defined for this test case as the set of conditions that yields the most widely spaced set of bands after electrophoretic separation, with the most even signal intensity between the bands.
• Two elements of the cleavage reaction that significantly affect the stability of the nucleic acid structures are the temperature at which the cleavage reaction is performed and t concentration of salt in the reaction solution. Likewise, other factors affecting nucleic acid structures, such as. formamide. urea or extremes in pH may be used.
• the initial test typical will comprise reactions performed at four temperatures (50°C, 55°C, 60°C and 65°C) in thre different salt concentrations (0 mM, 25 mM and 50 mM) for a total of twelve individual reactions. It is not intended that the present invention be limited by the salt utilized.
• the s utilized may be chosen from potassium chloride, sodium chloride, etc. with potassium chlori being a preferred salt.
• a master mix containing a DNA substrate, buffer and salt is prepared.
• suitable buffers include 3 [N-Morpholino]propanesulfonic acid (MOPS), pH 6.5 to 9.0. with pH 7.2 to 8.4 being particularly preferred and other "Good" biological buffers such as tris[Hydroxymethyl]aminomethane (Tris) or N,N-bis[2-Hydroxyethyl]glycine (Bicine). pH 6. to 9.0. with pH 7.5 to 8.4 being particularly preferred.
• the nucleic acid substrate is RNA.
• the pH of the buffer is reduced to the range of 6.0 to 8.5. with pH 6.0 to 7.0 being particularly preferred.
• Tris buffers When manganese is to used as the divalent cation in the reaction, th use of Tris buffers is not preferred. Manganese tends to precipitate as manganous oxide in Tris if the divalent cation is exposed to the buffer for prolonged periods (such as in incubations of greater than 5 minutes or in the storage of a stock buffer). When manganese is to be used as the divalent cation, a preferred buffer is the MOPS buffer.
• the mix includes enough detectable DNA for 5 digests (e.g., approximately 500 fmoles of 5 " biotinylated DNA or approximately 100 fmoles of 32 P-5 " end labeled DNA) in 30 ⁇ l of IX CFLPTM buffer ( 10 mM MOPS. pH 7.2 to 8.2) with 1.7 mM MnCL or MgCL (the final concentration of the divalent cation will be 1 mM).
• concentrations of the divalent cation may be used if appropriate for the cleavage agent chosen (e.g., E. coli DNA polymerase I is commonly used in a buffer containing 5 mM MgG 2 ).
• the "25 mM KG” mix includes 41.5 mM KG in addition to the above components; the "50 mM KG” mix includes 83.3 mM KG in addition to the above components.
• the mixes are distributed into labeled reaction tubes (0.2 ml. 0.5 ml or 1.5 ml "Eppendorf style microcentrifuge tubes) in 6 ⁇ l aliquots. overlaid with light mineral oil or a similar barrier, and stored on ice until use.
• Sixty microliters of an enzyme dilution cocktail is assembled, comprising a 5 " nuclease at a suitable concentration in IX CFLPTM buffer without MnCL.
• Preferred 5 * nucleases and concentrations are 25 to 100 ng of the GeavaseTMBN enzyme, with 25 ng being particularly preferred or 5 units of Taq DNA polymerase (or another eubacterial Pol A-type DNA polymerase).
• Suitable amounts of a similar structure- specific cleavage agent in IX CFLPTM buffer without MnCL may also be utilized.
• Elevated temperatures can be used to bring structures to the brink of instability, so that the effects of small changes in sequence are maximized, and revealed as alterations in the cleavage pattern within the target substrate, thus allowing the cleavage reaction to occur at that point. Consequently, it is often desirable to run the reaction at an elevated temperature (i.e.. above 50°C).
• reactions are performed at 50°C. 55°C. 60°C and 65°C.
• a trio of tubes at each of the three KG concentrations are brought to
• reaction 95°C for 5 seconds, then cooled to the selected temperature.
• the reactions are then started immediately by the addition of 4 ⁇ l of the enzyme cocktail.
• a duplicate trio of tubes may be included (these tubes receiving 4 ⁇ l of IX CFLPTM buffer without enzyme or MnCL). to assess the nucleic acid stability in these reaction conditions. All reactions proceed for 5 minutes, and are stopped by the addition of 8 ⁇ l of 95% formamide with 20 mM EDTA an 0.05% xylene cyanol and 0.05% bromophenol blue. Reactions may be assembled and store on ice if necessary. Completed reactions are stored on ice until all reactions in the series ha been performed.
• nuclei acids up to approximately 1.5 kb. or native or denaturing agarose gels for larger molecules.
• the nuclei acids may be visualized as described above, by staining, autoradiography (for radioisotopes) or by transfer to a nylon or other membrane support with subsequent hybridization and/or nonisotopic detection.
• the patterns generated are examined by the criteria described above and a reaction condition is chosen for the performance of the variant comparison CFLPsTM.
• a "no enzyme" control allows the assessment of the stability of the nucleic acid substrate under particular reaction conditions. In this instance, the substrate is placed in a tube containing all reaction components except the enzyme and treated the same as the enzyme-containing reactions. Other control reactions may be run.
• a wild-type substrate m be cleaved each time a new mutant substrate is tested.
• a previously characterized mutant may be run in parallel with a substrate suspected of containing a different mutation.
• Previously characterized substrates allow for the comparison of the cleavage pattern produced by the new test substrate with a known cleavage pattern. In this manner, alterations in the new test substrate may be identified.
• the preferred method for redistributing the signal is to alter the reaction conditio to increase structure stability (e.g., lower the temperature of the cleavage reaction, raise the monovalent salt concentration); this allows other less stable structures to compete more effectively for cleavage.
• the double- stranded substrate is prepared such that it contains a single end-label usi any of the methods known to the art.
• the molar amount of DNA used in the optimization reactions is the same as that use for the optimization of reactions utilizing single-stranded substrates.
• the most notable differences between the optimization of the CFLPTM reaction for single- versus double-stranded substrates is that the double-stranded substrate is denatured in distilled water without buffer, the concentration of MnCL in the reaction is reduced to 0.2 mM. the KG (or other monovalent salt) is omitted, and the enzyme concentration is reduced to 10 to 25 ng per reaction.
• the range of temperature is the more critical controlling factor for optimization of the reaction.
• a reaction tube containing the substrate and other components described below is set up to allow performance of the reaction at each of the following temperatures: 40°C. 45°C. 50°C. 55°C. 60°C. 65°C. 70°C. and 75°C.
• a mixture comprising the single end labelled double- stranded DNA substrate and distilled water in a volume of 15 ⁇ l is prepared and placed into a thin walled microcentrifuge tube.
• This mixture may be overlaid with light mineral oil or liquid wax (this overlay is not generally required but may provide more consistent results with some double-stranded DNA substrates).
• a 2 mM solution of MnCL is prepared.
• 5 ⁇ l of a diluted enzyme solution is prepared comprising 2 ⁇ l of 10X CFLPTM buffer ( 100 mM MOPS. pH 7.2 to 8.2. 0.5% Tween-20, 0.5% Nonidet P-40), 2 ⁇ l of 2 mM MnCL and 25 ng of GeavaseTM BN enzyme and distilled water to yield a final volume of 5 ⁇ l.
• the DNA mixture is heated to 95°C for 10 to 30 seconds and then individual tubes are cooled to the reaction temperatures to be tested (e.g., 40°C. 45°C. 50°C. 55°C. 60°C. 65°C. 70°C. and 75°C).
• the cleavage reaction is started by adding 5 ⁇ l of the dilute enzyme solution to each tube at the target reaction temperature.
• the reaction is incubated at the target temperature for 5 minutes and the reaction is terminated (e.g., by the addition of 16 ⁇ l of stop solution comprising 95% formamide with 10 mM EDTA and 0.05% xylene cyanol and 0.05% bromophenol blue).
• nucleic acids up to approximately 1.5 kb. or native or denaturing agarose gels for larger molecules.
• the nucleic acids may be visualized as described above, by staining, autoradiography (for radioisotopes) or by transfer to a nylon or other membrane support with subsequent hybridization and/or nonisotopic detection.
• the patterns generated are examined by the criteria described above and a reaction condition is chosen for the performance of the double-stranded CFLPTM. Control reactions may be run as described above to assess nucleic acid stability or to create patterns for reference.
• the MnCL concentration preferably will not exceed 0.25 mM. If the end label on the double-stranded DNA substrate disappears (i.e.. loses its 5' end label as judged by a loss of signal upon detection of the cleavage products), the concentration of MnCL may be reduced to 0.1 mM. Any EDTA present in the DNA storage buffer will reduce the amount of free Mn 2' in the reaction, so double-stranded DNA should be dissolved in water or Tris-HG with a EDTA concentration o 0.1 mM or less.
• the nucleic acid substrate When the nucleic acid substrate is labelled at one end (e.g.. with biotin or 2 P) all detectable fragments share a common end.
• concentration of the enzyme e.g.. GeavaseTM BN
• the length of the incubation have minimal influence on the distribution of signal intensity, indicating that the cleavage patterns are not partial digests of a single structure assumed by the nucleic acid substrate, but rather are relatively complete digests of a collection of stable structures formed by the substrate.
• longer DNA substrates greater than 250 nucleotides
• the enzyme concentration may be lowered in the cleavage reaction (for example, if 50 ng of the GeavaseTM BN enzyme were used initially and overdigestion was apparent, the concentration of enzyme may be reduced to 25, 10 or 1 ng per reaction).
• the concentration of enzyme may be reduced to 25, 10 or 1 ng per reaction.
• Mir and Mg 2 can be used in CFLPTM buffer, to attenuate the rate of cleavage.
• 0.2 mM MnCL is used in a CFLPTM reaction, as described above (with either a single- or double stranded nucleic acid substrate)
• the use of 1 mM Mg 2* in addition to the Mir * slows down the rate of cleavage: in the case of the 1059 bp amplicon seen in Figure 30.
• the rate of cleavage is reduced approximately three-fold (in the Mn 2 7Mg 24 mixture as compared to Mir ' alone).
• the 0.2 mM Mir /lmM Mg 2* mixture may be used in conjunction with a reaction time of 5 to 20 minutes.
• Cleavage products produced by cleavage of either single-or double-stranded substrates which contain a biotin label may be detected using the following nonisotopic detection method. The following description is exemplary only: the art knows alternative methods for the detection of biotin-labelled products. After electrophoresis of the reaction products, the gel plates are separated allowing the gel to remain flat on one plate.
• a positively charged nylon membrane include Nytran®Plus.
• 0.2 or 0.45 mm-pore size Schleicher and Schuell, Keene. NH), cut to size and pre-wetted in 0.5X TBE (45 mM tris- Borate, pH 8.3, 1.4 mM EDTA). is laid on top of the exposed gel. All air bubbles trapped between the gel and the membrane are removed (e.g., by rolling a 10 ml pipet firmly across the membrane). Two pieces of 3MM filter paper (Whatman) are then placed on top of the membrane, the other glass plate is replaced, and the sandwich is clamped with binder clips or pressed with books or weights. The transfer is allowed to proceed 2 hours to overnight (the signal increases with longer transfer).
• the membrane After transfer, the membrane is carefully peeled from the gel and allowed to air dry . Distilled water from a squeeze bottle can be used to loosen any gel that sticks to the membrane. After complete drying, the membrane is agitated for 30 minutes in 1.2X Sequenase Images Blocking Buffer (United States Biochemical. Cleveland. OH: avoid any precipitates in the blocking buffer by decanting or filtering); 0.3 ml of the buffer is used per cm 2 of membrane (e.g., 30 mis for a 10cm x 10cm blot).
• Blocking Buffer United States Biochemical. Cleveland. OH: avoid any precipitates in the blocking buffer by decanting or filtering
• a streptavidin-alkaline phosphatase conjugate (SAAP, United Stated Biochemical) is added at a 1 :4000 dilution directly to the blocking solution (avoid spotting directly on membrane), and agitated for 15 minutes.
• the membrane is rinsed briefly with dH,0 and then washed 3 times (5 minutes of shaking per/wash) in IX SAAP buffer (100 mM Tris-HG. pH 10; 50 mM NaCl) with 0.1% sodium dodecyl sulfate (SDS). using 0.5 ml buffer/cm 2 of membrane, with brief water rinses between each wash.
• the membrane is then washed twice in IX SAAP buffer (no SDS) with 1 mM
• MgCL drained thoroughly, and placed in a plastic heat-sealable bag. Using a sterile pipet tip. 0.05 ml/cm 2 of CDP-StarTM (Tropix. Bedford, MA) is added to the bag and distributed over the entire membrane for 5 minutes. The bag is drained of all excess liquid and air bubbles, sealed, and the membrane is exposed to X-ray film (e.g.. Kodak XRP) for 30 minutes. Exposure times are adjusted as necessary for resolution and clarity.
