WO2004067765A2 - Procede pour prendre des empreintes d'organismes a l'aide d'agents de coupure de brin - Google Patents

Procede pour prendre des empreintes d'organismes a l'aide d'agents de coupure de brin Download PDF

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WO2004067765A2
WO2004067765A2 PCT/US2004/002720 US2004002720W WO2004067765A2 WO 2004067765 A2 WO2004067765 A2 WO 2004067765A2 US 2004002720 W US2004002720 W US 2004002720W WO 2004067765 A2 WO2004067765 A2 WO 2004067765A2
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sequence
nicking
strand
oligonucleotide
nucleic acid
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PCT/US2004/002720
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WO2004067765A3 (fr
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Jeffrey Van Ness
David J. Galas
Lori K. Van Ness
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Keck Graduate Institute
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Publication of WO2004067765A3 publication Critical patent/WO2004067765A3/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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention is generally directed to compositions and methods for identifying any type of organism or individual, where the invention is based on creating and analyzing nucleotide sequence information characteristic of nucleic acids present in the organism or individual.
  • Nosocomial (hospital-based) infections have become one of the most serious problems in infectious disease. Staphylococcus aureus is exceeded only by Escherichia coli as a leading cause of nosocomial infections. See, for example, Brumfitt, W. et al., Drugs Exptl. Clin. Res. 76:205-214 (1990).
  • S. aureus methicillin-resistant S. aureus
  • Patients in the intensive care unit are very susceptible to bacterial infections, due to interventions such as respiratory tubes and indwelling catheters. E. coli and S.
  • aureus if introduced into surgical wounds, the blood stream or the urinary tract, cause serious, sometimes life-threatening infections.
  • a partial solution in most "nosocomial outbreaks" is simply identifying the source of the infection. That is, is the infectious agent coming from a common source (e.g., an infected nurse or doctor, or an instrument such as a respirator) or is there some other reason for the sudden emergence of a single type of bacterial infection.
  • Interspersed repetitive DNA sequence elements have been characterized extensively in eucaryotes although their function still remains largely unknown. The conserved nature and interspersed distribution of these repetitive sequences have been exploited to amplify unique sequences between repetitive sequences by the polymerase chain reaction. Additionally, species-specific repetitive DNA elements have been used to differentiate between closely related murine species. Prokaryotic genomes are much smaller than the genomes of mammalian species (approximately 10 6 versus 10 9 base pairs of DNA, respectively). Since these smaller prokaryotic genomes are maintained through selective pressures for rapid DNA replication and cell reproduction the non- coding repetitive DNA should be kept to a minimum unless maintained by other selective forces. For the most part prokaryotes have a high density of transcribed sequences. Nevertheless, families of short intergenic repeated sequences occur in bacteria.
  • repetitive sequences have been demonstrated in many different bacterial species. Reports of novel repeated sequences in the eubacterial genera, Escherichia, Salmonella, Deinococcus, Calothrix, and Neisseria, and the fungi, Candida albicans and Pneumocystis carinii, illustrate the presence of dispersed extragenic repetitive sequences in many organisms.
  • One such family of repetitive DNA sequences in eubacteria is the Repetitive Extragenic Palindromic (REP) elements.
  • the consensus REP sequence for this family includes a 38-mer sequence containing six totally degenerate positions, including a 5 bp variable loop between each side of the conserved stem of the palindrome.
  • the present invention fulfills this and other related needs.
  • the present invention is generally directed to compositions and methods for identifying any type of organism or individual using nucleic acid- based fingerprinting.
  • the method relies on the creation of a family of nucleic acid or oligonucleotide fragments formed by action of nicking agents on a nucleic acid sample.
  • the nicking reaction is preformed in the presence of a polymerase and one or more (preferably all four of the natural) deoxyribonucleoside triphosphates.
  • the nucleic acid or oligonucleotide fragments or portions thereof are created in higher concentration and are therefore more amenable to characterization.
  • a family also referred to herein as a pattern, of nucleic acid or oligonucleotide fragments of known characteristics (e.g., mass/charge ratios) are produced, which identify unambiguously an organism or individual.
  • the readout of the fingerprinting assay is preferably matrix-assisted-laser-desorption ionization (MALDI) or liquid chromatography time-of-flight (LC-TOF) mass spectrometry, however other characterization methods may be used as well.
  • MALDI matrix-assisted-laser-desorption ionization
  • LC-TOF liquid chromatography time-of-flight
  • a method has been devised according to the present invention in which a set of oligonucleotides are linearly amplified from template structures pre-existing in genomic DNA that can be used to initiate their own exponential amplification.
  • a set of oligonucleotides are linearly amplified from template structures pre-existing in genomic DNA that can be used to initiate their own exponential amplification.
  • short oligonucleotides are linearly amplified in the presence of a nicking agent that recognizes the nicking agent recognition sequences.
  • the products from the linear amplification referred to herein as "initiating oligonucleotide" or “initiator,” can then be coupled to a method for exponentially amplifying the initiating oligonucleotides in true chain reactions.
  • the linear and the exponential amplification reactions can be made into a homogenous assay in which 10 8 - 1O 9 — fold amplification can be achieved in as little as 3 minutes.
  • the linear amplification reaction, the exponential amplification, or both may be performed under isothermal conditions (e.g., at 60°C).
  • the exponential or string reaction is composed of two reaction components: a first amplification reaction that replicates the initiating oligonucleotide and a second amplification reaction that replicatess the complement of the initiating oligonucleotide.
  • a first amplification reaction that replicates the initiating oligonucleotide
  • a second amplification reaction that replicatess the complement of the initiating oligonucleotide.
  • two template oligonucleotides are used, a first template that comprises a sequence complementary to the initiating oligonucleotide, and a second template that comprises a sequence complementary to the complement of the initiating oligonucleotide.
  • the first template may anneal to the complement of the initiating oligonucleobe and be used as a template for amplifying the initiating oligonucleotide
  • the second template may anneal to the initiating oligonucleotide and be used as a template for amplifying the complement of the initiating oligonucleotide
  • a useful example is taken from E. coli K12 in which 55 unique oligonucleotides can be generated from genomic DNA without the use of pre- synthesized probes or primers.
  • the read-out is ideally done by mass spectrometry (LC-TOF or MALDI) but can also be accomplished by other means, e.g., using real-time fluorimetry or "self-amplifying arrays". Foreknowledge of the sequence of the individual or organism is not necessary as it is possible to generate the fragments de novo from genomic DNA.
  • the methods described here permit the creation an assay panel of diagnostic oligonucleotides that can identify any organism or individual.
  • the present invention is advantageous over previous methods for identifying bacterial species.
  • the present invention provides a novel approach to using nicking agent recognition sites within a genomic DNA to directly fingerprint bacterial (as well as viral, fungal, in fact, all prokaryotic and eukaryotic genomes).
  • the unique patterns of oligonucleotides generated by a nicking agent recognition sequence identify different bacterial species and strains.
  • the present invention may produce polymorphic oligonucleotide fragments that contain genetic variations (e.g., single nucleotide polymorphisms, deletions, insertions, variable repeats) from eukaryotic genomes. The characterization of these polymorphic oligonucleotide fragments enables the identification of individual organism from which a nucleic acid sample is obtained.
  • the present invention provides a method comprising: a) providing a nucleic acid sample; b) treating the nucleic acid sample with components under nicking conditions, where the components comprise: i) a nicking agent; and the conditions cause the nicking agent to nick the nucleic acid sample to thereby produce a family of initiating oligonucleotide fragments; c) subjecting one or more members of the family of initiating oligonucleotide fragments to a characterization process to thereby provide results; and d) identifying a source for the nucleic acid sample based on the results of the characterization process.
  • the components of the above method further comprise: ii) a polymerase; and iii) a deoxyribonucleoside triphosphate.
  • the components of the above methods may further comprise: iv) a template oligonucleotide comprising from 3' to 5':
  • A a first nucleotide sequence that is at least substantially complementary to a nucleotide sequence present in one or more members of the family of initiating oligonucleotide fragments;
  • (C) a second nucleotide sequence.
  • the template is immobilized.
  • sequence iv)(B) is a sequence of an antisense strand of the nicking agent recognition sequence
  • sequence iv)(A) is at least substantially identical to sequence iv)(C).
  • the components of the above methods may further comprising v) a second template oligonucleotide, comprising, from 3' to 5':
  • A a first nucleotide sequence that is at least substantially identical to a nucleotide sequence present in the one or more members of the family of initiating oligonucleotide fragments;
  • sequence iv)(B) is a sequence of a sense strand of the nicking agent recognition sequence.
  • sequence iv)(A) is exactly identical to sequence iv)(C).
  • template iv), template v), or both may be immobilized.
  • the components may further comprise a restriction endonuclease.
  • the one or more members of the family of initiating oligonucleotide fragments are 6-16 nucleotides in length.
  • the nicking agent is a nicking endonuclease, such as N.BstNB I or N.AIw I.
  • the polymerase is exo " Vent polymerase,
  • the characterization process is performed at least partially by a technique selected from the group consisting of luminescence spectroscopy or spectrometry, fluorescence spectroscopy or spectrometry, mass spectrometry (such as MALDI or LC-TOF), liquid chromatography, fluorescence polarization, and electrophoresis.
