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WO1998055653A1 - Detection d'acide nucleique - Google Patents

Detection d'acide nucleique Download PDF

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Publication number
WO1998055653A1
WO1998055653A1 PCT/US1998/011457 US9811457W WO9855653A1 WO 1998055653 A1 WO1998055653 A1 WO 1998055653A1 US 9811457 W US9811457 W US 9811457W WO 9855653 A1 WO9855653 A1 WO 9855653A1
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WIPO (PCT)
Prior art keywords
nucleic acid
dna polymerase
primer
nadh
ppi
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PCT/US1998/011457
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English (en)
Inventor
Steven Creighton
Larry Gold
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Nexstar Pharmaceuticals, Inc.
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Publication date
Application filed by Nexstar Pharmaceuticals, Inc. filed Critical Nexstar Pharmaceuticals, Inc.
Priority to AU78136/98A priority Critical patent/AU7813698A/en
Publication of WO1998055653A1 publication Critical patent/WO1998055653A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

Definitions

  • This invention is directed to a novel method for the highly selective detection of one or more specific target nucleic acid sequences in a sample composition that may contain a large number of nucleic acids. Included within the scope of this invention is a diagnostic kit for detecting the presence of target nucleic acid sequences in a sample composition that may contain such targets.
  • PCR polymerase chain reaction
  • Nucleic acid detection schemes e.g., PCR, 3SR, SDA, Southern hybridization
  • Hybridization of the probes to nearly complementary sequences which would lead to false positives — is suppressed by increasing the stringency of the hybridization.
  • Stringency is typically controlled with temperature, although other variables, such as ionic strength, will affect stringency.
  • the goal of a stringent hybridization protocol is to produce stable hybrids if and only if the target nucleic acid is present in the media.
  • nucleic acid detection schemes require a procedure to macroscopically register the presence of the hybrids.
  • Direct methods like Southern hybridization use the presence of a tag (radioactive, enzymatic, fluorescent) affixed to the probe to signal the presence of a hybrid. Since un-hybridized probes constitute a huge background, direct methods invariably require immobilizing the target to a solid phase so that un-hybridized probes can be washed away. Direct methods suffer from long assay times (up to 1 day) and relatively low sensitivity, with higher sensitivity coming at the cost of longer assays and the use of highly radioactive probes.
  • the amplified target sequence can bind ethidium bromide (EtBr) and give a fluorescent signal. Since un- hybridized probes do not bind EtBr and do not lead to amplified target, they do not provide a background signal and the assay can be run in the liquid phase.
  • the assay in principle has no sensitivity problems since, theoretically, a single target sequence can be detected.
  • PCR is initiated by the formation of hybrids between the target nucleic acid and synthetic complementary primers.
  • a class of naturally occurring enzymes is then used to "extend" the hybrid.
  • a recognizable feature is formed at the point where a double stranded nucleic acid is directly adjacent to a single stranded region — the rest of the target sequence.
  • DNA polymerases are able to recognize this feature and, in the presence of the appropriate deoxy nucleotide triphosphate (dNTP) that is complementary to the first nucleic acid residue of the single stranded region, will link the appropriate complementary residue to the 3' end of the primer.
  • dNTP deoxy nucleotide triphosphate
  • the DNA polymerase facilitated reaction creates the monophosphate link between the 3' end of the primer and the dNTP, thus releasing the inorganic species, pyrophosphate (PPi). So long as the appropriate dNTP is present, the chain will "grow" by the DNA polymerase catalyzed introduction of new residues. This reaction, which occurs naturally, and the existence, characterization and purification of DNA polymerases have been known for many years.
  • 3' ⁇ 5' exonuclease activity is the ability to cleave the 3' nucleic acid residue of a hybridized primer. This activity, therefore, reverses the reaction catalyzed by DNA 5'- 3' polymerase activity by removing a deoxy nucleotide-monophosphate (dNMP).
  • dNMP deoxy nucleotide-monophosphate
  • the 3' ⁇ 5' exonuclease domain exists on the same polypeptide chain as the DNA polymerase, but is a distinct domain; in other cases, the 3'-5' exonuclease is a separate polypeptide that is non-covalently associated with the polymerase.
  • Certain DNA polymerases have enhanced 3' ⁇ 5' exonuclease activity while others — naturally or by engineering — have no ability to remove the 3' nucleotide. It is believed that utilizing a DNA polymerase with significant 3'-5' exonuclease activity allows for a reduction in misincorporation of nucleotides. Hence this activity is commonly referred to as 3 5' proofreading exonuclease activity.
  • Systems can be engineered where a hybrid is formed between a primer and a template, and the composition of the dNTPs present is controlled in order to create a steady-state or "idling" condition.
  • the composition of the dNTPs present is controlled in order to create a steady-state or "idling" condition.
  • the first nucleic acid residue on the template 5' to the 3' terminus of the primer is an adenine
  • deoxy thymidine triphosphate (dTTP) must be present for the DNA polymerase reaction to proceed (Equation 1).
  • the next 5' residue on the template is a guanidine and the solution does not contain any deoxy cytosine triphosphate (dCTP)
  • an additional 3' residue will not be added to the 3' end of the primer sequence.
  • PPi pyrophosphate
  • Some such detection systems rely on coupled enzyme reactions that convert PPi into ATP, and then convert ATP into light using the firefly luciferin/luciferase system.
  • ELIDA pyrophosphate
  • the reactions that take place in ELIDA are as follows.
  • NAD Phosphorylase Pyrophosphorylase
  • the intensity of the light output can be analyzed to determine when more than one identical sequence is found adjacent to another. Because it was necessary to quantify the light output of the assay, conditions were selected to minimize or eliminate idling. Thus, a DNA polymerase was selected that had no 3' ⁇ 5' proofreading exonuclease activity. By contrast, the instant invention depends critically on the use of a DNA polymerase that does posses 3' ⁇ 5' proofreading exonuclease activity.
  • FIGURE 1 illustrates an overview of the central subject method.
  • Nucleic Acid in a Sample Composition (A) is hybridized under stringent conditions to one or more Probe Primers. At least one of the Probe Primers is conjugated to a group, such as biotin, that will allow that primer to be attached to a suitably functionalized Solid Phase Support (B). Following hybridization, the Target Nucleic Acid is partitioned from the Sample Composition by the addition of a Solid Phase
  • the Probe Primer is biotinylated and the Solid Phase Support comprises streptavidin or avidin conjugated beads (C).
  • C streptavidin or avidin conjugated beads
  • a DNA Polymerase and a dNTP are added.
  • the dNTP is the next nucleotide that will be added to the 3' end of the Probe Primer by a 5' ⁇ 3' DNA Polymerase using the Target Nucleic Acid as a template.
  • the DNA Polymerase possesses 3' ⁇ 5' Proofreading Exonuclease activity, and repeatedly incorporates and then excises the dNTP reside from the 3' terminus of the Probe Primer.
  • the net result is the production of dNMP and PPi (C).
  • PPi is converted to NADH
  • NADH is converted to light by the bacterial luciferase system.
  • Figure 2 illustrates the use of an Imprint of the Target Nucleic Acid.
  • the Target Nucleic Acid is hybridized to an Imprint Primer (IP) that is labelled with a biotin group (B) to permit attachment of the Imprint Primer to a streptavidin-coated bead (S).
  • IP Imprint Primer
  • B biotin group
  • S streptavidin-coated bead
  • the Imprint Primer is extended under stringent conditions to form a copy of the Target Nucleic Acid. This copy may be formed from Nuclease Resistant Nucleic Acid Residues.
  • the Sample Composition may be degraded by nuclease treatment. In either case, the beads are then isolated from the Sample Composition.
  • One or more Probing Primers are hybridized to the Imprint (sites P1-P3), and a dNTP and a DNA Polymerase are added to establish Idling.
  • the PPi produced by Idling is converted into NADH. and the NADH is consumed to generate light.
  • Figure 3 illustrates a Solid Phase Support-bound primer binding to a complementary nucleic acid.
  • Figure 4 illustrates a primer-target hybrid attached to a Solid Phase Support that is engaged and extended by a DNA polymerase.
  • Figure 5 illustrates the Idling phenomenon, and that the net result of Idling is the production of dNMP and PPi.
  • Figure 6 illustrates the different mass transport capabilities of bead columns and macroporous monolithic media.