• X-ray film e.g.. Kodak XRP
• nucleic acid substrate tested in the CFLPTM system has produced a reproducible pattern of fragments.
• the sensitivity and specificity of the cleavage reaction make this method of analysis very suitable for the rapid screening of mutations in cancer diagnostics, tissue typing, genetic identity, bacterial and viral typing, polymorphism analysis, structure analysis, mutant screening in genetic crosses, etc. It could also be applied to enhanced RNA analysis, high level multiplexing and extension to longer fragments.
• One distinct benefit of using the GeavaseTM reaction to characterize nucleic acids is that the pattern of cleavage products constitutes a characteristic finge ⁇ rint. so a potential mutant can be compared to previously characterized mutants without sequencing. Also, the place in the fragment pattern where a change is observed gives a good indication of the position of the mutation. But it is noted that the mutation need not be at the precise site of cleavage, but only in an area that affects the stability of the structure.
• Tumor suppressor genes control cellular proliferation and a variety of other processes important for tissue homeostasis.
• the p53 gene encodes a regulator of the cell cycle machinery that can suppress the growth of cancer cells a well as inhibit cell transformation (Levine. Annu. Rev. Biochem. 62:623 [1993]).
• Tumor suppressor mutations that alter or obliterate normal p53 function are common.
• Mutations in the p53 tumor suppressor gene are found in about half of all cases of human cancer making alterations in the p53 gene the most common cancer-related genetic change known at the gene level.
• the p53 gene encode a 53-kD nuclear phosphoprotein. comprising 393 amino acids, which is involved in the control of cellular proliferation.
• Mutations in the p53 gene are generally (greater than 90%) missense mutations which cause a change in the identity of an amino acid rather than nonsense mutations which cause inactivation of the protein.
• the gene encoding the p53 protein is large, spanning 20.000 base pairs, and is divided into 1 1 exons (see Figure 4).
• the ability to scan the large p53 gene for the presence of mutations has important clinical applications.
• the presenc of a tumor p53 mutation is associated with a poor prognosis.
• p53 mutation has been shown to be an independent marker of reduced survival in lymph node-negative breast cancers, a finding that may assist clinicians in reaching decisions regarding more aggressive therapeutic treatment.
• Lowe and co-workers have demonstrated that the vulnerability of tumor cells to radiation or chemotherapy is greatly reduced by mutations which abolish p53-dependent apoptosis [Lowe et al . Cell 74:957 (1995)].
• Regions of the p53 gene from approximately 10.000 tumors have been sequenced in the last 4 to 5 years, resulting in characterization of over 3.700 mutations of which approximately 1,200 represent independent p53 mutations (i.e., point mutations, insertion or deletions).
• a database has been compiled and deposited with the European Molecular Biology Laboratory (EMBL) Data Library and is available in electronic form [Hollstein. M. et al. ( 1994) Nucleic Acids Res. 22:3551 and Cariello. N.F. et al. (1994) Nucleic Acids Res. 22:3549].
• EMBL European Molecular Biology Laboratory
• an IBM PC compatible software package to analyze the information in the database has been developed. [Cariello et al. Nucl. Acids Res.
• the point mutations in the database were identified by DNA sequencing of PCR-amplified products. In most cases, preliminary screening for mutations by SSCP or DGGE was performed. Analysis of the p53 mutations shows that the p53 gene contains 5 hot spot regions
• HSR most frequently mutated in human tumors that show a tight correlation between domains of the protein that are evolutionary highly conserved (ECDs) and seem to be specifically involved in the transformation process (see Figure 4; the height of the bar represent the relative percentage of total mutations associated with the five HSRs).
• ECDs evolutionary highly conserved
• the five HSRs are confined to exons 5 to 8 and account for over 85% of the mutations detected.
• nucleic acid comprising p53 gene sequences are prepared.
• the nucleic acid may comprise 0 genomic DNA, RNA or cDNA forms of the p53 gene.
• Nucleic acid may be extracted from variety of clinical samples [fresh or frozen tissue, suspensions of cells (e.g.. blood), cerebral spinal fluid, sputum, etc.] using a variety of standard techniques or commercially available kits. For example, kits which allow the isolation of RNA or DNA from tissue samples are available from Qiagen. Inc. (Chatsworth, CA) and Stratagene (LaJolla. CA). respectivel .
• Total RNA may be isolated from tissues and tumors by a number of methods known to thos skilled in the art and commercial kits are available to facilitate the isolation.
• t RNeasy® kit Qiagen Inc.. Chatsworth, CA
• t RNeasy® kit provides protocol, reagents and plasticware to permit the isolation of total RNA from tissues, cultured cells or bacteria, with no modificati to the manufacturer ' s instructions, in approximately 20 minutes. Should it be desirable, in t
• the polyadenylated RNAs in the mixture may be specifically isolated by binding to an oligo-deoxythymidine matrix, through the use of a kit such as the Oligotex® kit (Qiagen). Comparable isolation kits for both of these steps are available through a number of commercial suppliers.
• RNA may be extracted from samples, including biopsy specimens,
• RNA _->D conveniently by lysing the homogenized tissue in a buffer containing 0.22 M NaCl. 0.75 m MgCL. 0.1 M Tris-HCl, pH 8.0, 12.5 mM EDTA. 0.25% NP40. 1% SDS. 0.5 mM DTT. 500 u/ml placental RNAse inhibitor and 200 ⁇ g/ml Proteinase K. Following incubation at 37°C for 30 min, the RNA is extracted with phenol xhloroform (1 :1 ) and the RNA is recovered by ethanol precipitation. Since the majority of p53 mutations are found within exons 5-8. it is convenient as a first analysis to examine a PCR fragment spanning this region.
• PCR fragments spanning exons 5- 8 may be amplified from clinical samples using the technique of RT-PCR (reverse transcription-PCR); kits which permit the user to start with tissue and produce a PCR product are available from Perkin Elmer (Norwalk. CT) and Stratagene (LaJolla. CA).
• the RT-PCR technique generates a single-stranded cDNA corresponding to a chosen segment of the coding region of a gene by using reverse transcription of RNA; the single-stranded cDNA is then used as template in the PCR.
• an approximately 600 bp fragment spanning exons 5-8 is generated using primers located in the coding region immediately adjacent to exons 5 and 8 in the RT-PCR.
• the PCR amplified segment is then subjected to the CFLP reaction and the reaction products are analyzed as described above in section VIII.
• Fragments suitable for CFLP analysis may also be generated by PCR amplification of genomic DNA.
• DNA is extracted from a sample and primers corresponding to sequences present in introns 4 and 8 are used to amplify a segment of the p53 gene spanning exons 5-8 which includes introns 5-7 (an approximately 2 kb fragment).
• primers may be chosen to amplify smaller (1 kb or less) segments of genomic DNA or alternatively a large PCR fragment may be divided into two or more smaller fragments using restriction enzymes.
• a library containing the CFLP pattern produced by previously characterized mutations may be provided. Comparison of the pattern generated using nucleic acid derived from a clinical sample with the patterns produced by cleavage of known and characterized p53 mutations will allow the rapid identification of the specific p53 mutation present in the patient ' s tissue.
• the comparison of CFLP patterns from clinical samples to the patterns present in the library may be accomplished by a variety of means. The simplest and least expensive comparison involves visual comparison. Given the large number of unique mutations known at the p53 locus. visual (i.e., manual) comparison may be too time-consuming, especially when large numbers of clinical isolates are to be screened.
• the CFLP patterns or "bar codes" may be provided in an electronic format for ease and efficiency in comparison.
• Electronic entry may comprise storage of scans of gels containing the CFLP products of the reference p53 mutations (using for example, the GeneReader and Gel Doctor Fluorescence Gel documentation system (BioRad. Hercules, CA) or the ImageMaster (Pharmacia Biotech. Piscataway, NJ).
• the banding pattern may be stored as the signal collected from the appropriate channels during an automated run [examples of instrumentation suitable for such analysis and data collection include fluorescence-based gel imagers such as fluoroimagers produced by Molecular Dynamics and Hitachi or by real-time electrophoresis detection systems such as the ABI 377 or Pharmacia ALF DNA Sequencer].
• fluorescence-based gel imagers such as fluoroimagers produced by Molecular Dynamics and Hitachi or by real-time electrophoresis detection systems such as the ABI 377 or Pharmacia ALF DNA Sequencer.
• the p53 bar code library is generated using reverse genetics. Engineering of p53 mutations ensures the identity and purity of each of the mutations as each engineered mutatio is confirmed by DNA sequencing.
• the individual p53 mutations in p53 bar code library are generated using the 2-step "recombinant PCR" technique [Higuchi, R. (1991 ) In Ehrlich, H.A.
• Figure 5 provides a schematic representation of one method of a 2-step recombinant PCR technique that may be used for the generation of p53 mutations.
• the template for the PCR amplifications is the entire human p53 cDNA gene.
• PCR 1 an oligonucleotide containing the engineered mutation
• oligo A an oligonucleotide containing the engineered mutation
• oligo B an oligonucleotide containing a 5 " arm of approximately 20 non-complementary bases
• the resulting amplification product will contain the mutation at its extreme 5' end and a foreign sequence at its 3 " end.
• the 3 " sequence is designed to include a unique restriction site (e.g., Eco ⁇ ) to aid in the directional cloning o ⁇ ⁇ the final amplification fragment (important for purposes of sequencing and archiving the DN containing the mutation).
• the product generated in the upstream or first PCR may be gel purified if desired prior to the use of this first PCR product in the second PCR: however gel purification is not required once it is established that this fragment is the only species amplified in the PCR.
• PCR 2 The small PCR fragment containing the engineered mutation is then used to direct a second round of PCR (PCR 2).
• PCR 2 the target DNA is a larger fragment
• oligo C a primer complementary to a region of the target DNA upstream of the locus of the engineered mutation
• oligo D a primer complementary to the 5' noncomplementary sequence of the small product of PCR 1
• a second unique restriction site can be engineered into oligo C (e.g., HindlU).
• DNA stocks of each mutant can be maintained in the form of large scale PCR preparations. This permits distribution of either bacteria harboring plasmids containing a given mutation or a PCR preparation to be distributed as individual controls in kits containing reagents for the scanning of p53 mutations in clinical samples or as part of a supplemental master p53 mutation library control kit.
• An alternative 2-step recombinant PCR is diagrammed in Figure 6. and described in Example 30.
• two mutagenic oligonucleotides one for each strand, are synthesized. These oligonucleotides are substantially complementary to each other but are opposite in orientation.. That is. one is positioned to allow amplification of an "upstream" region of the DNA, with the mutation inco ⁇ orated into the 3 " proximal region of the upper, or sense strand, while the other is positioned to allow amplification of a "downstream" segment with the intended mutation inco ⁇ orated into the 5 ' proximal region of the upper, or sense strand.
• this method does allow rapid recombinant PCR to be performed using existing end primers, and without the introduction of foreign sequences. In summary, this method is often used if only a few recombinations are to be performed. When large volumes of mutagenic PCRs are to be performed, the first described method is preferable as the first method requires a single oligo be synthesized for each mutagenesis and only recombinants ar amplified.
• kits designed for the identification of p53 mutations in clinica samples is the inclusion of the specific primers to be used for generating PCR fragments to b analyzed for CFLP. While DNA fragments from 100 to over 1500 bp can be reproducibly and accurately analyzed for the presence of sequence polymo ⁇ hisms by this technique, the precise patterns generated from different length fragments of the same input DNA sequence will of course vary. Not only are patterns shifted relative to one another depending on the length of the input DNA. but in some cases, more long range interactions between distant regions of long DNA fragments may result in the generation of additional cleavage products not seen with shorter input DNA products.
• the simplest and most direct method of analyzing the DNA fragments produced in the CFLPTM reaction is by gel electrophoresis. Because electrophoresis is widely practiced and easily accessible, initial efforts have been aimed at generating a database in this familiar format. It should, however, be noted that resolution of DNA fragments generated by CFLPTM analysis is not limited to electrophoretic methods. Mass spectrometry, chromatography. fluorescence polarization, and chip hybridization are all approaches that are currently being refined and developed in a number of research laboratories. Once generated, the CFLPTM database is easily adapted to analysis by any of these methods. There are several possible alternatives available for detection of CFLP patterns. A critical user benefit of CFLP analysis is that the results are not dependent on the chosen method of DNA detection.