  • a technique selected from the group consisting of luminescence spectroscopy or spectrometry, fluorescence spectroscopy or spectrometry, mass spectrometry (such as MALDI or LC-TOF), liquid chromatography, fluorescence polarization, and electrophoresis.
  • the present invention provides a template oligonucelotide for amplifying a portion of a target nucleic acid, wherein
  • the portion of the target is 6-16 nucleotides in length and flanked by
  • (C) a second nucleotide sequence that is at least substantially identical to the first nucleotide sequence.
  • the first and third nicking enzyme recognition sequences are identical to each other.
  • the first, second and third nicking enzyme recognition sequences are identical to each other.
  • some or all of the nicking enzyme recognition sequences are recognizable by N.BstNB I.
  • the 3' terminus of the template oligonucleotide is blocked.
  • the 3' terminus of the template oligonucleotide is immobilized.
  • the portion of the target is selected from the products listed in Tables 1 , 2, 4, 5, and 9.
  • the portion of the target comprises a genetic variation (e.g ⁇ single nucleotide polymorphism).
  • the present invention provides a composition for amplifying a portion of a target nucleic acid, comprising a first template oligonucleotide and a second template oligonucleotide, wherein
  • the portion of the target is 6-16 nucleotides in length and flanked by
  • the first template oligonucleotide comprises from 5' to 3': (A) a first nucleotide sequence,
  • nicking enzyme recognition sequence (B) a sequence of a sense strand of a fourth nicking agent recognition sequence, and (C) a second nucleotide sequence that is at least substantially identical to the portion of the target.
  • the first, third and fourth nicking enzyme recognition sequences are identical to each other.
  • the first, second, third and fourth nicking enzyme recognition sequences are identical to each other. In certain embodiments, some or all of the nicking enzyme recognition sequence are recognizable by N.BstNB I.
  • the 3' termini of the first and second templates are blocked.
  • the 3' terminus of the first, the 3' terminus of the second, or both termini are immobilized.
  • the portion of the target is selected from the products listed in Tables 1 , 2, 4, 5 and 9.
  • the portion of the target comprises a genetic variation (e.g., single nucleotide polymorphism).
  • the present invention provides a composition comprising:
  • the nicking agent is N.BstNB I or N.AIw I.
  • the composition may further comprise a restriction endonuclease selected from Bel I, Bsa Bl, Bsm I, Bsr I and Bsr D1 , wherein the at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50 fragments are selected from the products listed in Table 9.
  • the present invention provides a kit for identifying the source of a nucleic acid sample, comprising one or two template oligonucleotides as described above for amplifying a portion of a genomic DNA of an organism suspected to be the source of the nucleic acid sample, wherein the portion of the genomic DNA is 6-16 nucleotides in length and flanked by
  • A a sequence of one strand of a first nicking enzyme recognition sequence
  • B a sequence of one strand of a second nicking enzyme recognition sequence, or a sequence of one strand of a restriction enzyme recognition sequence.
  • the kit may further comprise a nicking enzyme that recognizes one or more nicking enzyme recognition sequences in the template(s).
  • kits may further comprise a DNA polymerase and/or one or more deoxyribonucleoside triphosphate.
  • the portion of the genomic DNA is selected from the products listed in Tables 1 , 2, 4, 5 and 9.
  • the portion of the genomic DNA comprises a single nucleotide polymorphism.
  • the present invention provides an array, comprising
  • Figure 1 schematically shows the cycle of the synthesis and release of an amplified short oligonucletide.
  • the recognition site for the enzyme N.BstNB I (5'-GAGTC-3') and the specific nicking site four bases downstream on this strand.
  • the oligonucleotide produced is indicated in blue, the primer in green and the template in red.
  • the lengths of the exemplary template and amplified oligonucleotides are shown in the upper left drawing.
  • Figure 2 is a diagram of the reaction scheme for the exponential amplification of oligonucleotides.
  • the segments in red represent the sequence complement of the oligonucleotide sequence to be amplified, the signal sequence (shown in blue).
  • the amplification template, ⁇ consists of two copies of the signal complement flanking the nicking enzyme recognition site shown as a light blue box, and a spacer sequence, shown as a green segment.
  • the signal oligonucleotide (labeled ⁇ ) is produced in the linear amplification cycle for each amplification template created.
  • Figure 3 is a schematic representation of a template oligonucleotide used in a replicator type of amplification reactions.
  • Figures 4a and 4b show the time of flight spectra of the multiplexed amplification reaction in the presence of short oligonucleotides generated from the genomic DNA of the E. coli strains K12 and 0157, respectively.
  • Figure 5a and 5b show the MALDI spectra of the multiplexed amplification reaction in the presence of short oligonucleotides generated from the genomic DNA of the E. coli strains K12 and O157, respectively.
  • Figure 6 shows real time fluorescence detection of the oligonucleotide amplification by an MJ Opticon I.
  • the time of amplification is plotted on the X axis versus accumulated fluorescence on the Y axis.
  • Each curve from left to right represents a serial dilution of 3-fold.
  • the starting concentration of the trigger was 0.01 picomoles/microliter and the last dilution (far right curve (bottom curve on figure)) was 1.9 x 10 "7 picomoles/microliter. This represents a dilution range of about 20,000-fold (3 9 ).
  • Figure 7 is a schematic diagram showing the ping-pong amplification reaction cycle.
  • Figure 8 is a schematic diagram showing the application of the ping-pong amplification reaction cycle in discriminating genetic variations.
  • the present invention provides methods for identifying any type of organism or individual using polynucleotide-based fingerprinting.
  • the method relies on the creation of a family of polynucleotides formed by action of nicking agents on a nucleic acid sample.
  • a nucleic acid sample may be nicked by a nicking agent to produce various nicked nucleic acid fragments.
  • the resulting nicked fragments are then characterized to determine the identity of the organism from which the nucleic acid sample was obtained or derived.
  • a nucleic acid sample may be nicked by a nicking agent in the presence of a DNA polymerase.
  • the presence of the DNA polymerase allows linear amplification of the nicked fragments or portions thereof, thus facilitates the characterization of such fragments.
  • These fragments may be further amplified by coupling the linear amplification reaction with an exponential amplification reaction.
  • Such an exponential amplification greatly increases the speed and the sensitivity of the identification methods.
  • the nicked fragments may be characterized to determine the presence or absence of a particular fragment unique, or characteristic, to a specific species, subspecies, or strain. The conclusion made from the presence or absence of that particular fragment may be further verified by determining the presence or absence of one or more other fragments also unique, or characteristic, to the specific species, subspecies, or strain.
  • the presence or absence of particular fragments in a nicking reaction mixture of a nucleic acid sample need not be determined. Rather, the pattern formed by the resulting nicked fragments (e.g., a mass spectrum of the nicked fragments) is characterized and compared with a standard pattern known for a particular species, subspecies, or strain.
  • the standard pattern may be generated by performing the nicking reaction with a nucleic acid sample from the particular species, subspecies or strain under conditions identical to that of the nucleic acid sample.
  • the standard pattern may be generated based on the known nucleic acid sequence of the particular species, subspecies, or strain.
  • the present invention uses the presence of closely located nicking agent recognition sequences.
  • short oligonucleotide fragments e.g., 6-16 nucleotides long
  • Short oligonucleotide fragments may also be generated or amplified by the use of a nicking agent in combination with a restriction enzyme if one or more recognition sequences of the nicking agent are located closely to the recognition sequence(s) of the restriction enzyme.
  • the resulting short oligonucleotide fragments may be easily separated from other larger fragments. Such separation simplifies the pattern generated from the characterization of the nucleic acid fragments in a nicking reaction mixture.
  • short oligonucleotide fragments may easily be exponentially amplified to increase the speed and sensitivity of the present methods. Short oligonucleotide fragments are also more suitable to certain characterization technologies, such as LC-TOF and MALDI. In some embodiments, the nicked fragments may also contain genetic variations useful to distinguish among individual organisms.
  • Fingerprinting refers to the identification of a source of nucleic acid based on analysis of the nucleic acid according to the methods described herein. For instance, fingerprinting may be applied to the identification of a bacterial strain from its characteristic pattern of oligonucleotides produced by action of a nicking agent (e.g., N.BstNB I). This characteristic pattern is the strain's genomic "fingerprint", which is determined by the sequence of the strain's genomic DNA.
  • a nicking agent e.g., N.BstNB I
  • nucleic acid when a location in a nucleic acid is "5' to” or “5' of a reference nucleotide or a reference nucleotide sequence, this means that it is between the 5' terminus of the reference nucleotide or the reference nucleotide sequence and the 5' phosphate of that strand of the nucleic acid. Further, when a nucleotide sequence is "directly 3' to” or “directly 3' of a reference nucleotide or a reference nucleotide sequence, this means that the nucleotide sequence is immediately next to the 3' terminus of the reference nucleotide or the reference nucleotide sequence.
  • nicking refers to the cleavage of only one strand of a fully double-stranded nucleic acid molecule or a double-stranded portion of a partially double-stranded nucleic acid molecule at a specific position relative to a nucleotide sequence that is recognized by the enzyme that performs the nicking.
  • the specific position where the nucleic acid is nicked is referred to as the "nicking site" (NS).