  • Figure 7 depicts certain amplifications possible in the conversion of PPi to light using the NADH generating system and the bacterial luciferase system.
  • Figure 8 illustrates the detection of Ml 3 template in the presence of a large excess of human genomic DNA according to the method of the invention.
  • Figure 9 depicts one embodiment of chemistry useful in attaching a nucleic acid to a Solid Phase Support, and also a cartridge-type macroporous monolith.
  • the present invention includes a method for detecting a target nucleic acid in a solution or composition.
  • the method can also be used to detect the presence of multiple target nucleic acids simultaneously, and can be used to directly sequence the detected target nucleic acid.
  • the methods of the subject invention will find utility in any application where it is necessary to detect the presence or absence of one or more specific nucleic acid sequences. Typical uses include, but are not limited to, detection of pathogenic organisms and viruses, diagnosis of genetic diseases, forensic analysis of bodily fluids, analysis of food substances, and environmental testing. Also included within the scope of this invention is a diagnostic kit that utilizes the method of the invention to detect a target nucleic acid in a composition that may contain such target.
  • At least a portion of the sequence of the target nucleic acid is known.
  • One or more primers, complementary to sequences within the target nucleic acid are contacted with the sample composition so that hybrids are formed between the target and the primers.
  • the sequences of the primers are chosen such that the identity of the nucleotide on the target that is immediately 5' to where the primers hybridize is known.
  • a DNA polymerase and the specific dNTP complementary to the first residue of the target 5' to the hybrid is added to the solution.
  • the DNA polymerase is one that has substantial proofreading 3'- 5' exonuclease activity. Therefore, in solutions where hybrids have been formed — indicating that the sample composition contains the target nucleic acid ⁇ DNA polymerase will catalyze the conversion of dNTP to dNMP and pyrophosphate (PPi). If an idling state is reached, the concentration of dNMP and PPi will increase with time. Reaction conditions can be designed such that the production of PPi and dNMP depends linearly on time and the concentration of the hybrids in the sample. In sample compositions not containing the target nucleic acid, there should be no generation of either dNMP or PPi.
  • the method of the invention is the "idling" of the DNA polymerase/Exonuclease reactions generating dNMP and PPi that provides "amplification” or “signal enhancement" of the hybridization event.
  • the abovementioned steps are performed on a copy or "imprint" of the target nucleic acid.
  • the copy is formed by first adding a primer to the sample composition, wherein the primer hybridizes to a portion of the target.
  • the primer is then extended in the presence of a DNA polymerase and dNTPs to form a copy of the region of the target nucleic acid where the idling reactions will take place.
  • the primer used to make the imprint may include some tag which will allow the imprint to be isolated from the reaction mixture.
  • the primer may be biotinylated to allow the imprint to be attached to a streptavidin- coated solid phase support.
  • the imprint may be isolated from the sample composition prior to establishing idling conditions. This greatly reduces the background signal produced by contaminating non-target nucleic acids.
  • the imprint may optionally be comprised wholly or partly of nuclease resistant nucleic acid residues, and then treated with nucleases. This reduces the background even further as only the imprint will survive nuclease treatment.
  • the method of this invention also includes means for sensitively measuring the generation of dNMP or PPi to indicate the presence of the target nucleic acid in the sample composition. Any method for detecting PPi and/or dNMP is contemplated in the instant invention. In some embodiments, the generation of PPi is detected to give rise to a positive reading.
  • the generation of PPi is measured by an enzymatic system that first converts PPi to NADH, and then consumes the NADH to generate light via the bacterial luciferase system.
  • This system uses a well known series of enzymatically-catalysed reactions to convert PPi to NADH.
  • the NADH produced can be assayed in a variety of ways well known in the art. Firstly, NADH can be assayed in the bacterial luciferase bioluminescence system that is well known in the art; this is the preferred method of NADH detection in the invention. Secondly, the NADH can be detected directly through fluorescent assays. Thirdly, NADH can be detected by electrochemical assays. Additionally, NADH can be assayed by a variety of colorimetric assays known in the art.
  • PPi generation can also be measured by converting adenosine 5' phosphosulfate to adenosine triphosphate (ATP) in the presence of the enzyme ATP sulfurylase.
  • PPi generation may be measured by converting nicotinamide dinucleotide into nicotinamide mononucleotide, with the concomitant generation of ATP, in the presence of NAD pyrophosphorylase.
  • the ATP thus formed is combined with firefly luciferin in the presence of the firefly enzyme luciferase, and light is generated.
  • the dNTPs in the DNA Polymerase reaction mixture must be removed prior to detecting the ATP, as all dNTPs can activate the firefly luciferase to some extent.
  • This invention includes a novel method for the detection of a target nucleic acid sequence.
  • the basis for the detection scheme of this invention is the formation of a hybrid between the target nucleic acid sequence and a primer sequence complementary to the target.
  • the presence of the hybrid is detected based on the formation of pyrophosphate (PPi) and/or deoxy nucleoside monophosphate (dNMP) in the presence of a DNA polymerase and the appropriate deoxy nucleoside triphosphate (dNTP).
  • PPi pyrophosphate
  • dNMP deoxy nucleoside monophosphate
  • dNTP deoxy nucleoside triphosphate
  • the detection of hybrid formation ⁇ and thus the detection of the target nucleic acid sequence ⁇ is accomplished by detecting the presence of PPi. Further in the preferred embodiment, the presence of PPi is detected by assays that rely on a well known enzyme system that converts PPi to NADH. The NADH is then consumed to generate light using the bacterial luciferase system. This detection system has the sensitivity to detect as few as 100 PPi molecules in a sample. The methods described herein have the potential to detect a single copy of a target nucleic acid.
  • the idling conditions are established on a copy of the target nucleic acid that is synthesized in the sample composition, and then attached to a solid phase support. Creating this copy, which can be isolated from the sample composition by virtue of its association with a solid support, greatly enhances the sensitivity of the central method.
  • Nucleic Acid refers to oligonucleotides including either
  • Modifications include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid bases or the nucleic acid as a whole.
  • modifications include, but are not limited to. 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine. substitution of 5- bromo or 5-iodo-uracil, backbone modifications, methylations. unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like.
  • Target Nucleic Acid refers to any nucleic acid that it is desired to be detected by the present invention.
  • the Target Nucleic Acid is genomic DNA, genomic R A or a specific mRNA.
  • the presence of the Target Nucleic Acid in a sample composition is a marker for a specific disease or medical condition. For example, markers to diseases are known in D53 and sas mutations and for sickle cell anemia.
  • Sample Composition refers to any heterogenous or homogenous solution being tested for the presence of the Target Nucleic Acid.
  • the Sample Composition may be a biological fluid (i.e., blood, serum, urine etc.) or it may be any prepared solution that may contain the Target Nucleic Acid.
  • the Sample Composition may contain other nucleic acids, as well as any other components, including, but not limited to, proteins, peptides, carbohydrates and any other components, so long as the components of the Sample Composition do not interrupt the ability of the Primer to hybridize with the Target Nucleic Acid.
  • Probe Primer refers to a nucleic acid sequence that is complementary to at least a portion of the Target Nucleic Acid.
  • the Probe Primer is comprised of Nucleic Acid residues, and may be from 3-100 nucleotides long. In the preferred embodiment, the Primer is about 6-30 nucleotides long. In some embodiments, the Probe Primer is comprised in whole or in part of Nuclease Resistant Nucleic Acid Residues and/or Thermostable Nucleic Acid Residues. The use of
  • Thermostable Nucleic Acid Residues increases the ability to eliminate mismatches between the Probe Primer and nucleic acid sequences similar but not identical to those found in the Target Nucleic Acid.
  • the Probe Primer is used to establish Idling on an Imprint copy of the Target Nucleic Acid.