• DNA fragments may be labeled with a radioisotope (e.g.. a ' 2 P or 35 S-labeled nucleotide) placed at either the 5' or 3 * end of the nucleic acid or alternatively the label may be distributed throughout the nucleic acid (i.e.. an internally labeled substrate).
• the label may be a nonisotopic detectable moiety, such as a fluorophore which can be detected directly, or a reactive group which permits specific recognition by a secondary agent.
• CFLP patterns have been detected by immunostaining, biotin-avidin interactions, autoradiography and direct fluorescence imaging.
• fluorescence-based detection methods may be preferred. It is important to note, however, that any of the above methods may be used to generate CFLP bar codes to be input into the database.
• fluorescence-based schemes offer a noteworthy additional advantage in clinical applications.
• CFLP allows the analysis of several samples in the same tube and in the same lane on a gel.
• This "multiplexing" permits rapid and automated comparison of a large number of samples in a fraction of the time and for a lower cost than can be realized through individual analysis of each sample.
• This approach opens the door to several alternative applications.
• a researcher could decide to double, triple or quadruple (up to 4 dyes have been demonstrated to be detectable and compatible in a single lane in commercially available DNA sequencing instrumentation such as the ABI 373/377) the number of samples run on a given gel.
• the analyst may include a normal p53 gene sample in each tube, and each gel lane, along with a differentially labeled size standard, as a internal standard to verify both the presence and the exact location(s) of a pattern difference(s) between the normal p53 gene and putative mutants.
• HCV infection is the predominant cause of post-transfusion non-A. non-B (NANB) hepatitis around the world.
• HCV is the major etiologic agent of hepatocellular carcinoma (HCC) and chronic liver disease world wide. HCV infection is transmitted primarily to blood transfusion recipients and intravenous drug users although maternal transmission to offspring and transmission to recipients of organ transplants have been reported.
• the genome of the positive-stranded RNA hepatitis C virus comprises several regions including 5 ' and 3 ' noncoding regions (i.e., 5 ' and 3 ' untranslated regions) and a polyprotein coding region which encodes the core protein (C).
• C core protein
• El and E2/NS1 envelope glycoproteins
• NS2-NS5b nonstructural glycoproteins
• HCV type has also been associated with differential efficacy of treatment with interferon. with Group 1 infected individuals showing little response [Kanai et al. Lancet 339:1543 (1992) and Yoshioka et al, Hepatology 16:293
• MDR-TB Multi-drug resistant tuberculosis
• tuberculosis has always been difficult to diagnose because of the extremely long generation time of Mycohacterium tuberculosis as well as the environmental prevalence of other, faster growing mycobacterial species.
• the doubling time of M. tuberculosis is 20-24 hours, and growth by conventional methods typically requires 4 t 6 weeks to positively identify M. tuberculosis [Jacobs. Jr. et al.. Science 260:819 ( 1993) and
• tuberculosis to be positively identified more rapidly than by classical methods: detection time have been reduced from greater than 6 weeks to as little as two weeks (culture-based methods) or two days (nucleic acid-based methods). While culture-based methods are currently in wide-spread use in clinical laboratories, a number of rapid nucleic acid-based methods that can be applied directly to clinical samples are under development. For all of th techniques described below, it is necessary to first "decontaminate" the clinical samples, such as sputum (usually done by pretreatment with N-acetyl L-cysteine and NaOH) to reduce contamination by non-mycobacterial species [Shinnick and Jones, supra.]
• PCR polymerase chain reaction
• the Amplified M. tuberculosis Direct Test (AMTDT; Gen-Probe) relies on Transcription Mediated Amplification [TMA; essentially a self-sustained sequence reaction (3SR) amplification] to amplify target rRNA sequences directly from clinical specimens.
• TMA Transcription Mediated Amplification
• the rRNA Once the rRNA has been amplified, it is then detected by a dye-labeled assay such as the PACE2. This assay is highly subject to inhibition by substances present in clinical samples.
• the Cycling Probe Reaction CPR; ID Biomedical. This technique, which is under development as a diagnostic tool for detecting the presence of M. tuberculosis, measures the accumulation of signal probe molecules.
• the signal amplification is accomplished by hybridizing tripartite DNA-RNA-DNA probes to target nucleic acids, such as M. tuberculosis-specific sequences.
• RNA portion of the chimeric probe is degraded, releasing the DNA portions, which accumulate linearly over time to indicate that the target sequence is present [Yule, Bio/Technology 12: 1335 ( 1994)].
• the need to use of RNA probes is a drawback, particularly for use in crude clinical samples, where RNase contamination is often rampant.
• the traditional method used to determine antibiotic resistance is the direct proportion agar dilution method, in which dilutions of culture are plated on media containing antibiotics and on control media without antibiotics. This method typically adds an additional 2-6 weeks to the time required for diagnosis and characterization of an unknown clinical sample [Jacobs. Jr.. supra].
• the Luciferase Reporter Mycobacteriophage (LRM) assay was first described in 1993 [Jacobs. Jr. et al. Science 260:819 (1993)].
• LRM Luciferase Reporter Mycobacteriophage
• a mycobacteriophage containing a cloned copy of the luciferase gene is used to infect mycobacterial cultures.
• the expressed luciferase produces photons, easily distinguishable by eye o by a luminometer. allowing a precise determination of the extent of mycobacterial growth in the presence of antibiotics. Once sufficient culture has been obtained (usually 10-14 days post-inoculation), the assay can be completed in 2 days.
• DNA-based methods Genetic loci involved in resistance to isoniazid. rifampin. streptomycin, fluoroquinolones, and ethionamide have been identified [Jacobs. Jr.. supra; Heym et al, Lancet 344:293 (1994) and Morris et al. J. Infect. Dis. 171 :954 ( 1995)].
• PCR-SSCP PCR-Single Stranded Conformational Polymorphism
• PCR-ddF PCR-dideoxyfinge ⁇ rinting
• CFLPTM method of the present invention provides an approach that relies on structure specific cleavage to generate distinct collections of DNA fragments.
• This method is highly sensitive (>98%) in its ability to detect sequence polymorphisms, and requires a fraction of the time, skill and expense of the techniques described above.
• CFLPTM fluoroquinoline
• Identification and typing of bacterial pathogens is critical in the clinical management of infectious diseases. Precise identity of a microbe is used not only to differentiate a disease state from a healthy state, but is also fundamental to determining whether and which antibiotics or other antimicrobial therapies are most suitable for treatment.
• Traditional methods of pathogen typing have used a variety of phenotypic features, including growth characteristics, color, cell or colony mo ⁇ hology, antibiotic susceptibility, staining, smell and reactivity with specific antibodies to identify bacteria.
• Organisms can be further differentiated by using the RFLP method described above, in which the genomic DNA is digested with one or more restriction enzymes before electrophoretic separation and transfer to a nitrocellulose or nylon membrane support.
• Probing with the species-specific nucleic acid probes will reveal a banding pattern that, if it shows variation between isolates, can be used as a reproducible way of discriminating between strains.
• these methods are susceptible to the drawbacks outlined above: hybridization-based assays are time-consuming and may give false or misleading results if the stringency of the hybridization is not well controlled, and RFLP identification is dependent on the presence of suitable restriction sites in the DNA to be analyzed.
• PCR finge ⁇ rinting the size of a fragment generated by PCR is used as an identifier.
• VNTRs regions containing variable numbers of tandem repeated sequences
• the number of repeats, and thus the length of the PCR amplicon. ca be characteristic of a given pathogen, and co-amplification of several of these loci in a single reaction can create specific and reproducible finge ⁇ rints. allowing discrimination between closely related species.
• the target of the amplification does not display a size difference, and the amplified segment must be further probed to achieve more precise identification. This may be done on a solid support, in a fashion analogous to the whole-genome hybridization described above, but this has the same problem with variable stringency as that assay.
• the interior of the PCR fragment may be used as a template for a sequence-specific ligation event.
• single stranded probes to be ligated are positioned along the sequence of interest on either side of an identifying polymorphism, so that the success or failure of the ligation will indicate the presence or absence of a specific nucleotide sequence at that site.
• primers that recognize conserved regions of bacterial ribosomal RNA genes allow amplification of segments of these genes that include sites of variation.
• the variations in ribosomal gene sequences have become an accepted method not only of differentiating between similar organisms on a DNA sequence level, but their consistent rate of change allows these sequences to be used to evaluate the evolutionary relatedness of organisms.
• the present invention allows the amplification products derived from these sequences to be used to create highly individual barcodes (i.e., cleavage patterns), allowing the detection of sequence polymorphisms without prior knowledge of the site, character or even the presence of said polymorphisms. With appropriate selection of primers, amplification can be made to be either all-inclusive (e.g..
• ribosomal genes are highly conserved ribosomal sequences
• the primers can be chosen to be very specific for a given genus, to allow examination at the species and subspecies level. While the examination of ribosomal genes is extremely useful in these characterizations, the use of the CFLPTM method in bacterial typing is not limited to these genes. Other genes, including but not limited to those associated with specific growth characteristics, (e.g., carbon source preference, antibiotic resistance, resistance to methycillin or antigen production), or with particular cell mo ⁇ hologies (such as pilus formation) are equally well suited to the CFLPTM assay.
• specific growth characteristics e.g., carbon source preference, antibiotic resistance, resistance to methycillin or antigen production
• cell mo ⁇ hologies such as pilus formation
• nucleic acid is extracted from the sample.
• the nucleic acid may be extracted from a variety of clinical samples [fresh or frozen tissue, suspensions of cells (e.g., blood), cerebral spinal fluid, sputum, urine, etc.] using a variety of standard techniques or commercially available kits. For example, kits which allow the isolation of RNA or DNA from tissue samples are available from Qiagen. Inc.
• QlAamp Blood kits permit the isolation of DNA from blood (fresh, frozen or dried) as well as bone marrow, body fluids or cell suspensions.
• QIAamp tissue kits permit the isolation of DNA from tissues such as muscles, organs and tumors.
• Samples which contain relatively few copies of the material to be amplified can be added directly to a PCR. Blood samples have posed a special problem in PCRs due to the inhibitory properties of red blood cells.
• the red blood cells must be removed prior to the use of blood in a PCR; there are both classical and commercially available methods for this pu ⁇ ose [e.g., QIAamp Blood kits, passage through a Chelex 100 column (BioRad). etc.].
• Extraction of nucleic acid from sputum the specimen of choice for the direct detection of M. tuberculosis, requires prior decontamination to kill or inhibit the growth of other bacterial species. This decontamination is typically accomplished by treatment of the sample with N- acetyl L-cysteine and NaOH (Shinnick and Jones, supra). This decontamination process is necessary only when the sputum specimen is to be cultured prior to analysis.
• Centigrade Centigrade); g (gravitational field); vol (volume); w/v (weight to volume); v/v (volume to volume); BSA (bovine serum albumin); CTAB (cetyltrimethylammonium bromide); HPLC (high pressure liquid chromatography); DNA (deoxyribonucleic acid); IVS (intervening sequence); p (plasmid); ⁇ l (microliters); ml (milliliters); ⁇ g (micrograms); pmoles (picomoles); mg (milligrams); MOPS (3-[N-Mo ⁇ holino]propanesulfonic acid); M (molar): mM (milliMolar); ⁇ M (microMolar); nm (nanometers): kdal (kilodaltons); OD (optical density); EDTA (ethylene diamine tetra-acetic acid); FITC (fluorescein isothiocyanate); SDS (sodium dodec
• Tris buffer titrated with boric acid rather than HG and containing EDTA i.e., Tris buffer titrated with boric acid rather than HG and containing EDTA
• PBS phosphate buffered saline
• PPBS phosphate buffered saline containing 1 mM PMSF
• PAGE polyacrylamide gel electrophoresis
• Tween polyoxyethylene-sorbitan: Boehringer Mannheim (Boehringer Mannheim, Indianapolis, IN); Dynal (Dynal A.S.. Oslo. Norway); Epicentre (Epicentre Technologies, Madison. WI); National Biosciences (National Biosciences. Plymouth, MN); New England Biolabs (New England Biolabs. Beverly, MA); Novagen
• DNAPTaq is able to amplify many, but not all. DNA sequences.
• One sequence that cannot be amplified using DNAPTw/ is shown in Figure 7 (Hai ⁇ in structure is SEQ ID NO: 15.
• PRIMERS are SEQ ID NOS: 16- 17.) This DNA sequence has the distinguishing characteristic of being able to fold on itself form a hairpin with two single-stranded arms, which correspond to the primers used in PCR.