  • NA nicking agent
  • Nicking agents include, but are not limited to, a nicking endonuclease (e.g., N.BstNB I) and a restriction endonuclease (e.g., Hinc II) when a completely or partially double-stranded nucleic acid molecule contains a hemimodified recognition/cleavage sequence in which one strand contains at least one derivatized nucleotide(s) that prevents cleavage of that strand (i.e., the strand that contains the derivatized nucleotide(s)) by the restriction endonuclease.
  • a nicking endonuclease e.g., N.BstNB I
  • a restriction endonuclease e.g., Hinc II
  • NE nicking endonuclease
  • a NE Unlike a restriction endonuclease (RE), which requires its recognition sequence to be modified by containing at least one derivatized nucleotide to prevent cleavage of the derivatized nucleotide-containing strand of a fully or partially double-stranded nucleic acid molecule, a NE typically recognizes a nucleotide sequence composed of only native nucleotides and cleaves only one strand of a fully or partially double-stranded nucleic acid molecule that contains the nucleotide sequence.
  • RE restriction endonuclease
  • nucleotide refers to adenylic acid, guanylic acid, cytidylic acid, thymidylic acid or uridylic acid.
  • a "derivatized nucleotide” is a nucleotide other than a native nucleotide.
  • NARS nicking agent recognition sequence
  • RERS Restriction endonuclease recognition sequence
  • a “hemimodified RERS,” as used herein, refers to a double-stranded RERS in which one strand of the recognition sequence contains at least one derivatized nucleotide (e.g., ⁇ -thio deoxynucleotide) that prevents cleavage of that strand (i.e., the strand that contains the derivatized nucleotide within the recognition sequence) by a RE that recognizes the RERS.
  • derivatized nucleotide e.g., ⁇ -thio deoxynucleotide
  • a NARS is a double-stranded nucleotide sequence where each nucleotide in one strand of the nucleotide is complementary to the nucleotide at its corresponding position in the other strand.
  • the nucleotide of a NARS in the strand containing a NS nickable by a NA that recognizes the NARS is referred to as a "sequence of the sense strand of the NARS” or a "sequence of the sense strand of the double-stranded NARS,” while the nucleotide of the NARS in the strand that does not contain the NS is referred to as a "sequence of the antisense strand of the NARS" or a "sequence of the antisense strand of the double-stranded NARS.”
  • a NERS is a double- stranded nucleotide sequence of which one strand is exactly complementary to the other strand
  • the nucleotide of a NERS located in the strand containing a NS nickable by a NE that recognizes the NERS is referred to as a "sequence of a sense strand of the NERS” or a "sequence of the sense strand of the double- stranded NERS”
  • the nucleotide of the NERS located in the strand that does not contain the NS is referred to a "sequence of the antisense strand of the NERS" or a "sequence of the antisense strand of the double-stranded NERS.”
  • the recognition sequence and the nicking site of an exemplary nicking endonuclease, N.BstNB I are shown below with V to indicate the cleavage site and N to indicate any nucleotide:
  • the sequence of the sense strand of the N.BstNB I recognition sequence is 5'- GAGTC-3', whereas that of the antisense strand is 5'-GACTC-3'.
  • the sequence of a hemimodified RERS in the strand containing a NS nickable by a RE that recognizes the hemimodified RERS is referred to as "the sequence of the sense strand of the hemimodified RERS” and is located in "the sense strand of the hemimodified RERS” of a hemimodified RERS- containing nucleic acid
  • the sequence of the hemimodified RERS in the strand that does not contain the NS i.e., the strand that contains derivatized nucleotide(s)
  • the sequence of the antisense strand of the hemimodified RERS is located in "the antisense strand of the hemimodified RERS" of a hemimodified RERS-containing nucleic acid.
  • a NARS is an at most partially double-stranded nucleotide sequence that has one or more nucleotide mismatches, but contains an intact sense strand of a double-stranded NARS as described above.
  • the hybridized product includes a NARS, and there is at least one mismatched base pair within the NARS of the hybridized product, then this NARS is considered to be only partially double- stranded.
  • NARSs may be recognized by certain nicking agents (e.g., N.BstNB I) that require only one strand of double-stranded recognition sequences for their nicking activities.
  • N.BstNB I may contain, in certain embodiments, an intact sense strand, as follows,
  • N indicates any nucleotide
  • N at one position may or may not be identical to N at another position, however there is at least one mismatched base pair within this recognition sequence.
  • the NARS will be characterized as having at least one mismatched nucleotide.
  • a NARS is a partially or completely single-stranded nucleotide sequence that has one or more unmatched nucleotides, but contains an intact sense strand of a double-stranded NARS as described above.
  • the hybridized product includes a nucleotide sequence in the first strand that is recognized by a NA, i.e., the hybridized product contains a NARS, and at least one nucleotide in the sequence recognized by the NA does not correspond to, i.e., is not across from, a nucleotide in the second strand when the hybridized product is formed, then there is at least one unmatched nucleotide within the NARS of the hybridized product, and this NARS is considered to be partially or completely single-stranded.
  • NARSs may be recognized by certain nicking agents (e.g., N.BstNB I) that require only one strand of double-stranded recognition sequences for their nicking activities.
  • N.BstNB I may contain, in certain embodiments, an intact sense strand, as follows,
  • N indicates any nucleotide, 0-4 indicates the number of the nucleotides "N," a "N” at one position may or may not be identical to a “N” at another position), which contains the nucleotide of the sense strand of the double-stranded recognition sequence of N.BstNB I.
  • at least one of G, A, G, T or C is unmatched, in that there is no corresponding nucleotide in the complementary strand. This situation arises, e.g., when there is a "loop" in the hybridized product, and particularly when the sense sequence is present, completely or in part, within a loop.
  • nucleic acid amplification reaction refers to the process of making more than one copy of a nucleic acid molecule (A) using a nucleic acid molecule (T) that comprises a sequence complementary to the nucleotide of nucleic acid molecule A as a template.
  • a first nucleic acid sequence is "at least substantially identical" to a second nucleic acid sequence when the complement of the first sequence is able to anneal to the second sequence to form at least a transient duplex under certain reaction conditions (e.g., conditions for amplifying nucleic acids).
  • the first sequence is exactly identical to the second sequence, that is, the nucleotide of the first sequence at each position is identical to the nucleotide of the second sequence at the same position, and the first sequence is of the same length as the second sequence.
  • a first nucleic acid sequence is "at least substantially complementary" to a second nucleic acid sequence when the first sequence is able to anneal to the second sequence to form at least a transient duplex under certain reaction conditions (e.g., conditions for amplifying nucleic acids).
  • the first sequence is exactly or completely complementary to the second sequence, that is, each nucleotide of the first sequence is complementary to the nucleotide of the second sequence at its corresponding position, and the first sequence is of the same length as the second sequence.
  • a transient duplex between a first nucleic acid sequence and a second nucleic acid sequence is formed when under given reaction conditions, the 3' terminal group of the first nucleic acid sequence (if unblocked) may be extended by a DNA polymerase using the second nucleic acid sequence as a template; or the 3' terminal group of the second nucleic acid sequence (if unblocked) may be extended by a DNA polymerase using the first nucleic acid sequence as a template.
  • at least 80% of the nucleotides of the first nucleic acid in a region of at least 8 nucleotides are complementary to the nucleotides of the second nucleic acid at their corresponding positions.
  • At least 85%, 90%, 95%, 97%, 98%, or 99% of the nucleotides of the first nucleic acid in a region of at least 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, or 18 nucleotides are complementary to the nucleotides of the second nucleic acid at their corresponding positions.
  • a nucleotide in one strand (referred to as the "first strand") of a double-stranded nucleic acid located at a position "corresponding to" another position (e.g., a defined position) in the other strand (referred to as the "second strand") of a double-stranded nucleic acid refers to the nucleotide in the first strand that is complementary to the nucleotide at the corresponding position in the second strand.
  • a position in one strand (referred to as the "first strand") of a double-stranded nucleic acid corresponding to a nicking site within the other strand (referred to as the "second strand”) of a double- stranded nucleic acid refers to the position between the two nucleotides in the first strand complementary to those in the second strand between which nicking occurs.
  • isothermal conditions refers to a set of reaction conditions where the temperature of the reaction is kept essentially constant (i.e., at the same temperature or within the same narrow temperature range wherein the difference between an upper temperature and a lower temperature is no more than about 20°C) during the course of the amplification.
  • a reaction is carried out under conditions where the difference between an upper temperature and a lower temperature is no more than 15°C, 10°C, 5°C, 3°C, 2°C or 1°C.
  • Exemplary temperatures for isothermal amplification include, but are not limited to, any temperature between 50°C to 70°C or the temperature range between 50°C to 70°C, 55°C to 70°C, 60°C to 70°C, 65°C to 70°C, 50°C to 55°C, 50°C to 60°C, or 50°C to 65°C.
  • the terms "polymorphism” and “genetic variation,” as used herein, refer to the occurrence of two or more genetically determined alternative sequences or alleles in a small region (i.e., one to several (e.g., 2, 3, 4, 5, 6, 7, or 8) nucleotides in length) in a population. The allelic form occurring most frequently in a selected population is referred to as the wild type form. Other allelic forms are designated as variant forms. Diploid organisms may be homozygous or heterozygous for allelic forms.
  • the genetic variation is a "single- nucleotide polymorphism" (SNP), which refers to any single nucleotide sequence variation, preferably one that is common in a population of organisms and is inherited in a Mendelian fashion.