  • the Probe Primer includes a tag which allows the Probe Primer, and the Target Nucleic Acid that is hybridized thereto, to be removed from the reaction medium.
  • the Probe Primer may be biotin-labelled and used to establish Idling directly on a Target Nucleic Acid while the Probe Primer is associated with a streptavidin-coated Solid Phase Support.
  • Imprint Primer refers to a nucleic acid sequence that is complementary to at least a portion of the Target Nucleic Acid 3' to where the Probe Primers Hybridize.
  • the Imprint Primer is comprised of nucleic acids, and may be from 3-100 nucleotides long.
  • the Imprint Primer may be comprised wholly or partly of Nuclease Resistant Nucleic Acid Residues and/or Thermostable Nucleic Acid Residues.
  • Thermostable Nucleic Acid Residues such as PNA, increases the ability to eliminate mismatches between the Primer and nucleic acid sequences similar but not identical to those found in the Target Nucleic Acid.
  • the Imprint Primer is about 6-30 nucleotides long.
  • the Imprint Primer is labelled at one or more sites with molecules that permit attachment to a Solid Support. Such attachment can be accomplished by a number of methods known to those skilled in the art.
  • the Imprint Primer is biotin- labeled, and will bind to a streptavidin-coated Solid Support.
  • Imprint refers to a copy of the Target Nucleic Acid made by hybridizing an Imprint Primer to a Target Nucleic Acid in the Sample Composition, followed by extending the 3' end of the hybridized Imprint Primer with Nucleic Acid residues and a DNA Polymerase.
  • the Imprint contains the sequences that will be hybridized to the Probe Primers.
  • the Imprint Primer is extended with Nuclease Resistant Nucleic Acid Residues to form a nuclease resistant Imprint.
  • the Imprint may also be comprised wholly or partly of Thermostable Nucleic Acid Residues. The fidelity of Imprint formation can be increased by any of the methods well known in the art.
  • betaine ((CH 3 ) 3 N ⁇ -CH 2 -COO e ) can be included in order to promote the isothermal denaturation of Nucleic Acids in the Sample Composition. This will prevent GC rich Target Nucleic Acids from being relatively poorly denatured prior to addition of the Imprint Primers.
  • DNA Polymerase refers to a family of enzymes known to those skilled in the art. DNA Polymerases are enzymes that recognize the junction between single-stranded and double-stranded nucleic acids created by the hybridization of primer to a Target Nucleic Acid. DNA Polymerases useful in the present invention include, but are not limited to, T4 DNA Polymerase, T7 DNA
  • T7 DNA Polymerase T7 DNA Polymerase :Thioredoxin, thermostable DNA Polymerase from Pyrococcus woesei, Klenow Fragment DNA Polymerase.
  • Preferred DNA Polymerases have 3' ⁇ 5' Proofreading Exonuclease activity.
  • the preferred DNA Polymerases of the present invention are T4 DNA Polymerase, T7 DNA Polymerase, T7 DNA
  • thermostable DNA Polymerase from Pyrococcus woesei.
  • reagents can be included in the buffer containing the DNA Polymerase in order to stabilize the enzyme or maximize its activity.
  • the addition of co-solvents can act to stabilize the activity of DNA Polymerases at elevated temperatures.
  • the disaccharide trehalose is used to thermostabilize or thermoactivate DNA Polymerases that are normally inactive at elevated temperatures (Carnici et al. 1998. Proc. Natl. Acad. Sci. 95: 520-524; inco ⁇ orated specifically by reference herein).
  • AMV Reverse Transcriptase can function efficiently at 60°C.
  • the osmolyte betaine is known to confer thermostability to a number of proteins.
  • Solid Phase Support refers to any of a number of supports compatible with the reagents of the present invention.
  • Solid Phase Supports can take the form of beads, filters, plugs or plates.
  • Solid Phase Supports are typically made of inert materials that are functionalized on their surface to allow for the attachment of Primers.
  • Preferred Solid Phase Supports include beads, Macroporous Supports, and micro fabricated planar surfaces, commonly known in the art as "biochips".
  • the Solid Phase Support is a streptavidin-coated paramagnetic bead that can be isolated from reagent solutions by the application of a magnetic field.
  • Thermostable Nucleic Acid Residues refers to modified nucleic acid residues that are stable under increased temperature conditions. Examples of Thermostable Nucleic Acid Residues are 2'F RNA and PNA.
  • Nuclease Resistant Nucleic Acid Residues refers to modified nucleic acid residues that are stable in the presence of enzymes with nuclease activity. Enzymes with nuclease activity include DNase I and Exo III, and certain DNA Polymerases. The use of any Nuclease Resistant Nucleic Acid Residue is contemplated in the instant invention. In one preferred embodiment, the Nuclease Resistant Nucleic Acid Residues are phosphorothioate Nucleic Acid residues (dNMP ⁇ S). The Imprint is made to contain dNMP ⁇ S by giving the Polymerases which copy the initial Sample Composition dNTP S instead of dNTP. Most Polymerases will accept this substitution without loss of efficiency and fidelity.
  • dNMP ⁇ S Nuclease Resistant Nucleic Acid Residues
  • Macroporous Supports refers to a class of Solid Phase Support materials characterized by a permanent porous structure formed during their preparation that persists in the dry state. These materials are comprised of cross- linked polymers (See, F. Svec et al, Science 273:205 (1996), incorporated herein by reference).
  • PPi Detection Assay refers to any assay or method for detecting the formation or presence of pyrophosphate (PPi) in an aqueous solution.
  • PPi can be detected by a variety of analytical methods, including, but not limited to: 1) luminescent, 2) fluorescent, 3) colorimetric, 4) light absorption, and 5) electrochemical. Any enzymatic system that can convert PPi into light is included within the scope of the subject invention.
  • the preferred method of PPi detection uses a coupled series of enzyme reactions to convert PPi into NADH; the NADH is then consumed to generate light in preferred embodiments.
  • the NADH is detected through electrochemical assays, colorimetric assays, or fluorometric assays known in the art.
  • Other examples of sensitive PPi Detection Assays involve the use of enzymes that catalyze the conversion of PPi to ATP, followed by the generation of light from the ATP using the firefly luciferin/luciferase assay well known in the art.
  • One example of such a coupled assay is ELIDA (See, Nyren et al. Analytical Biochem. 208 : 17 ( 1993), inco ⁇ orated herein by reference).
  • Still further examples include, but are not limited to, the NAD pyrophosphorylase-catalyzed reaction of PPi and NAD to form NADH and ATP, the adenylate-cyclase-catalyzed reaction of PPi and cyclic AMP to form ATP, the ADP-Glucose Pyrophosphorylase-catalyzed reaction of ADP- glucose and PPi to form ATP and 1-phosphoglucose, and the poly (A) polymerase- catalyzed reaction of Poly (A) n and PPi to form Poly (A) n+1 and ATP.
  • a spectrophotometric method that is a coupled assay in which the addition of inorganic pyrophosphatase initially cleaves the pyrophosphate into two molecules of phosphate, and the phosphate is then detected by the conversion of 2- amino G-mercapto-7-methyl prime ribonucleoside to 2-amino-G-mercapto-7-methyl prime by prime nucleoside phosphorylase (See, Upson et al, Analytical Biochem. 243:41 (1996), inco ⁇ orated herein by reference).
  • Idling refers to a coupled enzymatic process wherein DNA Polymerase catalyzes the linkage of a dNTP to a DNA at a point of hybridization to generate PPi, while the 3'- 5' Proofreading Exonuclease activity of the DNA Polymerase removes the linked dNMP.
  • the net reaction when a hybrid system is Idling is the conversion of dNTP to dNMP and PPi. Idling can occur indefinitely if the appropriate dNTP concentration is maintained in solution and the PPi product is removed. Idling only occurs when a hybrid is formed between the Probe Primer and the Target Nucleic Acid. In order to maintain idling, the complementary dNTP 5' to the end of the newly formed linkage is not included in the solution. Idling will only occur when the DNA Polymerase has 3'- 5' Exonuclease activity.
  • 3' ⁇ 5' Exonuclease Activity and “3'-5' Proofreading Exonuclease Activity” as defined herein refers to the ability to catalyze the cleavage of the last 5' nucleic acid residue of the Primer that is part of a hybrid formed between the Primer and the Target Nucleic Acid sequence.
  • Certain DNA polymerases have enhanced 3'- 5' Exonuclease activity. Typically DNA 5'-* 3 'polymerase activity and 3' ⁇ 5' Exonuclease Activity are found as distinct domains of the same polypeptide on the same gene. For example, E. coli pol I and T4 DNA Polymerases contain 5' ⁇ 3' polymerase and 3' ⁇ 5' Exonuclease Activity on a single polypeptide chain.
  • the present invention for detecting the presence of a Target Nucleic Acid in a Sample Composition involves the combination of a number of known principles and reactions.
  • the basic steps of the method include 1) formation of a hybrid between the Target Nucleic Acid and the Probe Primer; 2) addition of DNA Polymerase reagents and establishment of Idling conditions in which PPi and dNMPs are produced if the hybrid is formed; and 3) detection of either or both of PPi and dNMP.