• DNAP7 -7 and DNAPStf were obtained from The Biotechnology Center at the University of Wisconsin-Madison.
• the DNAP Tag and DNAPStf were from Perkin Elmer (i.e. , AmpliTaq DNA polymerase and the Stoffel fragment of Amplitaq DNA poly ⁇ merase).
• the substrate DNA comprised the hai ⁇ in structure shown in Figure 7 cloned in a double-stranded form into pUC19.
• primers used in the amplification are listed as SEQ ID NOS: 16-17.
• Primer SEQ ID NO:17 is shown annealed to the 3 " arm of the hairpin struc ture in Fig. 7.
• Primer SEQ ID NO: 16 is shown as the first 20 nucleotides in bold on the 5 " arm of the hai ⁇ in in Fig. 7.
• Polymerase chain reactions comprised 1 ng of supercoiled plasmid target DNA. 5 pmoles of each primer. 40 ⁇ M each dNTP. and 2.5 units of DNAPTaq or DNAPStf. in a 50 ⁇ l solution of 10 mM Tris Cl pH 8.3.
• the ONAPTaq reactions included 50 mM KG and 1. mM MgCL. The temperature profile was 95°C for 30 sec. 55°C for 1 min. and 72°C for 1 min.. through 30 cycles. Ten percent of each reaction was analyzed by gel electrophoresis through 6% polyacrylamide (cross-linked 29:1) in a buffer of 45 mM Tris Borate, pH 8.3. 1. mM EDTA (0.5X TBE). The results are shown in Figure 8. The expected product was made by DNAPStf
• PCR was compared using the same two polymerases (Figure 9).
• the hai ⁇ in templates such as the one described in Figure 6, were made using DNAPStf and a 2 P-5 " -end-labeled primer
• the 5 " -end of the DNA was released as a few large fragments by DNAPTaq but not by DNAPStf.
• the sizes of these fragments show that they contain most or all of the unpaired 5 ' arm of the DNA.
• cleavage occurs at or near the base o the bifurcated duplex.
• These released fragments terminate with 3 ' OH groups, as evidenced by direct sequence analysis, and the abilities of the fragments to be extended by terminal deoxvnucleotidvl transferase.
• Figures 10-12 show the results of experiments designed to characterize the cleavage reaction catalyzed by DNAPTaq. Unless otherwise specified, the cleavage reactions com ⁇ prised 0.01 pmoles of heat-denatured, end-labeled hai ⁇ in DNA (with the unlabeled comple ⁇ mentary strand also present), 1 pmole primer (complementary to the 3 " arm) and 0.5 units of ONAPTaq (estimated to be 0.026 pmoles) in a total volume of 10 ⁇ l of 10 mM Tris-G. pH
• reactions were initiated at the final reaction temperature by the addition of either the MgCL or enzyme. Reactions were stopped at their incubation temperatures by the addition of 8 ⁇ l of 95% formamide containing 20 mM EDTA and 0.05% marker dyes (stop solution). The T n ⁇ calculations listed were made using the OligoTM primer analysis software from National Biosciences. Inc. These were determined using 0.25 ⁇ M as the DNA concentration, at either 15 or 65 mM total salt (the 1.5 mM MgCL in all reactions was given the value of 15 mM salt for these calculations).
• Figure 10 is an autoradiogram containing the results of a set of experiments and conditions on the cleavage site.
• Figure 10A is a determination of reaction components that enable cleavage. Incubation of 5 * -end-labeled hai ⁇ in DNA was for 30 minutes at 55°C. with the indicated components. The products were resolved by denaturing polyacrylamide gel electrophoresis and the lengths of the products, in nucleotides, are indicated.
• Figure 10B describes the effect of temperature on the site of cleavage in the absence of added primer. Reactions were incubated in the absence of KG for 10 minutes at the indicated temperatures. The lengths of the products, in nucleotides. are indicated.
• cleavage by DNAPTia ⁇ requires neither a primer nor dNTPs (see Fig. 10A).
• Nuclease activity can be uncoupled from polymerization.
• Nuclease activity requires magnesium ions, though manganese ions can be substituted, albeit with potential changes in specificity and activity.
• zinc nor calcium ions support the cleavage reaction. The reaction occurs over a broad temperature range, from 25 °C to 85 ° C. with the rate of cleavage increasing at higher temperatures.
• the primer is not elongated in the absence of added dNTPs.
• the primer influences both the site and the rate of cleavage of the hairpin.
• the change in the site of cleavage apparently results from disruption of a short duplex formed between the arms of the DNA substrate.
• the sequences indicated by underlining in Figure 7 could pair, forming an extended duplex. Cleavage at the end of the extended duplex would release the 1 1 nucleotide fragment seen on the Fig. 10A lanes with no added primer. Addition of excess primer (Fig. 10A. lanes 3 and
• cleavage occurs at the end of the substrate duplex (either the extended or shortened form, depending on the temperature) between the first and second base pairs.
• cleavage also occurs one nucleotide into the duplex.
• a gap of four or six nucleotides exists between the 3 ' end of the primer and the substrate duplex, the cleavage site is shifted four to six nucleotides in the 5 ' direction.
• Fig. 1 1 describes the kinetics of cleavage in the presence (Fig. 1 1A) or absence (Fig.
• Figs. 1 1 A and 1 IB indicate that the reaction appears to be about twenty times faster in the presence of primer than in the absence of primer. This effect on the efficiency may be attributable to proper alignment and stabilization of the enzyme on the substrate.
• Cleavage does not appear to be inhibited by long 3 ' arms of either the substrate strand target molecule or pilot nucleic acid, at least up to 2 kilobases. At the other extreme. 3 " arms of the pilot nucleic acid as short as one nucleotide can support cleavage in a primer- independent reaction, albeit inefficiently. Fully paired oligonucleotides do not elicit cleavage of DNA templates during primer extension.
• PCR primers that have unpaired 3' ends could act as pilot oligonucleotides to direct selective cleavage of unwanted templates during preincubation of potential template-primer complexes with DNAPTaq in the absence of nucleoside triphosphates.
• each DNA polymerase was assayed in a 20 ⁇ l reaction, using either the buffers supplied by the manufacturers for the primer-dependent reactions, or 10 mM Tris Cl. pH 8.5. 1.5 mM MgCL, and 20 mM KG. Reaction mixtures were at held 72°C before the addition of enzyme.
• Figure 12 is an autoradiogram recording the results of these tests.
• Figure 12A demonstrates reactions of endonucleases of DNAPs of several thermophilic bacteria. The reactions were incubated at 55°C for 10 minutes in the presence of primer or at 72°C for 30 minutes in the absence of primer, and the products were resolved by denaturing polyacrylamide gel electrophoresis. The lengths of the products, in nucleotides. are indicate
• Figure 12B demonstrates endonucleolytic cleavage by the 5 ' nuclease of DNAPEcl. The DNAPEcl and DNAP Klenow reactions were incubated for 5 minutes at 37°C.
• Figure 12B also demonstrates DNAPTaq reactions in the presence (+) or absence (-) of primer. These reactions were run in 50 mM and 20 mM KG. respectively, and were incubated at 55 ° C for 10 minutes.
• DNAPs from the eubacteria Thermus thermophilus and Thermus flavus cleave the substrate at the same place as DNAP Tag, both in the presence an absence of primer.
• DNAPs from the archaebacteria Pyrococcus furiosus and Thermococcus litoralis are unable to cleave the substrates endonucleolytically.
• the DNAPs from Pyrococcus furious and Thermococcus litoralis share little sequence homology with eubacterial enzymes (Ito et al, Nucl Acids Res. 19:4045 (1991 ); Mathur et al. Nucl Acids. Res.
• DNAPEcl also cleave the substrate, but the resulting cleavage products are difficult to detect unless the 3' exonuclease is inhibited.
• the amino acid sequences of the 5 ' nuclease domains of DNAPEc and DNAPTaq are about 38% homologous (Gelfand, supra).
• the 5 " nuclease domain of DNAPTaq also shares about 19% homology with the 5 " exonuclease encoded by gene 6 of bacteriophage T7 [Dunn et al. J. Mol Biol 166:477
• This nuclease which is not covalently attached to a DNAP polymerization domain is also able to cleave DNA endonucleolytically, at a site similar or identical to the site that i cut by the 5 " nucleases described above, in the absence of added primers.
• pilot oligonucleotide A partially complementary oligonucleotide termed a "pilot oligonucleotide" was hybridized to sequences at the desired point of cleavage.
• the non-complementary part of the pilot oligonucleotide provided a structure analogous to the
• oligonucleotides 19-12 (SEQ ID NO: 18) and 34-19 (SEQ ID NO: 19) have only 19 and 30 nucleotides, respectively, that are complementary to different sequences in the substrate strand.
• the pilot oligonucleotides are calculated to melt off their complements at about 50°C (19-12) and about 75°C (30-12). Both pilots have 12 nucleotides at their 3 ' ends, which act as 3' arms with base-paired primers attached.
• cleavage could be directed by a pilot oligonucleotide
• the transcleavage reactions, where the target and pilot nucleic acids are not covalently linked, includes 0.01 pmoles of single end-labeled substrate DNA, 1 unit of ONAPTaq and 5 pmoles of pilot oligonucleotide in a volume of 20 ⁇ l of the same buffers.
• Oligonucleotides 30-12 and 19-12 can hybridize to regions of the substrate DNAs that are 85 and 27 nucleotides from the 5 " end of the targeted strand.
• Figure 23 shows the complete 206-mer sequence (SEQ ID NO:26).
• the 206-mer was generated by PCR .
• the M13/pUC 24-mer reverse sequencing (-48) primer and the M13/pUC sequencing (-47) primer from New England Biolabs (catalogue nos. 1233 and 1224 respectively) were used (50 pmoles each) with the pGEM3z(f+) plasmid vector (Promega Co ⁇ .) as template (10 ng) containing the target sequences.
• the conditions for PCR were as follows: 50 ⁇ M of each dNTP and 2.5 units of Taq DNA polymerase in 100 ⁇ l of IX PCR Buffer (20 mM Tris-G, pH 8.3, 1.5 mM MgCL.
• RNA substrate made by T7 RNA polymerase in the presence of [ ⁇ - 2 P]UTP, corresponds to a truncated version of the DNA substrate used in Figure 13B. Reaction conditions were similar to those in used for the DNA substrates described above, with 50 mM KG; incubation was for 40 minutes at 55°C.
• the pilot oligonucleotide used is termed 30-0 (SEQ ID NO:20) and is shown in Figure 14A. The results of the cleavage reaction is shown in Figure 14B. The reaction was run either in the presence or absence of DNAPTaq or pilot oligonucleotide as indicated in Figur 14B.
• the 5 " nuclease of DNAPTaq is a structure-specific RNaseH that cleaves the RNA at a single site near the 5 " end of the heteroduplexed region. It is su ⁇ rising that an oligonucleotide lacking a 3 " arm is able to act as a pilot in directing efficient cleavage of an RNA target because such oligonucleotides are unable to direct efficient cleavage of DNA targets using native DNAPs.
• some 5 " nucleases of the present invention can cleave DNA in the absence of a 3' arm. In other words, a non-extendable cleavage structure is not required for specific cleavage with some 5' nucleases of the present invention derived from thermostable DNA polymerases.
• DNAPTth Another thermophilic DNAP, DNAPTth, is able to use RNA as a template, but only in the presence of Mn *" , so we predicted that this enzyme would not cleave RNA in the presence of this cation. Accordingly, we incubated an RNA molecule with an appropriate pilot oligonucleotide in the presence of DNAPTaq or DNAPTth. in buffer containing either M or Mn " . As expected, both enzymes cleaved the RNA in the presence of Mg ⁇ However.
• DNAPTaq. degraded the RNA in the presence of Mn " .
• Mn " Mn " .
• Thermostable DNA polymerases were generated which have reduced synthetic activity, an activity that is an undesirable side-reaction during DNA cleavage in the detection assay of the invention, yet have maintained thermostable nuclease activity.
• the result is a thermostable polymerase which cleaves nucleic acids DNA with extreme specificity.
• Type A DNA polymerases from eubacteria of the genus Thermus share extensive protein sequence identity (90% in the polymerization domain, using the Lipman-Pearson method in the DNA analysis software from DNAStar, WI) and behave similarly in both polymerization and nuclease assays.
• Thermus aquaticus and Thermus flavus (DNAPTfl) as representatives of this class.
• Polymerase genes from other eubacterial organisms such as Thermus thermophilus. Thermus sp.. Thermotoga maritima. Thermosipho africanus and Bacillus stearothermophilus are equally suitable.