  • SNP single- nucleotide polymorphism
  • the SNP is either of two possible bases and there is no possibility of finding a third or fourth nucleotide identity at an SNP site.
  • Sample sources Biological samples of the present invention include any sample that originates from an organism and that may contain a nucleic acid of interest (i.e., target nucleic acid). They may be provided by obtaining a blood sample, biopsy specimen, tissue explant, organ culture or any other tissue or cell preparation from a subject or a biological source.
  • the subject or biological source may be a human or non-human animal, a plant, a fungus, a bacterium, and virus.
  • the subject or biological source may be suspected of having, or being at risk for having, a genetic disease or a pathogen infection.
  • the subject or biological source may be a patient that has a genetic disease or a pathogen infection.
  • the subject or biological source may be a control subject that does not have a genetic disease or a pathogen infection.
  • a bacterial sample can be utilized as starting material, provided it contains or is suspected of containing a bacterial genome of interest.
  • a sample may be obtained from any source that may potentially be contaminated by bacteria.
  • the sample to be tested can be selected or extracted from any bodily sample such as blood, urine, spinal fluid, tissue, vaginal swab, stool, amniotic fluid or buccal mouthwash.
  • the sample can come from a variety of other sources.
  • the sample can be from a plant, fertilizer, soil, liquid or other horticultural or agricultural product.
  • the sample can be from fresh food or processed food (for example infant formula, seafood, fresh produce and packaged food).
  • the sample can be from liquid, soil, sewage treatment, sludge and any other sample in the environment considered or suspected of being contaminated by bacteria.
  • the sample is a mixture of material for example blood, soil and sludge
  • it can be treated with an appropriate reagent effective to open the cells and expose or separate the strands of nucleic acids.
  • this lysing and nucleic acid denaturing step will allow amplification to occur more readily.
  • the bacteria can be cultured prior to analysis and thus a pure sample obtained.
  • fingerprinting genomic DNA may also be used to characterize other DNA molecules (e.g., cDNA).
  • the methods according to the present invention may also be applicable to characterize cDNA expression patterns.
  • the nucleic acids isolated from a biological source may be directly used in a nicking reaction. Alternatively, they may be amplified via known methods (such as PCR) prior to being subjected to action of a nicking agent.
  • a nicking agent may be a nicking endonuclease (used interchangeably with “nicking enzyme") or a restriction endonuclease (used interchangeably with “restriction enzyme”).
  • a nicking endonuclease (NE) useful in the present invention may or may not have a nicking site that overlaps with its recognition sequence.
  • An exemplary NE that nicks outside its recognition sequence is N.BstNB I, which recognizes a unique nucleic acid sequence composed of 5'-GAGTC-3', but nicks four nucleotides beyond the 3' terminus of the recognition sequence.
  • the recognition sequence and the nicking site of N.BstNB I are shown below with V to indicate the cleavage site where the letter N denotes any nucleotide:
  • N.BstNB I may be prepared and isolated as described in U.S. Pat. No. 6,191 ,267, incorporated herein by reference in its entirety. Buffers and conditions for using this nicking endonuclease are also described in the '267 patent.
  • An additional exemplary NE that nicks outside its recognition sequence is N.Alwl, which recognizes the following double-stranded recognition sequence:
  • N.Alwl The nicking site of N.Alwl is also indicated by the symbol V'. Both NEs are available from New England Biolabs (NEB). N.Alwl may also be prepared by mutating a type I Is RE Alwl as described in Xu et al. (Proc. Natl. Acad. Sci. USA 98:12990-5, 2001).
  • NEs that nick within their NERSs include N.BbvCI-a and N.BbvCI-b.
  • the recognition sequences for the two NEs and the NSs are shown as follows:
  • Both NEs are available from NEB.
  • nicking endonucleases include, without limitation, N.BstSE I (Abdurashitov et al., Mol. Biol. (Mosk) 30: 1261-7, 1996), an engineered EcoR V (Stahl et al., Proc. Natl. Acad. Sci. USA 93: 6175-80, 1996), an engineered Fok I (Kim et al., Gene 203: 43-49, 1997), endonuclease V from Thermotoga maritima (Huang et al., Biochem.
  • Additional NEs may be obtained by engineering other restriction endonuclease, especially type lls restriction endonucleases, using methods similar to those for engineering EcoR V, Alwl, Fok I and/or Mly I.
  • a restriction endonuclease useful as a nicking agent can be any restriction endonuclease (RE) that nicks a double-stranded nucleic acid at its hemimodified recognition sequences.
  • Exemplary REs that nick their double- stranded hemimodified recognition sequences include, but are not limited to Ava I, Bsl I, BsmA I, BsoB I, Bsr I, BstN I, BstO I, Fnu4H I, Hinc II, Hind II and Nci I. Additional REs that nick a hemimodified recognition sequence may be screened by the strand protection assays described in U.S. Pat. No. 5,631 ,147.
  • a nicking agent may recognize a nucleotide sequence in a DNA-RNA duplex and nicks in one strand of the duplex. In certain other embodiments, a nicking agent may recognize a nucleotide sequence in a double-stranded RNA and nicks in on strand of the RNA.
  • nicking agents require only the presence of the sense strand of a double-stranded recognition sequence in an at least partially double- stranded substrate nucleic acid for their nicking activities.
  • N.BstNB I is active in nicking a substrate nucleic acid that comprises, in one strand, the sequence of the sense strand of its recognition sequence "5 - GAGTC-3'" of which one or more nucleotides do not form conventional base pairs (e.g., G:C, A:T, or A:U) with nucleotides in the other strand of the substrate nucleic acid.
  • N.BstNB I decreases with the increase of the number of the nucleotides in the sense strand of its recognition sequence that do not form conventional base pairs with any nucleotides in the other strand of the substrate nucleic acid. However, even if none of the nucleotides of "5'-GAGTC-3"' form conventional base pairs with the nucleotides in the other strand, N.BstNB I may still retain 10-20% of its optimum activity. Several factors may be considered when choosing a particular nicking agent for a fingerprinting assay according to the present invention.
  • nicking agent a nicking enzyme that would produce short unique oligonucleotides may be desirable.
  • a nicking enzyme with an optimum temperature similar to that of the DNA polymerase may be desirable.
  • the nicking reaction may be simply performed by incubating the nucleic acid sample with a nicking agent under appropriate conditions. Identifying such appropriate conditions are within the ordinary skill in the art. For instance, the nicking reaction may be performed at the optimum temperature of the nicking agent and in a buffer suitable for the nicking agent.
  • the nicking reaction mixture that contains nicked nucleic acid fragments may be directly characterized. The characterization may be performed by any known applicable methods, including but not limited to, liquid chromatography, electrophoresis, hybridization and mass spectrometry. The use of such methods may indicate the presence or absence of one or more particular fragments unique to a species, subspecies, strain, or individual organism from which the nucleic acid sample is suspected to be. Alternatively, the use of such methods produces a pattern of nicked fragments, which may be compared with the pattern generated from an organism from which the nucleic acid sample is suspected to be.
  • the nicking reaction is performed in the presence of a DNA polymerase so that nicked fragments or portions thereof may be linearly amplified.
  • the amplification produces a larger amount of single-stranded nucleic acid or oligonucleotides, which increases the sensitivity of the fingerprinting assays.
  • the 3' terminus at the nicking site is extended by a DNA polymerase, preferably being 5'->3' exonuclease deficient and having a strand displacement activity and/or in the presence of a strand displacement facilitator, displacing the strand that contains the 5' terminus produced by the nicking reaction.
  • the resulting extension product having a recreated NARS for the NA is nicked ("re-nicked") by the NA.
  • the 3' terminus produced at the NS by the re-nicking is then extended in the presence of the DNA polymerase, also displacing the strand that contains the 5' terminus produced by the nicking reaction.
  • the nicking-extension cycle is repeated, preferably multiple times, to accumulate/amplify the displaced strand that contains the 5' terminus produced by the nicking reaction.
  • DNA polymerases useful in the present invention may be any DNA polymerase that is 5'- ⁇ 3' exonuclease deficient but has a strand displacement activity.
  • DNA polymerases include, but are not limited to, exo " Deep Vent, exo " Bst, exo " Pfu, and exo " Bca.
  • Additional DNA polymerases useful in the present invention may be screened for or created by the methods described in U.S. Pat. No. 5,631 ,147, incorporated herein by reference in its entirety.
  • the strand displacement activity may be further enhanced by the presence of a strand displacement facilitator as described below.
  • a DNA polymerase that does not have a strand displacement activity may be used.
  • DNA polymerases include, but are not limited to, exo " Vent, Taq, the Klenow fragment of DNA polymerase I, T5 DNA polymerase, and Phi29 DNA polymerase.
  • the use of these DNA polymerases requires the presence of a strand displacement facilitator.
  • a "strand displacement facilitator" is any compound or composition that facilitates strand displacement during nucleic acid extensions from a 3' terminus at a nicking site catalyzed by a DNA polymerase.
  • Exemplary strand displacement facilitators useful in the present invention include, but are not limited to, BMRF1 polymerase accessory subunit (Tsurumi et al., J. Virology 67: 7648-53, 1993), adenovirus DNA-binding protein (Zijderveld and van der Vliet, J. Virology 68: 1158-64, 1994), herpes simplex viral protein ICP8 (Boehmer and Lehman, J. Virology 67: 711-5, 1993; Skaliter and Lehman, Proc. Natl. Acad. Sci. USA 91: 10665-9, 1994), single-stranded DNA binding protein (Rigler and Romano, J. Biol. Chem.