  • the basic method is depicted in Figure 1, and the individual steps are described below in detail:
  • Probe Primer that is complementaryto at least a portion of the sequence of said Target Nucleic Acid.
  • the contacting step is conducted under conditions where a hybrid is formed between the Primer and Target Nucleic Acid if the Target Nucleic Acid is present in the Sample composition ( Figure 1 A).
  • the conditions for hybridization are chosen so that the maximum possible stringency is achieved. Preferred embodiments use high temperatures, particular ionic strengths, and judicious choice of Probe Primer sequences to achieve high stringency. These factors are well known in the art, and are described in "Nucleic Acid Analysis", Ed. C. Dangler, Wiley-Liss 1997, the contents of which are specifically inco ⁇ orated herein by reference. Any method known in the art for enhancing the stringency of Nucleic Acid hybridization is contemplated. For example, isothermal denaturation can be promoted by the addition of betaine.
  • the Probe Primer is attached to a
  • the hybrid is easily partitioned from the non-Target Nucleic Acids in the Sample Composition by any of the methods known in the art.
  • the Probe Primer is labeled with one or more biotin groups.
  • the biotin-labelled hybrid may then be attached to a streptavidin-coated Solid Phase Support, preferably a supe ⁇ aramagnetic bead.
  • Such beads are commercially available (e.g., DYNABEADS ® from Dynal Inc.) As both hybrids and free Probe Primer bind to a streptavidin-coated Solid Phase Support, the number of biotin-binding sites on the Solid Phase Support is in excess of the number of Probe Primers initially added to the Sample Composition. If the Solid Phase Support comprises paramagnetic beads, then these can be separated from the Sample Composition by the application of a magnetic field.
  • the Probe Primer is partly or wholly comprised of Nuclease Resistant Nucleic Acid Residues. This prevents the Probe Primer from being digested by the 3' ⁇ 5' Exonuclease Activity of the DNA Polymerase during Idling.
  • the Probe Primer comprises Thermostable Nucleic Acid residues.
  • the Probe Primer may include Thermostable Nucleic Acid residues at the 5' end of the Probe Primer and regular DNA at its 3' end. This allows hybridization based on the Thermostable
  • the Probe Primer Extending the Probe Primer by the addition of at least one dNTP and at least one DNA polymerase. wherein pyrophosphate and dNMP is produced by Idling.
  • the hybrids formed between the Probe Primer and the Target Nucleic Acid are physically segregated from the remainder of the Sample Composition and washed to remove all components of the Sample Composition that do not bind to the Probe Primer ( Figure 1C).
  • the trapped hybrids are cleaved from the Solid Support before the addition of the appropriate dNTP and the DNA Polymerase that will catalyse the Idling reaction.
  • the DNA Polymerase catalyzes the template-dependent 5' ⁇ 3' extension of the Probe Primer.
  • the dNTP that is added is the nucleotide that will be the next one to be incorporated at the 3' terminus of the Probe Primer using the Target Nucleic Acid as a template.
  • the DNA Polymerase used in the Idling reaction is one with a substantial 3' ⁇ 5' Proofreading Exonuclease Activity in order that the dNTP is repeatedly inco ⁇ orated and excised.
  • Preferred DNA Polymerases include T4 DNA Polymerase, T7 DNA Polymerase: Thioredoxin and the thermostable DNA Polymerase from Pyrococcus woesei.
  • Idling is carried out at elevated temperatures in order to maximize the rate of PPi turnover.
  • T4 DNA Polymerase a 10 fold increase in the rate of Idling can be achieved by raising the temperature from 20°C to 45°C.
  • the stability of the enzyme at high temperatures can be enhanced by the addition of co-solvents, such as trehalose.
  • the Probe Primer used to form hybrids with the Target Nucleic Acid need not be the hybrid site for DNA polymerase action.
  • the hybrid could be dissociated.
  • One or more additional Probe Primers complementary to the Target Nucleic Acid can then be added to the solution before establishing Idling conditions.
  • additional Probe Primers into the solution so that Idling can occur at multiple sites on the Target Nucleic Acid. In both cases, this provides an additive means for amplifying the original single hybridization event.
  • the amount of PPi and dNMP formed would be increased by an order of magnitude.
  • the Idling reaction is quenched by either heat or EDTA, and the amount of PPi and/or dNMP is quantitated. The presence of either of these molecules indicates the presence of the Target Nucleic
  • the formation of pyrophosphate is used to detect the hybrid formation event.
  • the presence of pyrophosphate can be detected by a PPi Detection Assay.
  • the preferred PPi Detection Assay of the present invention uses a series of enzyme reactions to convert PPi into NADH; the NADH is then consumed by bacterial luciferase to generate light.
  • the PPi is converted into NADH by addition of an appropriate amount of UDP-Glucose and NAD in the presence of the enzymes UDP-Glucose Pyrophosphorylase, Phosphoglucomutase, and Glucose-6-Phosphate Dehydrogenase to form NADH and 6-P-Gluconolactone.
  • UDP-Glucose Pyrophosphorylase Phosphoglucomutase
  • Glucose-6-Phosphate Dehydrogenase to form NADH and 6-P-Gluconolactone.
  • the NADH produced in this system is then converted into light by using the bacterial luciferase system, well known in the art.
  • This coupled enzyme system comprises an oxidoreductase which catalyzes the reduction of FMN by NADH, and a luciferase which catalyzes the reaction of the resulting FMNH 2 and a long-chain aldehyde to form a carboxylic acid with the concomitant emission of light at 495nm.
  • the system is described by Hastings J., In: Methods in Enzymology New York, Academic Press 1978, 125-135. and by Jablonski, E. and DeLuca, M.
  • the oxidoreductase and luciferase that form the system can be isolated from a number of marine bacteria, including, but not limited to, Lucibacterium harveyi, Beneckea harveyi, Photobacterium fischerii and Vibrio fischerii.
  • the quantum yield of the reaction is between 0.2 and 0.27.
  • the choice of aldehyde depends on the specific species of luciferase that is being used, but for Photobacterium fischerii the best intensity is achieved using decanal.
  • bacterial luciferases are not activated by dNTPs, there is no need to perform additional steps to remove these nucleotides from the reaction mixtures.
  • this method of detecting PPi is superior to prior art methods involving the use of ATP generating systems and the firefly luciferase system.
  • the assay conditions used in converting PPi to NADH are compatible with the all the various DNA Polymerase reagents.
  • any method for determining NADH is contemplated in the subject application.
  • the fluorometric assays provide a very sensitive means for assaying NADH. They rely on the ability of NADH to emit 420nm blue light upon excitation with 320nm ultraviolet light. Techniques known in the art that use laser excitation have the capability of detecting single molecules of NADH in this way.
  • the presence of PPi is detected by using an ATP generating system to convert the PPi to ATP, followed by the conversion of the ATP to light by the firefly luciferin/lucifase system.
  • an ATP generating system to convert the PPi to ATP, followed by the conversion of the ATP to light by the firefly luciferin/lucifase system.
  • ELIDA a system that uses the enzyme NAD-Pyrophosphorylase to catalyse the conversion of PPi and NAD to ATP and NMN.
  • the mixture containing the Idling nucleic acid may be treated to remove unincorporated dNTPs prior to use of the PPi Detection Assay.
  • dNTPs can activate the firefly luciferin/luciferase system, albeit at a far reduced level compared to ATP.
  • the removal of dNTPs may be accomplished by adding an appropriate amount of glycerol, glucose, and the enzymes glycerokinase and hexokinase. These enzymes can rapidly transfer the terminal phosphate groups of the dNTPs to glycerol and glucose, resulting in the formation of dNDPs that will not activate firefly luciferase.
  • the sequence of the Probe Primer(s) is chosen, if possible, such that dATP is the only dNTP that must be added to establish Idling at each Probe Primer hybridization. Following the Idling reaction, dATP can be efficiently removed from the reaction mixture by adding the enzyme apyrase.
  • the other dNTPs dGTP, dCTP, and dTTP are not present at any time in the Idling reaction mixture.
  • the PPi detection occurs in a separate vessel from the reaction chamber where the Idling reactions take place.
  • the amount of PPi is quantitated by first converting all of the PPi in the quenched Idling reaction into NADH by means of the NADH-generating enzymatic systems described above. For example, in one embodiment, all of the PPi is converted to NADH by the addition of the necessary enzymes and reagents. Then, a predetermined amount of the bacterial luciferase reaction mixture is injected into the quenched Idling reaction in front of a luminometer.