• the DNA polymerases from these thermophilic organisms are capable of surviving and performing at elevated temperatures, and can thus be used in reactions in which temperature is used as a selection against non-specific hybridization of nucleic acid strands.
• the restriction sites used for deletion mutagenesis described below, were chosen for convenience. Different sites situated with similar convenience are available in the Thermus thermophilus gene and can be used to make similar constructs with other Type A polymerase genes from related organisms.
• the modified Taq polymerase gene was isolated as follows: The Taq DNA polymerase gene was amplified by polymerase chain reaction from genomic DNA from Thermus aquaticus. strain YT-1 (Lawyer el al, supra), using as primers the oligonucleotides described in SEQ ID NOS:13-14. The resulting fragment of DNA has a recognition sequence for the restriction endonuclease EcoP ⁇ at the 5' end of the coding sequence and a BgHl sequence at the 3' end.
• the pTTQ18 vector which contains the hybrid trp-lac (tac) promoter
• the tac promoter is under the control of the E. coli lac repressor. Repression allows the synthesis of the gene product to be suppressed until the desired level of bacterial growth has been achieved, at which point repression is removed by addition of a specific inducer. isopropyl-b-D-thiogalactopyranoside (IPTG).
• IPTG isopropyl-b-D-thiogalactopyranoside
• Bacterial promoters such as tac, may not be adequately suppressed when they are present on a multiple copy plasmid. If a highly toxic protein is placed under control of " such a promoter, the small amount of expression leaking through can be harmful to the bacteria.
• this construct yields a translational fusion product in which the first two residues of the native protein (Met-Arg) are replaced by three from the vector (Met-Asn-Ser). but the remainder of the natural protein would not change.
• the construct was transformed into the .IM109 strain of E. coli and the transformants were plated under incompletely repressing conditions that do not permit growth of bacteria expressing the native protein. These plating conditions allow the isolation of genes containing pre-existing mutations, such as those that result from the infidelity of Taq polymerase during the amplification process.
• DNA sequence analysis of the recombinant gene showed that it had changes in the polymerase domain resulting in two amino acid substitutions: an A to G change at nucleotide position 1394 causes a Glu to Gly change at amino acid position 465 (numbered according to the natural nucleic and amino acid sequences. SEQ ID NOS: l and 4) and another A to G change at nucleotide position 2260 causes a Gin to Arg change at amino acid position 754. Because the Gin to Gly mutation is at a nonconserved position and because the Glu to Arg mutation alters an amino acid that is conserved in virtually all of the known Type A polymerases. this latter mutation is most likely the one responsible for curtailing the synthesis activity of this protein.
• the nucleotide sequence for the clone 4B construct ( Figure 16B) is given in SEQ ID NO:21.
• the corresponding amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:21 is listed in SEQ ID NO:72.
• This construction yields another translational fusion product, in which the first two amino acids of DNAPTaq (Met-Arg) are replaced by 13 from the vector plus two from the PCR primer (Met-Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg-Ile-Asn-Ser) (SEQ ID NO:23).
• One way of destroying the polymerizing ability of a DNA polymerase is to delete all or part of the gene segment that encodes that domain for the protein, or to otherwise render the gene incapable of making a complete polymerization domain.
• Individual mutant enzymes may differ from each other in stability and solubility both inside and outside cells. For instance, in contrast to the 5 ' nuclease domain of DNAPEcl. which can be released in an active form from the polymerization domain by gentle proteolysis [Setlow and Kornberg. J Biol Chem. 247:232 (1972)], the Thermus nuclease domain, when treated similarly, becomes less soluble and the cleavage activity is often lost.
• Figure 16C The utTaq construct was digested with Pstl. which cuts once within the polymerase coding region, as indicated, and cuts immediately downstream of the gene in the multiple cloning site of the vector. After release of the fragment between these two sites, the vector was re-ligated, creating an 894-nucleotide deletion, and bringing into frame a stop codon 40 nucleotides downstream of the junction.
• the nucleotide sequence of this 5 " nuclease (clone 4C) is given in SEQ ID NO:9.
• the corresponding amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:9 is listed in SEQ ID NO:73.
• Figure 16D The mutTaq construct was digested with Nhel, which cuts once in the gene at position 2047.
• the resulting four-nucleotide 5 ' overhanging ends were filled in, as described above, and the blunt ends were re-ligated.
• the resulting four-nucleotide insertion changes the reading frame and causes termination of translation ten amino acids downstream of the mutation.
• the nucleotide sequence of this 5 " nuclease (clone 4D) is given in SEQ ID NO: 10.
• the corresponding amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 10 is listed in SEQ ID NO:74.
• Figure 16E The entire mutTaq gene was cut from pTTQ18 using EcoRl and Sail and cloned into pET-3c. as described above. This clone was digested with BstXl and Xcml. at unique sites that are situated as shown in Figure 16E. The DNA was treated with the Klenow fragment of DNAPEcl and dNTPs. which resulted in the 3 " overhangs of both sites being trimmed to blunt ends. These blunt ends were ligated together, resulting in an out-of- frame deletion of " 1540 nucleotides. An in-frame termination codon occurs 18 triplets past the junction site.
• nucleotide sequence of this 5' nuclease (clone 4E) is given in SEQ ID NO: l 1 [The corresponding amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: l 1 is listed in SEQ ID NO:75].. with the appropriate leader sequence given in SEQ ID NO:24 [The corresponding amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:24 is listed in SEQ ID NO:76. It is also referred to as the GeavaseTM BX enzyme.
• Figure 16F The entire mutTaq gene was cut from pTTQ18 using £coRI and Sail and cloned into pET-3c. as described above. This clone was digested with BsiX and BamHl. at unique sites that are situated as shown in the diagram. The DNA was treated with the Klenow fragment of DNAPEcl and dNTPs, which resulted in the 3 " overhang of the BstX site being trimmed to a blunt end. while the 5 " overhang of the BamHl site was filled in to make a blunt end. These ends were ligated together, resulting in an in-frame deletion of 903 nucleotides. The nucleotide sequence of the 5 ' nuclease (clone 4F) is given in SEQ ID
• SEQ ID NO: 12 It is also referred to as the GeavaseTM BB enzyme.
• the corresponding amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 12 is listed in SEQ ID NO:77.
• Figure 16G This polymerase is a variant of that shown in Figure 16E. It was cloned in the plasmid vector pET-21 (Novagen). The non-bacterial promoter from bacteriophage T7. found in this vector, initiates transcription only by T7 RNA polymerase. See Studier and
• the pET-21 vector also features a "His-Tag". a stretch of six consecutive histidine residues that are added on the carboxy terminus of the expressed proteins.
• the resulting proteins can then be purified in a single step by metal chelation chromatography. using a commercially available (Novagen) column resin with immobilized NT ions.
• the 2.5 ml columns are reusable, and can bind up to 20 mg of the target protein under native or denaturing (guanidine-HG or urea) conditions.
• E. coli (DES)pLYS cells are transformed with the constructs described above using standard transformation techniques, and used to inoculate a standard growth medium (e.g..
• T7 RNA polymerase is induced during log phase growth by addition of IPTG and incubated for a further 12 to 17 hours. Aliquots of culture are removed both before and after induction and the proteins are examined by SDS-PAGE. Staining with Coomassie Blue allows visualization of the foreign proteins if they account for about 3-5% of the cellular protein and do not co-migrate with any of the major host protein bands. Proteins that co-migrate with major host proteins must be expressed as more than 10% of the total protein to be seen at this stage of analysis. Some mutant proteins are sequestered by the cells into inclusion bodies.
• the solubilized protein is then purified on the Ni " column as described above. following the manufacturers instructions (Novagen).
• the washed proteins are eluted from the column by a combination of imidazole competitor (1 M) and high salt (0.5 M NaCl). and dialyzed to exchange the buffer and to allow denatured proteins to refold. Typical recoveries result in approximately 20 ⁇ g of specific protein per ml of starting culture.
• the DNAP mutant is referred to as the GeavaseTM BN enzyme and the sequence is given in SEQ ID NO:25.
• ID NO:25 is listed in SEQ ID NO:78.
• the DNA polymerase gene of Thermus flavus was isolated from the "T. flavus" AT-62 strain obtained from the American Type Tissue Collection (ATCC 33923). This strain has a different restriction map then does the T. flavus strain used to generate the sequence published by Akhmetzjanov and Vakhitov. supra. The published sequence is listed as SEQ ID NO:2. No sequence data has been published for the DNA polymerase gene from the AT-62 strain of T. flavus.
• Genomic DNA from T. flavus was amplified using the same primers used to amplif the T. aquaticus DNA polymerase gene (SEQ ID NOS: 13-14).
• the approximately 2500 base pair PCR fragment was digested with EcoRI and BamHl. The over-hanging ends were made blunt with the Klenow fragment of DNAPEcl and dNTPs.
• the resulting approximately 1 800 base pair fragment containing the coding region for the N-terminus was ligated into pET-3c as described above.
• This construct, clone 5B is depicted in Figure 18B.
• the wild type T. flavus DNA polymerase gene is depicted in Figure 18A. In Figure 18.
• the designation "3 " Exo” is used to indicate the location of the 3' exonuclease activity associated with Type A polymerases which is not present in DNAPTfl.
• the 5B clone has the same leader amino acid as do the DNAPTaq clones 4E and F which were cloned into pET-3c; it is not known precisely where translation termination occurs, but the vector has a strong transcription termination signal immediately downstream of the cloning site.
• Bacterial cells were transformed with the constructs described above using standard transformation techniques and used to inoculate 2 mis of a standard growth medium (e.g..
• thermostable proteins i.e. , the 5 " nucleases
• the precipitated E. coli proteins were then, along with other cell debris, removed by centrifugation.
• 1.7 mis of the culture were pelleted by microcentrifugation at 12.000 to 14,000 rpm for 30 to 60 seconds. After removal of the supernatant, the cells were resuspended in 400 ⁇ l of buffer A (50 mM Tris-HCl . pH 7.9. 50 mM dextrose. 1 mM
• EDTA re-centrifuged. then resuspended in 80 ⁇ l of buffer A with 4 mg/ml lysozyme. The cells were incubated at room temperature for 15 minutes, then combined with 80 ⁇ l of buffer B (10 mM Tris-HCl , pH 7.9, 50 mM KG. 1 mM EDTA. 1 mM PMSF. 0.5% Tween-20. 0.5% Nonidet-P40).
• the foreign protein may not be detected after the heat treatments due to sequestration of the foreign protein by the cells into inclusion bodies.
• inclusion bodies These are granules that form in the cytoplasm when bacteria are made to express high levels of a foreign protein, and they can be purified from a crude lysate, and analyzed SDS PAGE to determine their protein content. Many methods have been described in the literature, and one approach is described below. D. Isolation And Solubilization Of Inclusion Bodies
• a small culture was grown and induced as described above. A 1.7 ml aliquot was pelleted by brief centrifugation, and the bacterial cells were resuspended in 100 ⁇ l of Lysis buffer (50 mM Tris-HCl , pH 8.0. 1 mM EDTA, 100 mM NaCl). 2.5 ⁇ l of 20 mM PMSF were added for a final concentration of 0.5 mM, and lysozyme was added to a concentration of 1.0 mg/ml.
• Lysis buffer 50 mM Tris-HCl , pH 8.0. 1 mM EDTA, 100 mM NaCl
• 2.5 ⁇ l of 20 mM PMSF were added for a final concentration of 0.5 mM, and lysozyme was added to a concentration of 1.0 mg/ml.
• the cells were incubated at room temperature for 20 minutes, deoxycholic acid was added to 1 mg/ml (1 ⁇ l of 100 mg/ml solution), and the mixture was further incubated at 37 ° C for about 15 minutes or until viscous.
• DNAse 1 was added to 10 ⁇ g/ml and the mixture was incubated at room temperature for about 30 minutes or until it was no longer viscous.
• the inclusion bodies were collected by centrifugation at 14.000 rpm for 15 minutes at 4°C. and the supernatant was discarded.
• the pellet was resuspended in 100 ⁇ l of lysis buffer with l OmM EDTA (pH 8.0) and 0.5% Triton X-100. After 5 minutes at room temperature, the inclusion bodies were pelleted as before, and the supernatant was saved for later analysis.
• the inclusion bodies were resuspended in 50 ⁇ l of distilled water, and 5 ⁇ l was combined with SDS gel loading buffer (which dissolves the inclusion bodies) and analyzed electrophoretically. along with an aliquot of the supernatant.