  • trehalose is present in the amplification reaction mixture.
  • Additional exemplary DNA polymerases useful in the present invention include, but are not limited to, phage M2 DNA polymerase (Matsumoto et al., Gene 84: 247, 1989), phage PhiPRDI DNA polymerase (Jung et al., Proc. Natl. Acad. Sci.
  • a DNA polymerase that has a 5'->3' exonuclease activity may be used.
  • such a DNA polymerase may be useful for amplifying short nucleic acid fragments that automatically dissociate from the template nucleic acid after nicking.
  • a RNA-dependent DNA polymerase may be used.
  • a DNA-dependent DNA polymerase that extends from a DNA primer such as Avian Myeloblastosis virus reverse transcriptase (Promega) may be used.
  • a target mRNA need not be reverse transcribed into cDNA and may be directly mixed with a template nucleic acid molecule that is at least substantially complementary to the target mRNA.
  • oligonucleotide fragments e.g., 6-20 nucleotides in length.
  • short oligonucleotides are more suitable to certain characterization technologies, such as LC-TOF and MALDI.
  • short fragments may be easily and more efficiently amplified to increase the speed and sensitivity of the present methods.
  • Such fragments also allow the use of a DNA polymerase that does not have a strand displacement activity, or is not 5' to 3' exonuclease deficient.
  • nicking agent recognition sequences or one nicking agent recognition sequence and one restriction enzyme recognition sequence
  • the proximity is about 12 to 24 nucleotides.
  • N can be any nucleotide
  • the number of Ns in the upper strand that is between the sequence of the sense strand of the N.BstNB I recognition sequence (i.e., 5'-GAGTC-3') and the sequence of the antisense strand of the recognition sequence (i.e., 5'-GACTC-3') may be between 12 and 24.
  • the recessed 3'-hydroxyl of each amplification template is filled in by the polymerase, the nicking enzyme then again cleaves the newly extended strand, the resulting short single-stranded oligonucleotide immediately dissociates, and the cycle of nicking and filling is repeated multiple ⁇ times, resulting a linear amplification of the short single-stranded oligonucleotides.
  • the above reaction synthesizes short oligonucleotides whose cycle of reactions depends on the idea that, at the reaction temperature, oligonucleotides above a certain length form stable duplexes, while those below this length form unstable duplexes that dissociate readily.
  • the short oligonucleotide generated in the nicking reaction is below the threshold of stability, and is thereby released from the duplex.
  • the release of the short oligonucleotide from the duplex regenerates a 5' overhang, which may be again used as a template for synthesizing the short oligonucleotide.
  • a schematic representation of a linear amplification of short oligonucleotides using an N.BstNB I as the nicking agent is shown in Figure 1.
  • the identification of one or more pairs of nicking agent recognition sequence (or one nicking agent recognition sequence and one restriction enzyme recognition sequence) in close proximity in a genomic DNA with a known sequence may be performed by the use of applicable computer programs.
  • Such identification may facilitate the selection of a nicking agent for fingerprinting a nucleic acid sample suspected to be derived from the genomic DNA, as well as the identification of oligonucleotide fragments or patterns thereof that are unique to the genomic DNA.
  • These unique oligonucleotide fragments or patterns thereof expected to be present in a fingerprinting assay according to the present invention may be used as a standard for those generated from the nucleic acid sample. The comparison of the expected unique oligonucleotide fragments or patterns thereof with those generated from the nucleic acid sample would indicate whether the nucleic acid sample is derived from the genomic DNA.
  • Genome Identifier through a Nicking-enzyme generated Unique Mass Spectrum (GINUMS).
  • GINUMS further predicts the amplified short oligonucleotides of a genomic sequence produced in the linear amplification reactions described herein.
  • the search begins with the acquisition of the genomic sequence.
  • the analysis is simplified with "finished" genomic sequence, which is represented by one long sequence, rather than "draft" quality sequence, which is represented by a series of sequences, each a fragment of the overall sequence separated by gaps, although the analysis will work with any sequence or set of sequences.
  • sequence is stored in FASTA (or Pearson) format, but the program will work with any sequence format.
  • the sequence is read from either a file of a database, and stored into memory so that it may be searched later.
  • GINUMS searches the genomic sequence for three different patterns, each represented by a regular expression.
  • Regular expressions are simply patterns represented by a string of characters that are used to search text, in this case one long string of A's, C's, G's and T's representing one strand of the genomic sequence.
  • the three regular expressions are:
  • GINUMS searches the genomic sequence with each of the regular expressions one at a time, and then stores all of the matching segments of sequence (termed "hits" for the rest of this document) for each search into separate lists. Thus, all of the hits are composed of the recognition sequences separated by any 14 to 24 base pairs, which are determined by the genomic sequence.
  • the program has three lists of hits in memory, each list the product of searching the genomic sequence with one of the regular expressions. For each hit in all of the lists, GINUMS will determine the "product(s)" of the reaction (the sequence that would be amplified), the mass of the product(s) and the starting and ending position of that product in the genomic sequence.
  • the program searches genomic sequence for target sequences (that is, any sequence beginning with GGATC and ending with GACTC, separated by any 14 to 24 nucleotides).
  • the '.' character represents any one character, in this case any one A, T, C, or G. So, one way to search for each of these occurrences would be to construct 11 regular expressions and then search the sequence for each, such as:
  • GGATC GACTC 14 ,'s
  • GGATC GACTC 15.'s
  • the regular expression represents any string of any 2- 4 characters.
  • this regular expression represents any string that begins with GGATC, and ends with GACTC, separated by at least $min characters and at most $max characters, where $min and $max are integers, and $min ⁇ $max. Because the brackets are preceded by a '.', these can be any characters. Building the profiles for more than one organism is a simple as repeating the process for as many genomic sequences necessary, noting which masses where derived from which organism.
  • GINUMS is capable of inputting the genomic sequences of an unlimited number of organisms, each contained within one file, all contained in the same directory, and then returning two outputs.
  • the first output is a set of masses unique to each organism. This can be accomplished by searching for the existence of each mass in one organism in all of the masses for the other organisms. If that mass is found only in one organism, then it is unique for that organism.
  • GINUMS does not do this precisely (it uses the properties of a data structure called a hash), but the process is analogous.
  • the second output is the complete list of masses for each organism, and would allow the user to determine if each organism has a distinct "profile" (hence, the name), or set, of masses. These could be written either to file or into a database for permanent storage, so that they may be searched for in experimental data at a later time.
  • GINUMS uses the following model to determine the products (and their corresponding masses) of the amplification process on genomic sequence.
  • N represents any nucleotide
  • P represents the nucleotides in the product
  • the products of the reaction are S and S'.
  • short oligonucleotide fragments may also be generated or amplified by the use of a different nicking endonuclease alone or in combination with another nicking endoculease or restriction endonuclease (e.g., a type lls restriction endonuclease).
  • a linear amplification reaction may be first performed under the lower optimal temperature and then under the higher optimal temperature.
  • the amplified short fragments may also contain genetic variations. For instance, SNPs in human genomic DNA that are flanked by two N.BstNB I recognition sequences or an N.BstNB I recognition sequence and another restriction enzyme recognition sequence are shown in Table 9. The characterization of such short fragments would identify the SNPs within these fragments and facilitate the identification of the individual from which a nucleic acid sample is obtained.
  • the oligonucleotides linearly amplified as described above may be further exponentially amplified. These oligonucleotides are referred to as “initiating oligonucleotides” or “initiators.” Such exponential amplification greatly increases the production rate and amount of the initiating oligonucleotides.
  • the key idea for exponential amplification is to arrange it so that the oligonucleotide product of the linear reaction serves to create a new primer that in turn anneals to a target template and creates a new primer-template, which in turn produces more of the same oligonucleotide product, creating a chain reaction.
  • a simple scheme for exponentially amplifying a short oligonucleotide (also referred to "direct EXPAR") using N.BstNB I as the nicking agent is depicted in Figure 2.
  • the scheme is based on our observation that even though the product oligonucleotide is unstable as a duplex it will form a transient duplex molecule with its complement and this transient duplex can act as a primer for extension by the DNA polymerase. Once extension of the oligonucleotide has occurred the duplex is stabilized by the additional complementary duplex section and will not readily dissociate. Extending the primer thus creates a stable primer-template that will produce oligonucleotide products in a linear fashion.
  • oligonucleotides that we call amplification templates.
  • the key feature of these single-stranded oligonucleotides is that they contain two copies in tandem of the complement of the oligonucleotide product to be amplified, separated by the sequence of the antisense strand of the recognition sequence of N.BstNB I (i.e., 3'-CTCAG-5') and a four base spacer (on the 5' side).
  • This primed template will then continue to produce oligonucleotide product via the linear amplification cycle as described above (nicking after the four base spacer, dissociating the oligonucleotide and re-elongating the primer) as long as the enzymes remain active and dNTPs are available.
  • Another scheme for exponentially amplifying short oligonucleotides referred to as "the replicator type” or "ping-pong amplification scheme" uses two templates.
  • an initiating oligonucleotide of sequence S primes a first template oligonucleotide T1 (S is about 8 to 16 nucleotides in length) to form the following partially double-stranded nucleic acid molecule.