  • the sudden oxidoreductase/ luciferase-catalyzed reaction of all of the NADH results in the delivery of a "burst" of light which can be integrated for a period of time, typically 5 seconds, after injection.
  • a "burst" of light which can be integrated for a period of time, typically 5 seconds, after injection.
  • Systems that can perform this procedure automatically using multiwell plates are well known in the art.
  • the luciferase mixture in this embodiment preferably contains a large excess of the luciferase enzyme as the removal of the reaction products from the active sites of this enzyme is the rate-limiting step in light production. If the light signal is of sufficient size, then it is possible to document the signal on radiographic film.
  • the foregoing embodiments all involve the establishment of Idling conditions using a hybrid formed between the Target Nucleic Acid and one or more Probe Primers.
  • the Sample Composition is first denatured and then contacted with an Imprint Primer.
  • the Imprint Primer may be wholly or partly comprised of Nuclease Resistant Nucleic Acid Residues, and is labelled with a group (e.g., biotin) that permits subsequent attached to a suitably conjugated Solid Phase Support.
  • the Imprint Primer may also be comprised wholly or partly of Thermostable Nucleic Acid Residues.
  • the Imprint Primer sequence is chosen so that it hybridizes to the Target Nucleic Acid at a position that is 3' to the location of the sequences that will later be used to establish Idling.
  • a DNA Polymerase, dNTP molecules, dNTP ⁇ S molecules, Thermostable Nucleic Acid Residues, or combinations thereof are then added in order to form the Imprint by extension from the Imprint Primer.
  • Agents that enhance the fidelity of DNA synthesis, such as single stranded binding proteins may also be included in the reaction mixture.
  • the Imprint thus formed is labelled with biotin, for example, (due to inclusion of the biotinylated Imprint Primer) and comprises the sequences that will subsequently be recognized by the Probe Primer(s).
  • the Imprint is formed from Nuclease Resistant Nucleic Acid residues using Taq DNA Polymerase at temperatures around 70°C.
  • the Imprint is attached to a Solid Phase Support, and partitioned from the Sample Composition. In preferred embodiments this is achieved, as above, by the use of streptavidin-conjugated paramagnetic beads.
  • the isolated Solid Phase Support with attached Imprint is washed to remove any contaminating nucleic acids. In preferred embodiments, the washing is done under alkaline denaturing conditions in order to remove any non-specifically bound Nucleic Acids or nucleic acids that have hybridized to the Imprint, and to hydrolyze any contaminating ribonucleic acid molecules.
  • the washing may be optionally preceded or followed by the addition of nucleases, such as DNase I and Exo III. Nuclease treatment will further reduce the potential for contamination from non-Target Nucleic Acids, as only the Imprint Target Nucleic Acid is comprised of Nuclease Resistant Nucleic Acid Residues and is spared from the action of the nucleases.
  • nucleases such as DNase I and Exo III.
  • the isolated Imprint is then contacted with one or more Probe Primers, and Idling is established as described above.
  • the Idling reactions can be performed with the Imprint attached to the Solid Phase Support, or the Imprint may first be detached therefrom. In other embodiments, multiple rounds of Imprint formation can be used to achieve, if necessary, an even higher degree of specificity.
  • the Imprint can be dissociated from the Solid Phase Support.
  • a second biotinylated Imprint Prime which recognizes a sequence distinct from that recognized by the first Imprint Primer — can then be added to the solution containing the first Imprint, and a second Imprint can be formed and isolated as described above. This process can be repeated if necessary.
  • Nucleic Acid Residues in both the Imprint and the Probe Primer will permit the use of DNA Polymerases with substantial 3'-5' Proofreading Exonuclease Activity without concern that the Imprint or Probe Primer will be digested.
  • Imprint and Probe Primers that recognize distinct sequences adds an additional degree of specificity to the method. If a non- Target Nucleic Acid in the Sample Composition is fortuitously converted into an Imprint, due to some limited sequence homology with the true Target Nucleic Acid in the region used for Imprinting, then it will be highly unlikely that this Imprint will also contain the additional sites recognised by the Probe Primers. Therefore, only the true Imprint will serve as a template for Idling. Furthermore, the length of any mis-copied Imprints can be kept to a minimum by allowing the Imprint formation polymerization reaction to proceed only for the minimum amount of time needed to copy the sequences recognized by the Probe Primers.
  • the hybrid containing solution ⁇ which can be but does not necessarily have to be separated from the remainder of Sample Composition — is adjusted to contain a DNA Polymerase and all of the dNTP, or optionally dNTP ⁇ S, monomer units and synthesis of the complementary DNA sequence is completed. During the synthesis, a molecule of pyrophosphate is generated for each monomer unit added to the chain.
  • Detection-by-Synthesis proceeds by the detection of pyrophosphate or dNMP as described in the other embodiments where Idling is established to generate additional amounts of pyrophosphate or dNMP.
  • This embodiment may be particularly useful when the Sample Composition contains a relatively large number of copies of the Target Nucleic Acid.
  • the DNA Polymerase will have a substantial 3'->5' Proofreading Exonuclease activity. This increases the amount of PPi released during synthesis of complementary DNA, as the DNA Polymerase will repeatedly add and excise monomer units, even though true Idling will not take place as all 4 dNTPs are present. In this case, it will not be possible to use dNTP ⁇ S monomers, as they will prohibit Idling.
  • an endonuclease can be included in the DNA Polymerase reagent.
  • the endonuclease will nick the Nucleic Acid that is extended from the Imprint or Probe Primer.
  • the DNA Polymerase in this embodiment recognizes these nicks, and will begin DNA synthesis at the 3' end of each nick site, displacing any existing DNA as it proceeds. This phenomenon is well known in the art, and is referred to as "nick translation". This will dramatically increase the amount of DNA synthesis per template molecule, as many DNA
  • Polymerase molecules can simultaneously use one template. This will in turn lead to an increase in the amount of PPi liberated.
  • the various reactions in the Imprinting and Idling schemes take place on Solid Phase Supports.
  • Many of these embodiments may use the well-known biotin-avidin interaction as a means for attaching the appropriate Nucleic Acid to the Solid Phase Support.
  • the Nucleic Acid is labeled with one or more biotin groups, then this Nucleic Acid can bind to an avidin or streptavidin-coated bead.
  • this is only one of many possible ways known in the art of attaching Nucleic Acids to Solid Phase Supports.
  • methods are well known in the art that can be used to covalently attach a suitably labeled Nucleic Acid to an appropriately functionalized Solid Phase Support. Any method known in the art for attaching a Nucleic Acid to a Solid Support is contemplated in the present invention.
  • Solid Phase Supports can be varied depending on the application.
  • Preferred embodiments use Solid Phase Supports in the form of beads to which the various reagents are added. In the most preferred embodiment, super- paramagnetic beads are used. These beads can be rapidly separated from reagents by the application of a magnetic field.
  • Other Solid Phase Supports contemplated in the subject invention take the form of microfabricated planar surfaces, known in the art as "biochips".
  • biochips microfabricated planar surfaces
  • the various primers used in this invention can be attached to spatially discrete locations on said biochips, using any of the methods provided in the art. This can permit the simultaneous detection of multiple Target Nucleic Acids.
  • cartridges upon which the appropriate Nucleic Acids are immobilized.
  • cartridges can be constructed which have Imprint Primers covalently attached. Reagents can be injected in such cartridges, and then eluted by injection of a wash buffer.
  • the present invention further includes a diagnostic kit for the detection of Target Nucleic Acids in a Sample Composition.
  • the kit includes at least one Probe Primer that is complementary to one or more portions of the Target Nucleic Acid Sequence.
  • the kit may optionally include one or more Imprint Primers that can be used to create an Imprint, if required, of the Target Nucleic Acid.
  • the Imprint and — if included ⁇ the Probe Primers are comprised of Nuclease Resistant Nucleic Acid Residues.
  • the Imprint Primers and an aliquot of the Probe Primers are labelled with a tag, such as biotin, that will allow them to attach to a Solid Phase Support.