• the cloned protein may be released to assay the cleavage and polymerase activities and the method of solubilization must be compatible with the particular activity. Different methods of solubilization may be appropriate for different proteins, and a variety of methods are discussed in Molecular Cloning (Sambrook et al. supra). The following is an adaptation we have used for several of our isolates.
• a fresh tube was prepared containing 180 ⁇ l of 50 mM KH 2 P0 4 , pH 9.5. 1 mM EDTA and 50 mM NaCl.
• a 2 ⁇ l aliquot of the extract was added and vortexed briefly to mix. This step was repeated until all of the extract had been added for a total of 10 additions.
• the mixture was allowed to sit at room temperature for 15 minutes, during which time some precipitate often forms. Precipitates were removed by centrifugation at 14,000 rpm, for 15 minutes at room temperature, and the supernatant was transferred to a fresh tube.
• a candidate modified polymerase is tested for 5 " nuclease activity by examining its ability to catalyze structure-specific cleavages.
• cleavage structure as used herein, is meant a nucleic acid structure which is a substrate for cleavage by the 5 " nuclease activity of a DNAP.
• a primer-directed cleavage ( Figure 19B) is performed because it is relatively insensitive to variations in the salt concentration of the reaction and can, therefore, be performed in whatever solute conditions the modified enzyme requires for activity; this is generally the same conditions preferred by unmodified polymerases; 2) a similar primer-directed cleavage is performed in a buffer which permits primer-independent cleavage, i.e., a low salt buffer, to demonstrate that the enzyme is viable under these conditions; and 3) a primer-independent cleavage ( Figure 19A) is performed in the same low salt buffer.
• the bifurcated duplex is formed between a substrate strand and a template strand as shown in Figure 19.
• substrate strand as used herein, is meant that strand of nucleic acid in which the cleavage mediated by the 5 ' nuclease activity occurs.
• the substrate strand is always depicted as the top strand in the bifurcated complex which serves as a substrate for 5 " nuclease cleavage ( Figure 19).
• template strand as used herein, is meant the strand of nucleic acid which is at least partially complementary to the substrate strand and which anneals to the substrate strand to form the cleavage structure.
• the template strand is always depicted as the bottom strand of the bifurcated cleavage structure ( Figure 19). If a primer (a short oligonucleotide of 19 to 30 nucleotides in length) is added to the complex, as when primer-dependent cleavage is to be tested, it is designed to anneal to the 3 " arm of the template strand ( Figure 19B). Such a primer would be extended along the template strand if the polymerase used in the reaction has synthetic activity.
• a primer a short oligonucleotide of 19 to 30 nucleotides in length
• the cleavage structure may be made as a single hairpin molecule, with the 3 " end of the target and the 5 " end of the pilot joined as a loop as shown in Figure 19E.
• a primer oligonucleotide complementary to the 3 " arm is also required for these tests so that the enzyme ' s sensitivity to the presence of a primer may be tested.
• Nucleic acids to be used to form test cleavage structures can be chemically synthesized, or can be generated by standard recombinant DNA techniques. By the latter method, the hairpin portion of the molecule can be created by inserting into a cloning vector duplicate copies of a short DNA segment, adjacent to each other but in opposing orientation. The double-stranded fragment encompassing this inverted repeat, and including enough flanking sequence to give short (about 20 nucleotides) unpaired 5 ' and 3 ' arms, can then be released from the vector by restriction enzyme digestion, or by PCR performed with an enzyme lacking a 5 ' exonuclease (e.g.. the Stoffel fragment of AmplitaqTM DNA polymerase. VentTM DNA polymerase).
• an enzyme lacking a 5 ' exonuclease e.g. the Stoffel fragment of AmplitaqTM DNA polymerase. VentTM DNA polymerase.
• test DNA can be labeled on either end. or internally, with either a radioisotope, or with a non-isotopic tag. Whether the hai ⁇ in DNA is a synthetic single strand or a cloned double strand, the DNA is heated prior to use to melt all duplexes. When cooled on ice. the structure depicted in Figure 19E is formed, and is stable for sufficient time to perform these assays.
• Reaction 1 To test for primer-directed cleavage (Reaction 1 ), a detectable quantity of the test molecule (typically 1 -100 fmol of j2 P-labeled hai ⁇ in molecule) and a 10 to 100-fold molar excess of primer are placed in a buffer known to be compatible with the test enzyme.
• a buffer known to be compatible with the test enzyme For Reaction 2. where primer-directed cleavage is performed under condition which allo primer- independent cleavage, the same quantities of molecules are placed in a solution that is the same as the buffer used in Reaction 1 regarding pH.
• enzyme stabilizers e.g.. bovine serum albumin, nonionic detergents, gelatin
• reducing agents e.g..
• test reactions are then exposed to enough of the enzyme that the molar ratio of enzyme to test complex is approximately 1 :1.
• the reactions are incubated at a range of temperatures up to. but not exceeding, the temperature allowed by either the enzyme stability or the complex stability, whichever is lower, up to 80°C for enzymes from thermophiles. for time sufficient to allow cleavage (10 to 60 minutes).
• the products of Reactions 1. 2 and 3 are resolved by denaturing polyacrylamide gel electrophoresis. and visualized by autoradiography or by a comparable method appropriate to the labeling system used. Additional labeling systems include chemiluminescence detection, silver or other stains. blotting and probing and the like.
• cleavage products is indicated by the presence of molecules which migrate at a lower molecular weight than does the uncleaved te structure. These cleavage products indicate that the candidate polymerase has structure- specific 5 " nuclease activity.
• a modified DNA polymerase has substantially the same 5 " nuclease activity as that of the native DNA polymerase.
• the results of the above-described tests are compared with the results obtained from these tests performed with the native DNA polymerase.
• substantially the same 5 ' nuclease activity we mean that the modified polymerase and the native polymerase will both cleave test molecules in the same manner. It is not necessary that the modified polymerase cleave at the same rate as the native DNA . polymerase.
• Some enzymes or enzyme preparations may have other associated or contaminating activities that may be functional under the cleavage conditions described above and that may interfere with 5 ' nuclease detection. Reaction conditions can be modified in consideration of these other activities, to avoid destruction of the substrate, or other masking of the 5 " nuclease cleavage and its products.
• the DNA polymerase I of E. coli (Pol I) in addition to its polymerase and 5 " nuclease activities, has a 3 * exonuclease that can degrade DNA in a 3 ' to 5 " direction.
• the mutant polymerase clones 4E (Taq mutant) and 5B (Tfl mutant) were examined for their ability to cleave the hai ⁇ in substrate molecule shown in Figure 19E.
• the substrate molecule was labeled at the 5 ' terminus with 32 P.
• Ten fmoles of heat-denatured, end-labeled substrate DNA and 0.5 units of DNAPTaq (lane 1 ) or 0.5 ⁇ l of 4e or 5b extract (Figure 20, lanes 2-7, extract was prepared as described above) were mixed together in a buffer containing 10 mM Tris-G, pH 8.5. 50 mM KG and 1.5 mM MgCL. The final reaction volume was 10 ⁇ l.
• Reactions shown in lanes 4 and 7 contain in addition 50 ⁇ M of each dNTP.
• Reactions shown in lanes 3. 4, 6 and 7 contain 0.2 ⁇ M of th primer oligonucleotide (complementary to the 3 ' arm of the substrate and shown in Figure 19E).
• Reactions were incubated at 55° C for 4 minutes. Reactions were stopped by the addition of 8 ⁇ l of stop solution per 10 ⁇ l reaction volume. Samples were then applied to 12% denaturing acrylamide gels. Following electrophoresis, the gels were autoradiographed.
• Figure 20 shows that clones 4E and 5B exhibit cleavage activity similar to that of the native DNAPTaq.
• Assay For Synthetic Activity The ability of the modified enzyme or proteolytic fragments is assayed by adding the modified enzyme to an assay system in which a primer is annealed to a template and DNA synthesis is catalyzed by the added enzyme.
• an assay system in which a primer is annealed to a template and DNA synthesis is catalyzed by the added enzyme.
• Many standard laboratory techniques employ such an assay. For example, nick translation and enzymatic sequencing involve extension of a primer along a DNA template by a polymerase molecule.
• an oligonucleotide primer is annealed to a single-stranded DNA template, e.g.. bacteriophage Ml 3 DNA. and the primer/template duplex is incubated in the presence of the modified polymerase in question, deoxynucleoside triphosphates (dNTPs) and the buffer and salts known to be appropriate for the unmodified or native enzyme. Detection of either primer extension (by denaturing gel electrophoresis) or dNTP inco ⁇ oration (by acid precipitation or chromatography) is indicative of an active polymerase.
• dNTPs deoxynucleoside triphosphates
• a label either isotopic or non- isotopic, is preferably included on either the primer or as a dNTP to facilitate detection of polymerization products.
• Synthetic activity is quantified as the amount of free nucleotide incorporated into the growing DNA chain and is expressed as amount incorporated per unit of time under specific reaction conditions.
• FIG. 21 Representative results of an assay for synthetic activity is shown in Figure 21.
• the synthetic activity of the mutant DNAPTaq clones 4B-F was tested as follows: A master mixture of the following buffer was made: 1.2X PCR buffer. 50 ⁇ M each of dGTP. dATP and dTTP. 5 ⁇ M dCTP and 0.125 ⁇ M ⁇ - 32 P-dCTP at 600 Ci/mmol. Before adjusting this mixture to its final volume, it was divided into two equal aliquots. One received distilled water up to a volume of 50 ⁇ l to give the concentrations above. The other received 5 ⁇ g of single-stranded M13mpl 8 DNA (approximately 2.5 pmol or 0.05 ⁇ M final concentration) and
• each reaction was mixed, then incubated at room temperature (approx. 22 ° C) for 5 minutes, then at 55T for 2 minutes, then at 72°C for 2 minutes. This step incubation was done to detect polymerization in any mutants that might have optimal temperatures lower than 72°C. After the final incubation, the tubes were spun briefly to collect any condensation and were placed on ice. One ⁇ l of each reaction was spotted at an origin 1.5 cm from the bottom edge of a polyethyleneimine (PEI) cellulose thin layer chromatography plate and allowed to dry.
• PEI polyethyleneimine
• chromatography plate was run in 0.75 M NaH 2 P0 4 . pH 3.5. until the buffer front had run approximately 9 cm from the origin. The plate was dried, wrapped in plastic wrap, marked with luminescent ink. and exposed to X-ray film. Incorporation was detected as counts that stuck where originally spotted, while the unincorporated nucleotides were carried by the salt solution from the origin.
• the ability of the 5' nucleases to cleave hairpin structures to generate a cleaved hairpin structure suitable as a detection molecule was examined.
• the structure and sequence of the hairpin test molecule is shown in Figure 22A (SEQ ID NO: 15).
• the oligonucleotide (the primer in Figure 22A. SEQ ID NO:22) is shown annealed to its complementary sequence on the 3' arm of the hai ⁇ in test molecule.
• the hai ⁇ in test molecule was single-end labeled with 2 P using a labeled T7 promoter primer in a polymerase chain reaction. The label is present on the 5 * arm of the hairpin test molecule and is represented by the star in Figure
• the cleavage reaction was performed by adding 10 fmoles of heat-denatured, end- labeled hairpin test molecule, 0.2 ⁇ M of the primer oligonucleotide (complementary to the 3 " arm of the hairpin), 50 ⁇ M of each dNTP and 0.5 units of DNAPTaq (Perkin Elmer) or 0.5 ⁇ l of extract containing a 5' nuclease (prepared as described above) in a total volume of 10 ⁇ l in a buffer containing 10 mM Tris-G, pH 8.5, 50 mM KG and 1.5 mM MgCL. Reactions shown in lanes 3. 5 and 7 were run in the absence of dNTPs.
• Figure 22B shows that altered polymerases lacking any detectable synthetic activity cleave a hai ⁇ in structure when an oligonucleotide is annealed to the single-stranded 3 ' arm of " the hairpin to yield a single species of cleaved product ( Figure 22B. lanes 3 and 4). 5 ' nucleases. such as clone 4D, shown in lanes 3 and 4, produce a single cleaved product even in the presence of dNTPs.
• thermostable DNA polymerases are capable of cleaving hai ⁇ in structures in a specific manner and that this discovery can be applied with success to a detection assay.
• the mutant DNAPs of the present invention are tested against three different cleavage structures shown in Figure 24A.
• Structure 1 in Figure 24A is simply single stranded 206-mer (the preparation and sequence information for which was discussed above).
• Structures 2 and 3 are duplexes: structure 2 is the same hai ⁇ in structure as shown in Figure 13A (bottom), while structure 3 has the hai ⁇ in portion of structure 2 removed.