  • T1 is about 8 to 16 nucleotides in length
  • the 3' terminus of T1 may be blocked by a phosphate group.
  • the upper strand of the above partially double-stranded nucleic acid molecule is elongated to form the following fully double-stranded nucleic acid molecule:
  • a nicking enzyme e.g., N.BstNB I
  • the lower strand is nicked to generate a 3' hydroxyl group and release an oligonucleotide blocked at the 3' terminus (un-productive oligonucleotide).
  • a nicking enzyme e.g., N.BstNB I
  • the resulting nicked structure is shown below:
  • the DNA polymerase uses sequence S as a template and fills in the recessed 3' hydroxyl group of the lower strand to produce the following double-stranded nucleic acid.
  • the nicking enzyme cleaves the lower strand, releasing an oligonucleotide having the sequence S' (which is completely complementary to the sequence S) as shown below:
  • the lower strand of the above partially double-stranded nucleic acid may be extended again to produce a fully double-stranded nucleic acid molecule.
  • the lower strand of the fully double-stranded nucleic acid may be nicked again to release another oligonucleotide having the sequence S'.
  • the above extension and nicking cycle may be repeated multiple times, resulting in the amplification of the oligonucleotide having the sequence S'.
  • This oligonucleotide (S') is capable of priming the oligonucleotide template T2 to follow a partially double-stranded nucleic acid molecule as shown below:
  • the upper strand of the above partially double-stranded nucleic acid molecule is elongated to form the following fully double-stranded nucleic acid molecule:
  • a nicking enzyme e.g., N.BstNB I
  • the lower strand is nicked to generate a 3' hydroxyl group and release an oligonucleotide blocked at the 3' terminus (un-productive oligonucleotide).
  • a nicking enzyme e.g., N.BstNB I
  • the resulting nicked structure is shown below:
  • the nicking enzyme cleaves the lower strand, releasing an oligonucleotide having the sequence S' (which is completely complementary to the sequence S) as shown below:
  • the lower strand of the above partially double-stranded nucleic acid may be extended again to produce a fully double-stranded nucleic acid molecule.
  • the lower strand of the fully double-stranded nucleic acid may be nicked again to release another oligonucleotide having the sequence S.
  • the above extension and nicking cycle may be repeated multiple times, resulting in the amplification of the oligonucleotide having the sequence S.
  • the two oligonucleotides having the sequences of S and S' are now capable of priming T1 and T2, respectively, and the exponential amplification is started.
  • S and S' are sufficiently short (e.g., 8-16 nucleotides in length) which prevents the triggers from forming a stable duplex in a reaction mixture under conditions for exponential amplification (e.g., 60°C).
  • This variation of exponential amplification has a substantial advantage of requiring a very high level of stringency of an oligonucleotide priming its template.
  • oligonucleotide e.g., an oligonucleotide having the sequence S or S'
  • the oligonucleotide has to be nearly perfectly based paired with its template for an exponential amplification reaction to start. In many cases, even a single mismatch in the oligonucleotide will inhibit the reaction.
  • T1 The first template oligonucleotide (T1 ).
  • T1 may be 24 to 60 nucleotides (including all the integer values therebetween), preferably 32-36 nucleotides in length.
  • the 3'-end of T1 may be blocked with, for example, a phosphate, an amine, a biotin, a dideoxy group or a fluorophore (that is, there is no free 3'-hydroxyl in T1 ) to prevent extension by a polymerase.
  • the region from the 3' terminus of T1 to the fifth nucleotide directly 3' to the 3' terminus of the sense strand of a nicking endonuclease recognition sequence (e.g., GAGTC) (i.e., Region l in Figure 6) may be 8, 9, 10, 11 , 12, 13, 14, 15 or 16 nucleotides in length and is completely (or at least substantially) complementary to the sequence S.
  • a nicking endonuclease e.g., N.BstNB I
  • sequence 3'-CTGAG-5' which is the sense strand of the recognition sequence for a nicking enzyme (e.g., N.BstNB I). Further in the 5' direction is about 10 to 20 nucleotides of any sequence (Region III in Figure 6). The sequence at the 5' end should not be complementary to any of the sequence at the 3'-end.
  • concentration of T1 is 0.001 to 1 micromolar if in solution. T1 can also be tethered to a solid support or covalently attached to any type of solid support.
  • T2 The second template oligonucleotide (T2). Similar to T1 , T2 may be 24 to 60 nucleotides (including all the integer values therebetween), preferably 32-36 nucleotides, in length.
  • the 3'-end of T2 may be blocked with, for example, a phosphate, an amine, a biotin, a dideoxy group or a fluorophore (that is, there is no free 3'-hydroxyl in T2) to prevent extension by a polymerase.
  • the region from the 3' terminus of T2 to the fifth nucleotide directly 3' to the 3' terminus of the sense strand of a nicking endonuclease recognition sequence (e.g.,
  • GAGTC may be 8, 9, 10, 11 , 12, 13, 14, 15 or 16 nucleotides in length and is at least substantially complementary to the sequence S'.
  • a nicking endonuclease e.g., N.BstNB I
  • CGAG the sense strand of the recognition sequence for a nicking enzyme
  • T2 The concentration of T2 is 0.001 to 1 micromolar if in solution. T2 can also be tethered to a solid support or covalently attached to any type of solid support.
  • a DNA polymerase such as exo " Vent, 9°N m TM, Taq, or Bst at a concentration of 0.002 to 20 units per microliter.
  • concentration of the polymerase is 0.02 to 0.5 units per microliter.
  • the enzyme is typically available commercially in 100 mM KCI, 0.1 mM EDTA, 10 mMTris-HCI (pH 7.4), 1 mM DDT, and 50% glycerol.
  • a nicking enzyme such as N.BstNB I (from New England Biolabs, (NEB), MA) at a concentration of 0.002 to 20 units per microliter.
  • concentration of the nicking enzyme is 0.02 to 0.5 units per microliter.
  • the enzyme is supplied in 50 mM KCI, 10 mMTris-HCI (pH 7.5), 0.1 mM EDTA, 1 mM DTT, 200 ug/ml BSA and
  • a salt e.g., MgCI 2 or MgSO 4 ) at 0.5 to 10 mM in concentration. Preferably the concentration is 2 to 6 mM.
  • a salt e.g., (NH 4 ) 2 SO 4
  • a salt e.g., KCI
  • a buffer e.g., Tris-HCl
  • pH 7-8 preferably 7.5 in the 10-50 mM range of concentrations, preferably 10 mM.
  • a reducing agent e.g., dithiothreitol (DTT)
  • a detergent e.g., Triton X-100
  • V/V 0.01 % to 1 % range
  • V/V 0.01 % to 1 % range
  • T1 and T2 may be blocked so that no free 3'-hydroxyl groups are available for extension.
  • T1 and T2 molecules may be immobilized in different regions of a solid substrate or different solid substrates (e.g., microbeads).
  • the ping-pong amplification reaction cycle is also schematically described in Figure 7.
  • the use of the ping-pong amplification reaction cycle in discriminating genetic variations is shown in Figure 8.
  • the fingerprinting assays described above may be carried out in various formats. For instance, the reactions may be performed in a mixture where all the components are soluble.
  • one or all of the template(s) can be covalently attached at the 3' end or the 5' end to a solid phase with the use of cross-linkers or spacers.
  • the solid phase includes (without limitation) nylon tip beads, fluted tips, microbeads, microplate wells, membranes, slides, arrays, and the materials of which the solid phase is made include glass, nylon 6/6, silica, plastics like polystyrene, polymers like poly(ethyleneimine), etc.
  • a replicator type of exponential amplification reaction may be performed using immobilized templates. More specifically, the first template (T1) molecules may be linked to beads, while the second template (T2) molecules are linked to different beads.
  • the beads linked with T1 molecules may be mixed with the beads linked with T2 molecules in a reaction mixture to amplify two oligonucleotides (i.e., S and S' as described above in the context of the replicator type of amplification reaction).
  • S and S' as described above in the context of the replicator type of amplification reaction.
  • such a reaction may be carried out to amplify multiple oligonucleotide sequences.
  • Beads linked with template molecules other than T1 and T2 molecules may be included in the reaction mixture so that oligonucleotides other than S and S' may also be amplified. It is also possible and advantageous to perform amplification reactions (e.g., direct EXPAR as described above) on arrays of immobilized oligonucleotides.
  • the arrays can be composed of elements separated spatially on a 2-dimensional solid support. Suitable solid supports include, but are not limited to, glass slides, wafers, beads, microbeads, rods, ribbons, nylon6/6, nylon parts, polymer-coated solid supports, wells, etc.
  • the arrays can be further assembled on a 3-dimensional solid support.
  • the amplification template (e.g., ⁇ in Figure 2) is immobilized to a solid support at its 5' end or its 3' end, preferably at its 3'- end. There may or may not be any spacer between the template oligonucleotide and the solid support.
  • the immobilized template when annealing to a trigger oligonucleotide, may be used as a template to amplify an oligonucleotide having a sequence identical to the trigger oligonucleotide.
  • the newly synthesized oligonucleotide then primes an adjacent template oligonucleotide in the element on (or in) the array and an exponential amplification reaction takes place. Oligonucleotide amplification is detected by employing a DNA binding dye that preferentially binds to double strand DNA (e.g., SYBR ® green).