  • a Solid Phase Support capable of binding to the labelled Probe Primers and Imprint Primers is also included. This preferably comprises streptavidin-conjugated supe ⁇ aramagnetic beads. Further included in the Diagnostic Kit is a solution containing at least one dNTP and at least one DNA Polymerase. The diagnostic kit also includes a PPi Detection Assay or means for detecting the presence of dNMP.
  • an Imprint copy of a Target Nucleic Acid is synthesized in the manner described above.
  • one or more Probe Primers are hybridized to the Imprint.
  • Each Probe Primer comprises sequences complementary to the Target
  • Nucleic Acid and further comprises one or more molecules of a PPi detection system enzyme.
  • the enzyme molecules can be covalently attached to the Probe Primers by any of the methods known in the art.
  • the Probe Primer may comprise one or more molecules of UDP-Glucose Pyrophosphorylase.
  • the Imprint is bound to Solid Phase Supports as described above to facilitate the separation of Probe-Imprint hybrids from the hybridization reaction.
  • the hybrids formed between the Imprint and the Probe Primer can be detected by incubating the Solid Phase Supports with PPi and the remainder of the reagents required to converted PPi into NADH: namely UDP-Glucose, Phosphoglucomutase, NAD, and Glucose-6-Phosphate Dehydrogenase.
  • NADH formed in this way may be converted into light using the bacterial luciferase system described above.
  • the enzymatic component that is attached to the Probe Primer is not included in the mixture of reagents that are added after Probe Primer hybridization. This technique may be equally well applied to the Target Nucleic Acid in the Sample Composition without first synthesizing an Imprint.
  • a number of basic principles are known to those skilled in the art that facilitate and enable the present invention. Several of these concepts are set forth below.
  • Solid-phase linked probes can efficiently (and specifically) capture complementary nucleic acids from solution.
  • the various primers are attached to a Solid Phase Support ( Figure 3).
  • the Solid Phase Support comprises supe ⁇ aramagnetic beads.
  • linking of the primers to a Solid Phase Support is not essential to the performance of the invention.
  • the hybrids formed can be separated from the Sample Composition without prior attachment of the primers to a Solid Phase Support.
  • the primers could contain a reactive moiety that would react to a Solid Phase Support subsequent to hybrid formation.
  • a linker is attached to the 3' end of the primer sequence.
  • a linker may be attached to the primers by any of the methods known to those skilled in the art.
  • the linker may be comprised of a short alkyl chain, a synthetic polymer such as PEG, a peptide or synthetic nucleic acid linker.
  • the linker can serve to help attach the Imprint Primer to the Solid Phase Support, and also serves to provide a physical separation between the Solid Phase Support and the primers, to allow full access to the primers to all of the components of the Sample Composition.
  • the hybrid is cleaved from the Solid Phase Support before establishing Idling conditions. This situation may be desirable in certain cases where the rate of Idling can be enhanced by allowing the hybrid to exist in solution.
  • the primer need not be attached to a Solid Phase Support at any time during the performance of the method of this invention.
  • pyrophosphate (PPi) released through DNA polymerase catalyzed dNTP inco ⁇ oration can be assayed easily and quickly at the 0.1 fmol level using a coupled luminescence assay.
  • the PPi generated during Idling is detected via the generation of NADH.
  • This assay comprises a coupled enzymatic system that generates light as a product.
  • the coupled reactions are as follows: a) dNTP + DNA n - DNA n+1 + PPi b) PPi + UDP-Glucose ⁇ UTP + Glucose- 1 -Phosphate c) Glucose- 1 -Phosphate - Glucose-6-Phosphate d) Glucose-6-Phosphate + NAD ⁇ NADH + 6- P-glucolactone e) NADH + FMN + H + ⁇ FMNH 2 + NAD f) FMNH 2 + O 2 + R-CHO ⁇ FMN +H 2 O + R-COOH + LIGHT wherein a) is catalyzed by a DNA Polymerase, b) is catalyzed by UDP- Glucose Pyrophor
  • the NADH generating reactions (a-d) and the luciferase reactions (e-f) can all be done in a one-pot system. In preferred embodiments, the reactions are done at separate times.
  • all of the PPi is converted to NADH using the UDP-Glucose Pyrophosphorylase/Phosphoglucomutase/Glucose-6-Phosphate Dehydrogenase enzymes in the presence of UDP-Glucose and NAD.
  • the oxidoreductase/luciferase reagents are injected into the NADH generation solution.
  • the efficiency photons/PPi-sec
  • the PPi generated during Idling is detected via an ATP- generating system, such as the ELIDA assay.
  • an ATP- generating system such as the ELIDA assay.
  • assays also comprise coupled enzyme systems that generate light as a product.
  • Luciferase ATP + Luciferin + O 2 -AMP + PPi + CO 2 + Oxy-luciferin
  • a related assay use NAD-Pyrophosphorylase to generate ATP from PPi through the following series of coupled reactions:
  • DNA Polymerase dNTP + DNA ⁇ - DNA n+l + PPi NAD-Pyrophosphorylase:PPi + NAD + ⁇ ATP + NMN +
  • Luciferase ATP + Luciferin + O 2 - AMP + PPi + CO 2 + Oxy- luciferin + light
  • APS Adenosine 5' phosphosulfate
  • NAD Nicotinamide dinucleotide
  • NMN Nicotinamide mononucleotide
  • Adenylate cyclase cyclic AMP + PPi -ATP
  • ADP-Glucose Pyrophosphorylase ADP-Glucose + PPi - ATP+1- phosphoglucose
  • Poly (A) Polymerase Poly (A) n + PPi - Poly (A) n+1 + ATP
  • One of the critical aspects of the present invention is the ability to reduce the production of background light. It is important, therefore, to utilize reagents that are free of contaminating nucleic acids and PPi. Methods are known to those skilled in the art, to simply remove these compounds from reaction solutions and reagents. For example, DNA Polymerase can be cleansed of contaminating nucleic acids (that may fortuitously form hybrids with the Probe Primer or the Imprint Primer) simply by incubating the enzyme at the reaction temperature in the presence of Mg ++ .
  • ATP generating systems that are used in conjunction with firefly luciferase is that any dNTP (especially dATP) in the Idling mixture will activate the luciferase system.
  • the preferred system described above for converting PPi into NADH. followed by the consumption of NADH by bacterial luciferase to generate light is far less prone to background artefacts than the ATP generating assays.
  • an ATP generating system it is possible to reduce the level of dNTPs through the use of the hexokinase/glycerokinase system described previously.
  • a single T4 polymerase molecule bound to a single probe-target complex can produce about lOPPi/sec using idling turnover.
  • Idling is a process whereby a DNA polymerase possessing a strong 3' ⁇ 5' Proofreading Exonuclease activity can be made to alternatively inco ⁇ orate a specific dNTP residue (releasing PPi) at a defined location on the target and then excise the newly inco ⁇ orated dNMP if the next templated dNTP is absent (Figure 5). Both of these reactions are rapid, around 10 sec "1 .
  • establishing an Idling condition is critical to the success of the present invention.
  • the DNA Polymerase selected will have strong 3'-5' Exonuclease Activity, be thermostable, and will Idle at high rate of turnover.
  • the primers either Imprint or Probe Primers—are attached to Macroporous Supports. Use of such supports enhances mass transport between the solution phase and the surface of the support — where the primers are attached.
  • the empty space in these macroporous systems consists of channels about 1 - 10 micrometers wide, the interstitial spaces present in bead based media are absent, and injected molecules are always very close to the solid phase (Figure 6)
  • Each PPi can in principle be converted to a NADH molecule with 100% efficiency in the presence of an excess of the necessary NADH generating enzymes and reagents.
  • Each of these NADH molecules can be used to activate a luciferase- complex to a state that has a 20% chance of emitting a photon.
  • a polymerase Idling at the lowest possible rate of IPPi/second is generating 0.2 photons/second.
  • a single Probe Primer will be the center of generation for the generation of 900 PPi in a typical 15 minute Idling reaction. If this PPi is converted to 900 NADH molecules prior to addition of the luciferase cocktail, then addition of the luciferase cocktail will result in the generation of approximately 180 photons.
  • Typical PMT backgrounds are about 1 pulse/sec. Thus, a single hybrid can generate an easily detectable signal. In embodiments where the PPi is used to generate ATP, the same calculation reveals that 15 minutes Idling at the lowest possible rate will provide about 800 photons.
  • each NADH molecule in the original solution can give rise to 5,000 to 10,000 NADH molecules in the final amplified mix when the cycling scheme is used.
  • a single T4 Polymerase Idling for 15 minutes at the lowest possible rate of lPPi/sec will produce between 900,000 and 1.8 million photons.
  • a co-enzyme cycling system may be useful in embodiments where the highest possible sensitivity is required.
  • - Source (1) is common to all Nucleic Acid detection technologies and can be minimized by appropriate protocols.