• the cleavage reactions comprised 0.01 pmoles of the resulting substrate DNA. and 1 pmole of pilot oligonucleotide in a total volume of 10 ⁇ l of 10 mM Tris-G, pH 8.3. 100 mM
• I is native Taq DNAP: II is native Tfl DNAP; III is the GeavaseTM BX enzyme shown in Figure 16E: IV is the GeavaseTM BB enzyme shown in Figure 16F: V is the mutant shown in Figure 18B; and VI is the GeavaseTM BN enzyme shown in Figure 16G.
• Structure 2 was used to "normalize" the comparison. For example, it was found that it took 50 ng of Taq DNAP and 300 ng of the GeavaseTM BN enzyme to giv similar amounts of cleavage of Structure 2 in thirty (30) minutes. Under these conditions native Taq DNAP is unable to cleave Structure 3 to any significant degree. Native Tfl DNA cleaves Structure 3 in a manner that creates multiple products.
• thermostable DNAPs including those of the present invention, have a true 5 ' exonuclease capable of nibbling the 5 ' end of a linear duplex nucleic acid structures.
• the 206 base pair DNA duplex substrate is again employed (see above).
• the cleavage reactions comprised 0.01 pmoles of heat-denatured, end-labeled substrate DNA (with the unlabeled strand also present).
• pilot oligonucleotide see pilot oligos in Figure 13A
• DNAPTaq 5 pmoles of pilot oligonucleotide
• GeavaseTM BB enzyme in the E. coli extract see above
• the faint band seen at 24 nucleotides is residual end- labeled primer from the PCR.
• the su ⁇ rising result is that the GeavaseTM BB enzyme under these conditions causes all of the label to appear in a very small species, suggesting the possibilit that the enzyme completely hydrolyzed the substrate.
• samples of the 206 base pair duplex were treated with either T7 gene 6 exonuclease (USB) or with calf intestine alkaline phosphatase (Promega), according to manufacturers " instructions, to produce either labeled mononucleotide (lane a of Figure 25B) or free j2 P-labeled inorganic phosphate (lane b of Figure 25B), respectively.
• T7 gene 6 exonuclease USB
• calf intestine alkaline phosphatase Promega
• the nibbling by the enzyme GeavaseTM BB is duplex dependent.
• single strands of the 206-mer were produced by 15 cycles of primer extension inco ⁇ orating ⁇ - 32 P labeled dCTP combined with all four unlabeled dNTPs. using an unlabeled 206-bp fragment as a template.
• Single and double stranded products were resolved by electrophoresis through a non-denaturing 6% polyacrylamide gel (29: 1 cross-link) in a buffer of " 0.5X TBE, visualized by autoradiography, excised from the gel. eluted by passive diffusion, and concentrated by ethanol precipitation.
• the cleavage reactions comprised 0.04 pmoles of substrate DNA. and 2 ⁇ l of the enzyme Geavase BB (in an E. coli extract as described above) in a total volume of 40 ⁇ l of "
• thermostable proteins i.e. , the 5 " nucleases. were isolated by crude bacterial cell extracts. The precipitated E. coli proteins were then, along with other cell debris, removed by centrifugation.
• cells expressing the GeavaseTM BN clone were cultured and collected (500 grams). For each gram (wet weight) of E. coli. 3 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, 1 mM EDTA. 100 ⁇ M NaCl) was added. The cells were lysed with 200 ⁇ g/ml lysozyme at room temperature for 20 minutes. Thereafter deoxycholic acid was added to make a 0.2% final concentration and the mixture was incubated 15 minutes at room temperature.
• the lysate was sonicated for approximately 6-8 minutes at 0°C.
• the precipitate was removed by centrifugation (39.000 ⁇ for 20 minutes).
• Polyethyleneimine was added (0.5%) to the supernatant and the mixture was incubated on ice for 15 minutes.
• the mixture was centrifuged (5,000# for 15 minutes) and the supernatant was retained.
• the supernatant was precipitated with 35% ammonium sulfate at 4°C for 15 minutes.
• Binding Buffer comprises: 40 mM imidazole. 4 M NaCl. 160 mM Tris-HCl, pH 7.9).
• the solubilized protein was then purified on the NP column (Novagen).
• Buffer was allowed to drain to the top of the column bed and the column was then loaded with the prepared extract.
• a flow rate of about 10 column volumes per hour is optimal for efficient purification. If the flow rate is too fast, more impurities will contaminate the eluted fraction.
• IX Elute Buffer (4X Elute Buffer comprises: 4 mM imidazole. 2 M NaCl. 80 mM Tris-HCl. pH 7.9). Protein is then reprecipitated with 35% Ammonium Sulfate as above. The precipitate was then dissolved and dialyzed against: 20 mM Tris. 100 mM KC 1. 1 mM EDTA). The solution was brought up to 0.1% each of Tween 20 and NP-40 and stored at 4°C.
• nucleases to cleave naturally occurring structures in nucleic acid templates (structure-specific cleavage) is useful to detect internal sequence differences in nucleic acids without prior knowledge of the specific sequence of the nucleic acid.
• mutations e.g.. single base changes (point mutations), small insertions or deletions, etc.
• the primer pair 5 ' biotin-CACCGTCCTCTTCAAGAAG 3' (SEQ ID NO:29) and 5 " fluorescein-CTGAATCTTGTAGATAGCTA 3' (SEQ ID NO:30) was used to prime the PCRs.
• the synthetic primers were obtained from Promega; the primers were labeled on the
• the target DNA for the generation of the 157 bp fragment of mutant G419R was a 339 bp PCR product (SEQ ID NO:31) generated using genomic DNA homozygous for the 41 mutation. Genomic DNA was isolated using standard techniques from peripheral blood leukocytes isolated from patients. This 339 bp PCR product was prepared as follows.
• the symmetric PCR reaction comprised 10 ng of genomic DNA from the 419 mutant. 100 pmoles of the primer 5 " biotin-GCCTTATTTTACTTTAAAAAT-3 ' (SEQ ID NO:32). 100 pmoles of the primer 5' fluorescein-TAAAGTTTTGTGTTATCTCA-3 " (SEQ ID NO:33). and 50 ⁇ M of each dNTP in IX PCR buffer.
• the primers of SEQ ID NOS:32 and 33 were obtained from Integrated DNA Technologies. Coralville, IA. A tube containing 45 ⁇ l of the above mixture was overlaid with two drops of light mineral oil and the tube was heated to 95 ° C for 1 min.
• Taq polymerase was then added as 1.25 units of enzyme in 5 ⁇ l of IX PCR buffer.
• the tube was heated to 94°C for 40 sec. cooled to 55°C for 50 sec. heated to 72°C for 70 sec for 29 repetitions with a 5 min incubation at 72 ° C after the last repetition.
• the PCR products were gel purified as follows. The products were resolved by electrophoresis through a 6% polyacrylamide gel (29:1 cross-link) in a buffer containing 0.5X TBE. The DNA was visualized by ethidium bromide staining and the 339 bp fragment was excised from the gel. The DNA was eluted from the gel slice by passive diffusion overnight into a solution containing 0.5 M NH OAc. 0.1% SDS and 0.1 M EDTA. The DNA was then precipitated with ethanol in the presence of 4 ⁇ g of glycogen carrier. The DNA was pelleted and resuspended in 40 ⁇ l of TE (10 mM Tris-G, pH 8.0. 0.1 mM EDTA).
• the purified 339 bp 419 PCR fragment was used as the target in an asymmetric PCR.
• the asymmetric PCR comprised 100 pmoles of the biotinylated primer of SEQ ID NO:32, 1 pmole of the fluoresceinated primer of SEQ ID NO:33. 50 ⁇ M of each dNTP, in IX PCR buffer.
• a tube containing 45 ⁇ l of " the above mixture was overlaid with two drops of light mineral oil and the tube was heated to 95 °C for 5 sec and then cooled to 70°C.
• Taq polymerase was then added as 1.25 units of enzyme in 5 ⁇ l of I X PCR buffer. The tube was heated to 95°C for 45 sec. cooled to 50 ° C for 45 sec. heated to 72°C for 1 min 15 sec for 30 repetitions with a 5 min incubation at 72 °C after the last repetition.
• the asymmetric PCR products were gel purified as follows. The products were resolved by electrophoresis through a 6% polyacrylamide gel (29: 1 cross-link) in a buffer containing 0.5X TBE. The DNA was visualized by ethidium bromide staining: the double- stranded DNA was differentiated from the single-stranded DNA due to the mobility shift commonly seen with single-stranded DNA produced from asymmetric PCR (in an asymmetric PCR both single-stranded and double-stranded products are produced: typically the single- stranded product will have a slower speed of migration through the gel and will appear closer to the origin than will the double-stranded product). The double-stranded 157 bp substrate corresponding to the 419 mutant (SEQ ID NO:28) was excised from the gel.
• the 157 bp wild-type fragment was generated by asymmetric PCR as described above for the 419 mutant with the exception that the target DNA was 10 ng of supercoiled pcTYR-
• NlTyr plasmid DNA contains the entire wild-type tyrosinase cDNA [Geibel. L.B., et al. (1991 ) Genomics 9:435].
• Cleavage reactions comprised 100 fmoles of the resulting double-stranded substrate DNAs (the substrates contain a biotin moiety at the 5 ' end of the sense strand) in a total volume of 10 ⁇ l of 10 mM MOPS, pH 8.2. 1 mM divalent cation (either MgCL or MnCL) and 1 unit of DNAPTaq.
• the reactions were overlaid with a drop of light mineral oil.
• reactions were heated to 95 "C for 5 seconds to denature the substrate and then the tubes were quickly cooled to 65 °C (this step allows the DNA assume its unique secondary structure by allowing the formation of intra-strand hydrogen bonds between complimentary bases).
• the reaction can be performed in either a thermocycler (MJ Research. Watertown. MA) programmed to heat to 95°C for 5 seconds then drop the temperature immediately to 65°C or alternatively the tubes can be placed manually in a heat block set at 95°C and then transferred to a second heat block set at 65 °C.
• reaction was incubated at 65 ° C for 10 minutes and was stopped by the addition of 8 ⁇ l of stop buffer. Samples were heated to 72 ° C for 2 minutes and 5 ⁇ l of each reaction were resolved by electrophoresis through a 10% polyacrylamide gel (19:1 cross-link), with 7
• SAAP streptavidin-alkaline phosphatase conjugate
• the lane marked "M” contains molecular weight markers.
• the marker fragments were generated by digestion of pUC19 with Haelll followed by the addition of biotinylated dideoxynucleotides (Boehringer Mannheim, Indianapolis. IN) to the cut ends using terminal transferase (Promega).
• Lanes 1 , 3 and 5 contain the reaction products from the incubation of the wild type 157 nucleotide substrate in the absence of the DNAPTaq enzyme (lane 1 ). in the presence of MgCL and enzyme (lane 3) or in the presence of " MnCL and enzyme (lane 5).
• 4 and 6 contains the reaction products from the incubation of the 157 nucleotide substrate derived from the 419 mutant in the absence of enzyme (lane 2). in the presence of " MgCL and enzyme (lane 4) or in the presence of MnCL and enzyme (lane 6).
• Figure 27 demonstrates that the use of MnCL rather than MgCL in the cleavage reaction results in the production of an enhanced cleavage pattern. It is desirable that the cleavage products are of different sizes so that the products do not all cluster at one end of the gel. The ability to spread the cleavage products out over the entire length of the gel makes it more likely that alterations in cleavage products between the wild type and mutant substrates will be identified.
• Figure 27 shows that when Mg 2 is used as the divalent cation, the majority of the cleavage products cluster together in the upper portion of the gel. In contrast when Mn 2* is used as the divalent cation, the substrate assumes structures which, when cleaved, generate products of widely differing mobilities. These results show that Mn 2' is the preferred divalent cation for the cleavage reaction.
• the ability of 5 ' nuclease to generate a cleavage pattern or "fingerprint" which is unique to a given piece of DNA was shown by incubating four similarly sized DNA substrates with the GeavaseTM BN enzyme.
• the four DNA substrates used were a 157 nucleotide fragment from the sense (or coding) strand of exon 4 of the wild-type tyrosinase gene (SEQ ID NO:34); a 157 nucleotide fragment from the anti-sense (or non-coding) strand of exon 4 of the wild-type tyrosinase gene (SEQ ID NO:35); a 165 nucleotide DNA fragment derived from pGEM3Zf(+) (SEQ ID NO:36) and a 206 nucleotide DNA fragment derived from pGEM3Zf(+) (SEQ ID NO:37).
• the DNA substrates contained either a biotin or fluorescein label at their 5' or 3' ends.