  • nucleic acids or oligonucleotides of the present invention are immobilized to a substrate to form an array.
  • an "array" refers to a collection of nucleic acids or oligonucleotides that are placed on a solid support in distinct areas. Each area is separated by some distance in which no nucleic acid or oligonucleotide is bound or deposited. In some embodiments, area sizes are 20 to 500 microns and the center to center distances of neighboring areas range from 50 to 1500 microns.
  • the array of the present invention may contain 2-9, 10-100, 101 -400, 401 - 1 ,000, or more than 1 ,000 distinct areas.
  • the nucleic acid or oligonucleotide may be immobilized to a substrate in the following two ways: (1) synthesizing the nucleic acids or the oligonucleotides directly on the substrate (often termed “in situ synthesis"), or (2) synthesizing or otherwise preparing the nucleic acid or the oligonucleotides separately and then position and bind them to the substrate (sometimes termed "post-synthetic attachment").
  • in situ synthesis the primary technology is photolithography. Briefly, the technology involves modifying the surface of a solid support with photolabile groups that protect, for example, oxygen atoms bound to the substrate through linking elements.
  • This array of protected hydroxyl groups is illuminated through a photolithographic mask, producing reactive hydroxyl groups in the illuminated areas.
  • a 3'-0- phosphoramidite-activated deoxynucleoside protected at the 5'-hydroxyl with the same photolabile group is then presented to the surface and coupling occurs through the hydroxyl group at illuminated areas.
  • the substrate is rinsed and its surface is illuminated through a second mask to expose additional hydroxyl groups for coupling.
  • a second 5'- protected, 3'-0-phosphoramidite-activated deoxynucleoside is present to the surface. The selective photo-de-protection and coupling cycles are repeated until the desired set of products is obtained.
  • carbodiimides are commonly used in three different approaches to couple DNA to solid supports.
  • the support is coated with hydrazide groups that are then treated with carbodumide and carboxy-modified oligonucleotide.
  • a substrate with multiple carboxylic acid groups may be treated with an amino- modified oligonucleotide and carbodumide.
  • Epoxide-based chemistries are also used with amine modified oligonucleotides.
  • the primary post-synthetic attachment technologies include ink jetting and mechanical spotting.
  • Ink jetting involves the dispensing of nucleic acids or oligonucleotides using a dispenser derived from the ink-jet printing industry.
  • the nucleic acid oligonucleotides are withdrawn from the source plate up into the print head and then moved to a location above the substrate.
  • the nucleic acids or oligonucleotides are then forced through a small orifice, causing the ejection of a droplet from the print head onto the surface of the substrate.
  • Mechanical spotting involves the use of rigid pins.
  • the pins are dipped into a nucleic acid or oligonucleotide solution, thereby transferring a small volume of the solution onto the tip of the pins. Touching the pin tips onto the substrate leaves spots, the diameters of which are determined by the surface energies of the pins, the nucleic acid or oligonucleotide solution, and the substrate.
  • Mechanical spotting may be used to spot multiple arrays with a single nucleic acid or oligonucleotide loading. Detailed description of using mechanical spotting in array fabrication may be found in the following patents or published patent applications: U.S. Patent Nos.
  • the substrate to which the nucleic acids or oligonucleotides of the present invention are immobilized to form an array is prepared from a suitable material.
  • the substrate is preferably rigid and has a surface that is substantially flat. In some embodiments, the surface may have raised portions to delineate areas. Such delineation separates the amplification reaction mixtures at distinct areas from each other and allows for the amplification products at distinct areas to be analyzed or characterized individually.
  • the suitable material includes, but is not limited to, silicon, glass, paper, ceramic, metal, metalloid, and plastics. Typical substrates are silicon wafers and borosilicate slides (e.g., microscope glass slides).
  • a particularly useful solid support is a silicon wafer that is usually used in the electronic industry in the construction of semiconductors.
  • the wafers are highly polished and reflective on one side and can be easily coated with various linkers, such as poly(ethyleneimine) using silane chemistry.
  • Wafers are commercially available from companies such as WaferNet, San Jose, CA.
  • WaferNet San Jose, CA.
  • one of ordinary skill in the art may vary the composition of immobilized molecules of the present array.
  • the T1 orT2 molecules of the present invention may or may not be immobilized to every distinct area of the array.
  • the nucleic acids or oligonucleotides in a distinct area of an array are homogeneous.
  • nucleic acids or oligonucleotides in every distinct area of an array to which the nucleic acids or oligonucleotides are immobilized are homogeneous.
  • homogeneous indicates that each nucleic acid or oligonucleotide molecule in a distinct area has the same sequence as another nucleic acid or oligonucleotide molecule in the same area.
  • nucleic acid or oligonucleotide in at least one of the distinct areas of an array are heterogeneous.
  • heterogeneous indicates that at least one nucleic acid or oligonucleotide molecule in a distinct area has a different sequence from another nucleic acid or oligonucleotide molecule in the area.
  • molecules other than the nucleic acids or oligonucleotides described above may also be present in some or all of distinct areas of an array.
  • a molecule useful as an internal control for the quality of an array may be attached to some or all of distinct areas of an array.
  • Another example for such a molecule may be a nucleic acid useful as an indicator of hybridization stringency.
  • composition of nucleic acids or oligonucleotides in every distinct area of an array is the same.
  • Such an array may be useful in determining genetic variations in a particular gene in a selected population of organisms or in parallel diagnosis of a disease or a disorder associated with mutations in a particular gene.
  • the immobilized nucleic acids or oligonucleotides of the present invention may contain oligonucleotide sequences that are at least substantially complementary or identical to various target nucleic acids.
  • target nucleic acids include, but are not limited to, genes associated with hereditary diseases in animals, oncogenes, genes related to disease predisposition, genomic DNAs useful for forensics and/or paternity determination, genes associated with or rendering desirable features in plants or animals, and genomic or episomic DNA of infectious organisms.
  • An array of the present invention may contain nucleic acids or oligonucleotides that are at least substantially complementary or identical to a particular type of target nucleic acids in distinct areas.
  • an array may have a nucleic acid or an oligonucleotide that is at least substantially complementary or identical to a first gene related to disease predisposition in a first distinct area, another nucleic acid or an oligonucleotide that is at least substantially complementary or identical to a second gene also related to disease predisposition in a second distinct area, yet another nucleic acid or an oligonucleotide that is at least substantially complementary or identical to a third gene also related to disease predisposition in a third distinct area, etc.
  • an array is useful to determine disease predisposition of an individual animal (including a human) or a plant.
  • an array may have nucleic acids or oligonucleotides that are at least substantially complementary or identical to multiple types of target nucleic acids categorized by the functions of the targets.
  • an array may contain nucleic acids or oligonucleotides that are at least substantially complementary or identical to a portion of a target nucleic acid that contains various potential genetic variations.
  • a first area of the array may contain immobilized nucleic acids or oligonucleotides that are at least substantially complementary or identical to a portion of a target gene that contains a genetic variation of one allele of the target.
  • a second area of the array may contain immobilized nucleic acids or oligonucleotides that are at least substantially complementary or identical to a portion of target gene that contains a genetic variation of another allele of the target.
  • the array may have additional areas that contain immobilized nucleic acids or oligonucleotides that are at least substantially complementary or identical to portions of the target gene that contains genetic variations of additional alleles of the target.
  • the immobilized nucleic acids or oligonucleotides must be stable and not dissociate during various treatment, such as hybridization, washing or incubation at the temperature at which an amplification reaction is performed.
  • the density of the immobilized nucleic acids or oligonucleotides must be sufficient for the subsequent analysis.
  • typically 1000 to 10 12 , preferably 1000 to 10 6 , 10 6 to 10 9 , or 10 9 to 10 12 ODNP molecules are immobilized in at least one distinct area.
  • the immobilization process should not interfere with the ability of immobilized nucleic acids or oligonucleotides required for exponential nucleic acid amplification.
  • the linker (also referred to as a "linking element") comprises a chemical chain that serves to distance the nucleic acids or oligonucleotides from the substrate.
  • the linker may be cleavable.
  • the substrate is coated with a polymeric layer that provides linking elements with a lot of reactive ends/sites.
  • a common example is glass slides coated with polylysine, which are commercially available.
  • Another example is substrates coated with poly(ethyleneimine) as described in
  • the array of the present invention enables the high throughput of various analyses to which the present nucleic acid amplification is applicable.
  • an array of T2 molecules may be used to amplify multiple target nucleic acids.
  • the reaction mixture or the products of an amplification reaction performed in the presence of a target nucleic acid may be pooled together and applied to the array of T2 molecules.
  • the reaction mixtures or the amplification products of different amplification reactions may be applied to distinct areas of the array.
  • Another round (“second round”) of amplification reactions may then be performed on the array in the present of a nicking agent that recognizes the nicking agent recognition sequence of which the antisense strand is present in the T2 molecules.
  • the amplification products of the second round of reactions performed on the array may be pooled together and analyzed. If the array (e.g., a microwell array) has distinct areas that are delineated by certain physical barriers, the amplification products of the second round of reactions in distinct arrays may be analyzed individually.
  • nucleic acid molecules of the present invention may be immobilized via the methods described above that are useful in preparing an array.
  • any methods known in the art may be used.
  • a target nucleic acid of the present invention may be immobilized by the use of a fixative or tissue printing. It may also be first isolated or purified and then transferred to a substrate that binds to nucleic acids or oligonucleotides, such as nitrocellulose or nylon membranes.