  • - Source (2) can be minimized by purification of the dNTP solution.
  • - Source (3) can be avoided by using the preferred assay system in which PPi is converted to NADH. If the ATP generating schemes are used, it can be minimized by adding a purification step between stages C and D to eliminate dNTP.
  • the spontaneous hydrolysis rate of the dNTP to form PPi is an impediment to the lowest limits of detection according to this invention.
  • the following procedure illustrates the use of the subject method for detecting a rare Target Nucleic Acid in a Sample Composition.
  • the method is used to detect the presence of M 13 DNA single-stranded DNA in the presence of a very large excess of human genomic DNA.
  • the Ml 3 primer is chosen such that dGTP is the nucleotide that must be added to permit Idling.
  • M13 Solution 0.5 nanomolar M13mp 18(+) strand DNA (Pharmacia) in 50 mM Tris-Cl/lmM EDTA/pH 7.5
  • Genomic DNA Solution 10 microgram/ml of purified human genomic DNA (Promega) in 50 mM
  • tubes B, D, E and F 2 microliter of Ml 3 Solution was added.
  • the tubes A-F were held at 70 °C for 5 minutes, then held at 40 °C.
  • 20 microliters of the Trap Bead Solution were added to each tube.
  • the tubes were allowed to sit at room temperature for 5 minutes with occasional mixing.
  • the beads in tubes A-F were then magnetically pelleted and washed three times in water. They were resuspended in 10 microliter Denaturing solution, and then magnetically pelleted again.
  • the supernatant was pipetted into new tubes (still labeled A-F) and 2 microliters of Neutralization solution was added to each tube.
  • 1 microliter of Primer solution was added to tubes C, D, E and F.
  • Tubes A-F were allowed to sit at 37°C for 1 minute, and then 10 microliters of Polymerase Mix (2x) was added to each tube and incubated for 5 minutes at room temperature. The reaction was quenched by holding each tube at 95 °C for 1 minute. Finally, 20 microliters of NAD Ppase/Luciferase mix (2x) was added to each tube, and the absolute levels of light emission were counted using Bioscan's Lumi-One Luminometer for 10 seconds after allowing 1 minute for the luminescence to come to equilibrium.
  • the Sample Composition is denatured by heating, and biotinylated Imprint
  • Primer is added. The mix is cooled to the polymerization temperature.
  • a DNA Polymerase typically a thermostable DNA Polymerase such as Taq, is added along with a suitable buffer, E. coli SSB, and phosphorothioate dNTPs (dNTP ⁇ S). Polymerization is allowed to proceed for a few minutes at an appropriate temperature, typically around 70°C.
  • Enough NaCl is added to the Imprint mix above to give a molarity of 2M. Then, avidin-coated paramagnetic beads are added to the mix and allowed to react with gentle shaking for 5 minutes. There is an excess of biotin-binding sites on the beads.
  • the beads are removed from the mix by the use of a magnet.
  • the beads are washed 3 times in 0.1N NaOH and optionally treated with DNase I or Exo III.
  • the beads are re-isolated, washed in buffer, and placed in 2M NaCl containing around 1 ⁇ M of each Probe Primer.
  • the beads are incubated at the desired temperature for a few minutes before removing them from this solution and washing once in TE buffer at 20°C.
  • the temperature chosen for hybridization depends on the melting temperature of the Probe Primer-Imprint interaction.
  • the beads are removed from the TE and placed in a DNA Polymerase mix consisting of lO ⁇ M of the Idling dNTP, lOnM DNA Polymerase (typically T4 DNA Polymerase, T7:Thioredoxin or the thermostable enzyme from Pyrococcus woesei), and the appropriate buffer components.
  • the reaction is allowed to proceed at the DNA Polymerase's optimal Idling temperature for a predetermined amount of time before the reaction is quenched with either heat or EDTA. The supernatant is then removed and used in the next phase.
  • the supernatant is adjusted to the following conditions: 50mM Tris-Cl, pH 8.0, O.lmM EDTA, l-5mM MgCl 2 , 500 ⁇ M ⁇ -NAD + , and ImM Uridine-5'- diphosphoglucose.
  • the enzymes are then added at the following concentrations: 0.25U/ml of uridine-5'-diphosphoglucose pyrophosphatase, 0.4U/ml of phosphoglucomutase, and 0.2U/ml of glucose-6-phosphate dehydrogenase.
  • the reactions are allowed to proceed at 37°C for 5 minutes.
  • the enzymes are then heat killed at 95°C for 3 minutes and the entire mix is spun through a PVDF protein absorbing membrane.
  • the NADH in the mix is quantitated by adding a bacterial luciferase cocktail that is set to deliver a "burst" of light to the mix in front of the luminometer by syringe injection.
  • the cocktail contains oxidoreductase, FMN, decanal, and luciferase in buffer components.
  • the components are added in the following amounts: O.lmL 0.01% mercaptoethanol, 1.3mL 0.1M sodium phosphate, pH 6.8, 0.2mL 0.42mM FMN, 0.05mL 0.1% decanal in methanol, and 0.2mL lOmg/ml enzyme preparation in distilled water (containing both oxidoreductase and luciferase from Photobacterium fischerii; available commercially from Worthington)
  • a rapid flow-through hybridization system can be constructed by immobilizing the Primer Probe in the pores of the monolith using a variety of possible chemistries ( Figure 9).
  • Figure 9 cartridges can be assembled which will trap the Target Nucleic Acid by hybridization.
  • the Sample Composition is injected into the column through the inlet, allowed to hybridize for a short time, then expelled through the outlet.
  • the trap column is exhaustively washed to remove all un-hybridized Nucleic Acid and other, possibly interfering, components from the Sample Composition.
  • a solution containing T4 DNA polymerase, buffer, and the single dNTP complementary to the next base on the Target Nucleic Acid is added to the column and left to incubate for about 10 min. at 37 C°.
  • each polymerase :probe-target complex will generate about 5,000 PPi molecules.
  • dNTPs Conversion of dNTPs to dNDPs.
  • the supernatant from Example 1 prior to addition of the Luciferin Luciferase components is adjusted to have 20% glycerol and 5% glucose. 1 unit each of hexokinase and glycerokinase are added and the mix is incubated for 2 minutes at 37°C. The enzymes are then heat killed at 95°C for 3 minutes and the entire mix is spun through a PVDF protein absorbing membrane. NAD to 30 ⁇ M and 0.2 units of NAD-pyrophosphorylase are added to the dNTP-free mix. The mix is allowed to incubate at 37°C for 2 minutes. The mix is then heated to 95°C for 1 minute, after which the Luciferin/Luciferase mixture may be added as described in Example 1.
  • the method of the instant invention allows for the ultra-sensitive and rapid determination of the presence or absence of any disease-causing or disease-associated nucleic acid in a sample.
  • a particular substance e.g., HIV, Salmonella etc
  • the method would make use of an Imprint Primer and a Probe Primer comprised of sequences that will specifically hybridize to the pathogenic sequences.
  • the method can be easily automated using robotic liquid-handling systems that dispense mixtures of the appropriate reagents into the wells of microtitre plates, wherein each well contains a different sample for testing. This will allow the subject invention to be used routinely in clinical diagnosis applications in which a large number of samples must be assayed.
  • the method may be used to determine the identity of one or more pathogenic contaminants in a sample in which it is known that a contaminant is present, but the identity thereof is unknown.
  • the sample would be contacted with a pool of Imprint and Probe Primers, according to the methods described above, in which each primer is specific for a particular pathogen that may be contained in the sample above.
  • the sequences of the Probe Primers would be chosen such that only one nucleotide need be added for Idling at each Probe Primer hybridization site. Idling conditions would be established, and the presence of PPi would be detected. If PPi is detected, then it is known that one or more of the suspected pathogenic contaminants is contained within the sample.
  • the Imprints would then be isolated from the Idling mixture, and contacted with a subset of the initial pool of primer; Idling conditions would again be established, and if PPi is detected, the process would be repeated with pools of progressively decreasing primer complexity. In this way, it is possible to narrow down and ultimately identify the pathogenic contaminant(s) contained within the sample.
  • Target Nucleic Acid and then sequence a further region of the same Target.
  • many viruses are capable of undergoing rapid mutation in selected regions of their genomes, e.g., strain variation in influenza virus and HIV virus.
  • the resulting new variants often have radically different properties, and are resistant to conventional treatments.
  • the current method can be combined with the method of Nyren, et al. (Anal. Biochem. 208:171-175 (1993)) and Ronaghi et al. (Anal. Biochem. 242:84-89 (1996), specifically inco ⁇ orated herein by reference) to first detect a viral Nucleic Acid and then rapidly sequence the variable regions thereof without the need to purify the Target Nucleic Acid and perform conventional Sanger sequencing.