• the substrates were made as follows. To produce the sense and anti-sense single-stranded substrates corresponding to exon 4 of the wild-type tyrosinase gene, a double-stranded DNA fragment, 157 nucleotides in length (SEQ ID NO:27), was generated using symmetric PCR. The target for the symmetric PCR was genomic DNA containing the wild-type tyrosinase gene.
• the symmetric PCR comprised 50-100 ng of genomic wild-type DNA, 25 pmoles each of primers SEQ ID NOS:42 and 43, 50 ⁇ M each dNTP and 1.25 units of Taq polymerase in 50 ⁇ l of IX PCR buffer.
• the reaction mixture was overlaid with two drops of light mineral oil and the tube was heated to 94°C for 30 sec. cooled to 50°C for 1 min, heated to 72°C for 2 min for 30 repetitions.
• the double-stranded PCR product was gel purified, precipitated and resuspended in 40 ⁇ l of TE buffer as described above in a).
• the single-stranded sense and anti-sense 157 nucleotide DNA fragments were generated using the above 157 bp wild-type DNA fragment (SEQ ID NO:27) in two asymmetric PCR reactions.
• the sense strand fragment was generated using 5 ⁇ l of the above purified 157 bp fragment (SEQ ID NO:27) as the target in an asymmetric PCR.
• the reaction mixtures for the asymmetric PCR were as above for the symmetric PCR with the exception that 100 pmoles of the biotin-labeled sense primer (SEQ ID NO:29) and 1 pmole of the fluorescein-labeled anti-sense primer (SEQ ID NO:30) was used to prime the reaction.
• the anti-sense fragment was generated using 5 ⁇ l of the above purified 157 bp fragment as the
• the reaction conditions for the asymmetric PCR were as above for the symmetric PCR with the exception that 1 pmole of the sense primer (SEQ ID NO:29) and 100 pmoles of the anti-sense primer (SEQ ID NO:30) was used to prime the reaction.
• the reaction conditions for the asymmetric PCR were 95°C for 45 sec. 50°C for 45 sec. 72°C for 1 min and 15 sec for 30 repetitions with a 5 min incubation at 72°C after the last repetition.
• the reaction products were visualized, extracted and collected as described above with the single stranded DNA being identified by a shift in mobility when compared to a double stranded DNA control.
• the single-stranded 165 nucleotide fragment from pGEM3Zf(+) (SEQ ID NO:36) was generated by asymmetric PCR.
• the PCR comprised 50 pmoles of 5 ' biotin-
• AGCGGATAACAATTTCACACAGGA-3' (SEQ ID NO:38: Promega) and 1 pmole of 5 ' - CACGGATCCTAATACGACTCACTATAGGG-3' (SEQ ID NO:39 Integrated DNA Technologies. Coralville, IA), 50 ⁇ M each dNTP. in IX PCR buffer. Forty-five microliters of this reaction mixture was overlaid with two drops of light mineral oil and the tube was heated to 95°C for 5 sec and then cooled to 70 ° C. Taq polymerase was then added at 1.25 units in 5 ⁇ l of IX PCR buffer. The tubes were heated to 95°C for 45 sec. cooled to 50°C for 45 sec.
• reaction products were visualized, extracted and collected as described above with the 164 nucleotide DNA fragment being identified by a shift in mobility when compared to a double stranded DNA control.
• the 206 nucleotide DNA fragment (SEQ ID NO:37) was prepared by asymmetric PCR. performed as described above, using 1 pmole of a double-stranded 206 bp PCR product (generated as described in Example 1C), and 50 pmoles of the primer 5 " - CGCCAGGGTTTTCCCAGTCACGAC-3' (SEQ ID NO:40).
• the tubes were heated to 95T for 45 sec cooled to 63°C for 45 sec. heated to 72 ° C for 1 min 15 sec for 15 repetitions with a 5 min incubation at 72°C after the last repetition.
• reaction products were visualized, extracted and collected as described above with the 206 nucleotide DNA fragment being identified by a shift in mobility when compared to a double stranded DNA control.
• the precipitated DNA was resuspended in 70 ⁇ l of TE buffer. Twenty-five microliters of the above product was biotinylated on the 3 ' end using 10-
• TdT terminal deoxynucleotidyl transferase
• the tubes were incubated at 37°C for 15 min followed by ethanol precipitation in the presence of 4 ⁇ g of glycogen.
• the DNA was ethanol precipitated a second time and then resuspended in 25 ⁇ l of TE.
• the cleavage reactions were carried out in a final volume of 10 ⁇ l of 10 mM MOPS, pH 8.2. with 1 mM MnCL using approximately 100 fmoles of substrate DNA and 250 ng of the enzyme GeavaseTM BN.
• Parallel reactions lacking the enzyme GeavaseTM BN were set up as above with the exception that one third as much DNA template was used (approximately 33 fmoles of each template) to balance the signal on the autoradiograph.
• Each substrate DNA was placed in a 200 ⁇ l thin wall microcentrifuge tube (BioRad.
• Hercules. CA in 5 ⁇ l of 10 mM MOPS. pH 8.2, with 2 mM MnCL. The solution was overlaid with one drop of light mineral oil. Tubes were brought to 95°C for 5 seconds to denature the substrates and then the tubes were quickly cooled to 65°C.
• Figure 28 shows the results of incubation of the four substrates described above in the presence or absence of the GeavaseTM BN enzyme.
• Four sets of reactions are shown.
• Set one contains the reaction products from the incubation of the 157 nucleotide sense strand fragment of the tyrosinase gene (SEQ ID NO:34) in the absence or presence of the GeavaseTM BN enzyme.
• Set two contains the reaction products from the incubation of the 157 nucleotide anti-sense strand fragment of the tyrosinase gene (SEQ ID NO:35) in the absence or presence of the GeavaseTM BN enzyme.
• Set three contains the reaction products from the incubation of the 165 base bottom strand fragment of the plasmid pGEM3Zf(+) (SEQ ID NO:36) in the absence or presence of the GeavaseTM BN enzyme.
• Set four contains the reaction products from the incubation of the 206 base top strand fragment of the plasmid pGEM3Zf(+) (SEQ ID NO:37) in the absence or presence of the GeavaseTM BN enzyme.
• Lanes marked "M” contain biotin-labeled molecular weight markers prepared as described above; the sizes of the marker fragments are indicated in Figure 28. In the absence of " the GeavaseTM BN enzyme, no cleavage of the substrates is observed.
• each substrate is cleaved generating a unique set of cleavage products.
• these cleavage products are resolved on a polyacrylamide gel. a unique pattern or fingerprint is seen for each substrate DNA.
• the GeavaseTM BN enzyme generates a unique collection of " cleavage products from each substrate. These unique cleavage patterns result from the characteristic conformation each substrate DNA assumes.
• the present invention contemplates the ability to generate a unique cleavage pattern for two or more DNA substrates of the same size as part of a method for the detection of genetic mutations.
• This method compares a normal (or wild type or non-mutated) substrate with a substrate from a patient suspected of having a mutation in that substrate.
• the two substrates would be of the same length and the cleavage reaction would be used to probe the patient
• DNA substrate for conformational changes relative to the pattern seen in the wild type control substrate DNA substrate for conformational changes relative to the pattern seen in the wild type control substrate.
• the ability of the GeavaseTM BN enzyme to cleave DNA substrates of the same size but which contain single base changes between the substrates is herein demonstrated.
• the human tyrosinase gene was chosen as a model system because numerous single point mutations have been identified in exon 4 of this gene [Spritz. R.A. ( 1994) Human Molecular Genetics 3: 1469]. Mutation of the tyrosinase gene leads to oculocutaneous albinism in humans. Three single-stranded substrate DNAs were prepared; the substrates contain a biotin label at their 5 ' end.
• the wild type substrate comprises the 157 nucleotide fragment from the sense strand of the human tyrosinase gene [(SEQ ID NO:34); Geibel. L.B.. et al. (1991 ) Genomics 9:435]. Two mutation-containing substrates were used.
• the 419 substrate (SEQ ID NO:41 ) is derived from the tyrosinase mutant G419R which contains a glycine (GGA) to arginine (AGA) substitution; this mutant differs from the wild-type exon 4 fragment by a single base change at nucleotide 2675 [King, R.A., et al. (1991 ) Mol. Biol. Med.
• the 422 substrate (SEQ ID NO:42) is derived from the tyrosinase mutant R422Q which contains an arginine (CGG) to glutamine (CAG) substitution; this mutant differs from the wild type exon 4 fragment by a single base change at nucleotide 2685 [Giebel. L.B.. et al. (1991 ) J.
• Single-stranded DNA containing a biotin label at the 5 ' end was generated for each substrate using asymmetric PCR as described in Example 8a with the exception that the single-stranded PCR products were recovered from the gel rather than the double-stranded products.
• the following primer pair was used to amplify each DNA (the 419 and 422 mutations are located internally to the exon 4 fragment amplified by the primer pair thus the same primer pair can be used to amplify the wild type and two mutant templates).
• the primer listed as SEQ ID NO:29 (sense primer) contains a biotin label at the 5 " end and was used in a 100-fold excess over the anti-sense primer of SEQ ID NO:30.
• the asymmetric PCR comprised 100 pmoles of SEQ ID NO:29 and 1 pmole of SEQ ID NO:30. and 50 ⁇ M each dNTP in IX PCR buffer.
• the reaction mixture 45 ⁇ l was overlaid with two drops of light mineral oil and the tubes were heated to 95°C for 5 sec then cooled to 70°C.
• Taq polymerase was then added as 1.25 units of enzyme in 5 ⁇ l of I X PCR buffer.
• the tubes were heated to 95°C for 45 sec. cooled to 50T for 45 sec. heated to 72°C for 1 min 15 sec for 30 repetitions with a 5 min incubation at 72 ° C after the last repetition.
• the single stranded PCR products were gel purified, precipitated and resuspended in 40 ⁇ l of TE buffer as described above.
• Lane 29 lanes marked “M” contain molecular weight markers prepared as described in Example 8. Lanes 1-3 contain the no enzyme control for the wild type (SEQ ID NO:
• Lane 4 contains the cleavage products from the wild type template.
• Lane 5 contains the cleavage products from the 419 mutant.
• Lane 6 contains the cleavage products from the 422 mutant.
• Figure 29 shows that a similar, but distinctly different, pattern of cleavage products is generated by digestion of the three template DNAs with the GeavaseTM BN enzyme. Note that in the digest of mutant 419. the bands below about 40 nucleotides are absent, when compared to wild-type, while in the digest of mutant 422 several new bands appear in the 53 nucleotide range. Although the three template DNAs differed in only one of the 157 nucleotides. a unique pattern of cleavage fragments was generated for each. Thus a single base change in a
• a cDNA clone containing either the wild-type [pcTYR-N 1 Tyr. Bouchard. B.. et al. ( 1989) J. Exp. Med. 169:2029] or 422 mutant [pcTYR-A422, Giebel. L.B.. et al ( 1991 ) 87: 1 1 19] tyrosinase gene was utilized as the target DNA in PCRs to generate the above substrate DNAs.
• the primer pair consisting of SEQ ID NOS:42 and 43 were used to generate a double stranded 157 bp DNA fragment from either the mutant of wild-type cDNA clone.
• the primer pair consisting of SEQ ID NO:29 and SEQ ID NO:49 was used to generate a double stranded 378 bp DNA fragment from either the wild-type or mutant cDNA clone.
• the primer pair consisting of SEQ ID NO:50 and SEQ ID NO:30 was used to generate a double stranded 1.059 kbp DNA fragment from either the wild-type or mutant cDNA clone.
• the primer pair consisting of SEQ ID NO:51 and SEQ ID NO:49 was used to generate a double stranded 1.587 kbp DNA fragment from either the wild-type or mutant cDNA clone.
• the sense strand primer contained a biotin label at the 5 ' end.
• PCR reactions were carried out as follows. One to two ng of plasmid DNA from the wild-type or 422 mutant was used as the target DNA in a 100 ⁇ l reaction containing 50 ⁇ M of each dNTP, 1 ⁇ M of each primer in a given primer pair, in IX PCR buffer. Tubes containing the above mixture were overlaid with three drops of light mineral oil and the tubes were heated to 94°C for 1 min, then cooled to 70°C. Taq polymerase was then added as 2.5 units of enzyme in 5 ⁇ l of IX PCR buffer. The tube was heated to 93°C for 45 sec. cooled to 52 for 2 min. heated to 72°C for 1 min 45 sec for 35 repetitions, with a 5 min incubation at 72°C after the last repetition.

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