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Abstract

La présente invention concerne un nouveau procédé permettant la détection hautement sélective d'une séquence nucléotidique cible spécifique dans une composition échantillon pouvant contenir un grand nombre d'acides nucléiques. On commence par former une copie de la séquence nucléotidique cible par extension à partir d'une première amorce complémentaire d'une partie de la séquence cible. On hybride alors cette copie de la séquence nucléotidique cible avec une deuxième amorce, la détection de la séquence nucléotidique cible étant basée sur la formation de pyrophosphate et/ou de désoxynucléotide-monophosphate (dNMP).
PCT/US1998/011457 1997-06-06 1998-06-03 Detection d'acide nucleique WO1998055653A1 (fr)

Priority Applications (1)

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AU78136/98A AU7813698A (en) 1997-06-06 1998-06-03 Nucleic acid detection

Applications Claiming Priority (4)

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US4888697P 1997-06-06 1997-06-06
US60/048,886 1997-06-06
US2710798A 1998-02-20 1998-02-20
US09/027,107 1998-02-20

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WO1998055653A1 true WO1998055653A1 (fr) 1998-12-10

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AU (1) AU7813698A (fr)
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000040750A1 (fr) * 1998-12-30 2000-07-13 Gyros Ab Procede de sequençage d'adn a l'aide d'un dispositif microfluidique
WO2000043540A1 (fr) * 1999-01-22 2000-07-27 Pyrosequencing Ab Procede de sequençage d'adn
US6210891B1 (en) * 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
WO2004035818A1 (fr) * 2002-10-21 2004-04-29 Biotage Ab Utilisation d'osmolytes afin de thermostabiliser de la luciferase et/ou apyrase
US6750018B2 (en) * 2000-09-28 2004-06-15 Hitachi, Ltd. Method for detecting nucleic acid mutation by detecting chemiluminiscence generated with by-product of complementary strand extension reaction
EP1435394A1 (fr) * 2002-12-12 2004-07-07 Fuji Photo Film Co., Ltd. Méthode pour mesurer un acide nucléique
EP1435393A1 (fr) * 2002-12-12 2004-07-07 Fuji Photo Film Co., Ltd. Méthode pour analyser l'expression des gènes par quantification d'acide pyrophosphorique
WO2004090167A1 (fr) * 2003-04-14 2004-10-21 Temasek Life Sciences Laboratory Limited Detection d'un acide nucleique cible par reaction de la polymerase, et detection enzymatique de pyrophosphate libere
WO2006010948A1 (fr) * 2004-07-29 2006-02-02 Lumora Limited Procede permettant de determiner la quantite d'acide nucleique matrice present dans un echantillon
US7371545B2 (en) 2003-01-14 2008-05-13 Lumora Limited Method for determining the amount of template nucleic acid present in a sample

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EP0362042A1 (fr) * 1988-09-26 1990-04-04 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procédé d'analyse d'une séquence spécifique d'ADN ou d'ARN, réactifs et nécessaires pour sa mise en oeuvre
US5221736A (en) * 1988-12-21 1993-06-22 Bionebraska, Inc. Sequential peptide and oligonucleotide syntheses using immunoaffinity techniques
US5534424A (en) * 1992-05-12 1996-07-09 Cemu Bioteknik Ab Chemical method for the analysis of DNA sequences

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
EP0362042A1 (fr) * 1988-09-26 1990-04-04 Institut National De La Sante Et De La Recherche Medicale (Inserm) Procédé d'analyse d'une séquence spécifique d'ADN ou d'ARN, réactifs et nécessaires pour sa mise en oeuvre
US5221736A (en) * 1988-12-21 1993-06-22 Bionebraska, Inc. Sequential peptide and oligonucleotide syntheses using immunoaffinity techniques
US5534424A (en) * 1992-05-12 1996-07-09 Cemu Bioteknik Ab Chemical method for the analysis of DNA sequences

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6210891B1 (en) * 1996-09-27 2001-04-03 Pyrosequencing Ab Method of sequencing DNA
US6258568B1 (en) 1996-12-23 2001-07-10 Pyrosequencing Ab Method of sequencing DNA based on the detection of the release of pyrophosphate and enzymatic nucleotide degradation
WO2000040750A1 (fr) * 1998-12-30 2000-07-13 Gyros Ab Procede de sequençage d'adn a l'aide d'un dispositif microfluidique
US6828100B1 (en) 1999-01-22 2004-12-07 Biotage Ab Method of DNA sequencing
WO2000043540A1 (fr) * 1999-01-22 2000-07-27 Pyrosequencing Ab Procede de sequençage d'adn
US6750018B2 (en) * 2000-09-28 2004-06-15 Hitachi, Ltd. Method for detecting nucleic acid mutation by detecting chemiluminiscence generated with by-product of complementary strand extension reaction
WO2004035818A1 (fr) * 2002-10-21 2004-04-29 Biotage Ab Utilisation d'osmolytes afin de thermostabiliser de la luciferase et/ou apyrase
EP1435394A1 (fr) * 2002-12-12 2004-07-07 Fuji Photo Film Co., Ltd. Méthode pour mesurer un acide nucléique
EP1435393A1 (fr) * 2002-12-12 2004-07-07 Fuji Photo Film Co., Ltd. Méthode pour analyser l'expression des gènes par quantification d'acide pyrophosphorique
US7371545B2 (en) 2003-01-14 2008-05-13 Lumora Limited Method for determining the amount of template nucleic acid present in a sample
WO2004090167A1 (fr) * 2003-04-14 2004-10-21 Temasek Life Sciences Laboratory Limited Detection d'un acide nucleique cible par reaction de la polymerase, et detection enzymatique de pyrophosphate libere
WO2006010948A1 (fr) * 2004-07-29 2006-02-02 Lumora Limited Procede permettant de determiner la quantite d'acide nucleique matrice present dans un echantillon
JP2008510455A (ja) * 2004-07-29 2008-04-10 ルモラ・リミテッド 試料中に存在するテンプレート核酸の量を決定するための方法
AU2005266143B2 (en) * 2004-07-29 2011-06-16 Lumora Limited Method for determining the amount of template nucleic acid present in a sample
CN101035908B (zh) * 2004-07-29 2011-11-16 路玛拉有限公司 确定样本中模板核酸数量的方法
US8309308B2 (en) 2004-07-29 2012-11-13 Lumora Limited Method for determining the amount of template nucleic acid present in a sample

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