  • the products of a nicking reaction, a linear amplification reaction or an exponential amplification reaction according to the present invention may be characterized by any applicable known methods. These methods include, but are not limited to mass spectrometry, fluorescence spectrometry, electrophoresis, liquid chromatography, hybridization and radiography. Certain exemplary methods are described in more detail below.
  • not all the amplified nucleic acids are characterized. In other words, in these embodiments, only certain amplified nucleic acids that meet a given criterion need be characterized. For instance, the amplified nucleic acid molecules may first be separated by liquid chromatography and only the fractions that contain short nucleic acid fragments are further characterized by, for example, mass chromatography.
  • the fingerprinting assays of the present invention can be read out in a number of ways but the most ideal is by mass spectrometry since a series of well-defined and characterized oligonucleotides are generated that have known mass/charge ratios (m/z).
  • Exemplary mass spectrometric analysis includes Matrix-Assisted Laser Desorpotion/lonization Mass Spectrometry (MALDI) and Time-of-Fight (TOF).
  • Matrix-Assisted Laser Desorption/lonization Mass Spectrometry is becoming an ever more popular technique for studying biomolecules (Hillenkamp et al., Anal. Chem. 63, 1193A-1203A, 1991 ). This technique ionizes high molecular weight biopolymers with minimal concomitant fragmentation of the sample material. This is typically accomplished via the incorporation of the sample to be analyzed into a matrix that absorbs radiation from an incident UV or IR laser. This energy is then transferred from the matrix to the sample resulting in desorption of the sample into the gas phase with subsequent ionization and minimal fragmentation.
  • MALDI-MS One of the advantages of MALDI-MS over ESI-MS is the simplicity of the spectra obtained: MALDI spectra are generally dominated by singly charged species. Typically, the gaseous ions generated by MALDI techniques are detected and analyzed by determining the time-of-flight (TOF) of these ions. While MALDI-TOF MS is not a high resolution technique, resolution can be improved by making modifications to such systems, e.g., by the use of tandem MS techniques, or by the use of other types of analyzers, such as Fourier transform (FT) and quadrupole ion traps.
  • FT Fourier transform
  • MALDI techniques have found application for the rapid and straightforward determination of the molecular weight of certain biomolecules (Feng and Konishi, Anal. Chem. 64, 2090-2095, 1992; Nelson, Dogruel and Williams, Rapid Commun. Mass Spectrom. 8, 627-631 , 1994). These techniques have been used to confirm the identity and integrity of certain biomolecules such as peptides, proteins, oligonucleotides, nucleic acids, glycoproteins, oligosaccharides and carbohydrates. Further, these MS techniques have found biochemical applications in the detection and identification of post-translational modifications on proteins.
  • Verification of DNA and RNA sequences that are less than 100 bases in length has also been accomplished using ESI with FTMS to measure the molecular weight of the nucleic acids (Little et al, Proc. Natl. Acad. Sci. USA 92, 2318-2322, 1995).
  • the matrix is an important feature of MALDI-MS.
  • analysis of nucleic acids by MALDI can be divided into two steps.
  • the first step involves preparing the sample by mixing the sample to be analyzed with a molar excess of a chemical commonly referred to as the "matrix.” See, e.g., Wu et al. Rapid Commun. Mass Spectrom. 7:142-146 (1993).
  • the primary purpose of the matrix is to promote ionization of the nucleic acid. Without the matrix, the nucleic acid molecule tends to fragment upon exposure to the laser energy, so that the mass and identity of the nucleic acid is difficult or impossible to determine.
  • matrix refers to a substance which absorbs radiation at a wavelength substantially corresponding to the pulse of laser energy used in the MALDI method, and where the matrix facilitates desorption and ionization of molecules.
  • a matrix may be any one of several small, light- absorbing chemicals that may be mixed in solution with a nucleic acid in such a manner so that, upon drying on a solid support (e.g., a sample plate or a probe element), the crystalline matrix-embedded analyte molecules are successfully desorbed by laser irradiation and ionized from the solid phase crystals into the vapor phase and accelerate as intact molecular ions.
  • the second step of the MALDI process involves desorption of the bulk portions of the solid sample by a short pulse of laser light.
  • the analyte- containing sample is added to (e.g., spotted onto) a coating of cationic polyelectrolyte, allowing the analyte (nucleic acid) to bind to the cationic polyelectrolyte.
  • This spot is then washed in order to purify the nucleic acid.
  • the spot is then treated with matrix (when the matrix is a liquid) or a solution of matrix (when the matrix is a solid).
  • the spot When the matrix is a solid, the spot should be allowed to dry in order to remove the solvent that was formerly used to dissolve the matrix in solution. Thereafter, this spot of nucleic acid and matrix can be subjected to MALDI-MS to provide a very strong signal due to the nucleic acid.
  • the present invention provides a solid support having a surface, where that surface is at least partially coated with a coating comprising cationic polyelectrolyte, where at least some of the cationic polyelectrolyte is in contact with nucleic acid and the nucleic acid is in contact with matrix.
  • the solid support is a plate, e.g., a stainless steel plate, and the cationic polyelectrolyte either forms a continuous coating across all or a significant portion of the surface, or is spotted onto the surface in distinct regions. Nucleic acid and matrix is then located in distinct regions on the surface, so as to provide an array-type appearance.
  • the surface may be a 96-well plate, with cationic polyelectrolyte, nucleic acid and matrix located in one, and preferably more than one, of the wells.
  • This array is then subjected to MALDI-MS, where the various regions are sequentially subjected to laser light, and the mass spectrum of the nucleic acid present in the spots is sequentially obtained.
  • the matrix should meet one or more of the following criteria, and preferably meets many or all of these criteria.
  • the matrix should be able to embed and isolate nucleic acid (e.g., by co-crystallization), it should be soluble in solvents compatible with nucleic acids, it should be stable under the vacuum used in MALDI, it should assist co-desorption of the nucleic acid upon laser irradiation, and it should promote ionization of the nucleic acid.
  • the matrix should comprise a chromophore that strongly absorbs in the wavelength of light being emitted by the laser. For instance, if the laser is an ultraviolet laser, then the matrix should have a chromophore that absorbs in the ultraviolet region.
  • Suitable matrices for nucleic acids have been identified as suitable matrices for nucleic acids, where these as well as other suitable matrix chemicals known in the art may be used in the methods and compositions of the present invention: 6-aza-2-thiothymine (ATT), glycerol, 2,4,6-trihydroxyacetophenone (THAP), picolinic acid (PA), 3-hydroxy picolinic acid (HPA), 2,5- dihiydroxybenzoic acid, anthranilic acid, nicotinic acid, and salicylamide. Mixtures of these chemicals are also suitable.
  • the matrix is a solid at room temperature.
  • the matrix may be a liquid chemical, where suitable liquid matrices are substituted or unsubstituted: (1 ) alcohols, including: glycerol, 1 ,2- or 1 ,3-propane diol, 1 ,2-, 1 ,3- or 1 ,4-butane diol, triethanolamine; (2) carboxylic acids including: formic acid, lactic acid, acetic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid and esters thereof; (3) primary or secondary amides including acetamide, propanamide, butanamide, pentanamide and hexanamide, whether branched or unbranched; (4) primary or secondary amines, including propylamine, butylamine, pentylamine, hexylamine, heptylamine, diethylamine and dipropylamine; (5) nitriles, hydrazine and hydrazide.
  • liquid matrices are particularly useful when the MALDI laser emits light in the infrared spectrum. It is reported that THAP works best for samples below 10kDa while HPA and PA are more appropriate for oligonucleotides above 10kDa. Acidic matrices, e.g., HPA, are preferred for single-stranded nucleic acids, while neutral matrices, e.g., glycerol and ATT, are preferred for double-stranded nucleic acids.
  • MS is particularly advantageous in those applications in which it is desirable to eliminate a size separation step prior to molecular weight determination. Sensitivities of MS may be achieved to at least to 1 amu. The smallest mass differences in nucleic acid bases is between adenine and thymidine which is 9 Daltons. Particularly preferred methodologies according to the present invention employ Liquid Chromatography-Time-of-Flight Mass Spectrometry (LC-TOF-MS).
  • LC-TOF-MS Liquid Chromatography-Time-of-Flight Mass Spectrometry
  • LC-TOF-MS is composed of an orthogonal acceleration Time- of-Flight (TOF) MS detector for atmospheric pressure ionization (API) analysis using electrospray (ES) or atmospheric pressure chemical ionization (APCI).
  • TOF Time- of-Flight
  • ES electrospray
  • APCI atmospheric pressure chemical ionization
  • LC-TOF-MS provides high mass resolution (5000 FWHM), high mass measurement accuracy (to within 5ppm) and very good sensitivity (ability to detect femtomolar amount of DNA polymer).
  • TOF instruments are generally more sensitive than quadrupoles, but are correspondingly more expensive.
  • LC-TOF-MS has a more efficient duty cycle since the current instruments can sequentially analyze one mass at a time while rejecting all others (this is referred to as single ion monitoring (SIM)).
  • SIM single ion monitoring
  • LC-TOF-MS samples all of the ions passing into the TOF analyzer at the same time. This results in higher sensitivity, provides quantitative data, which improves the sensitivity between 10 and 100 fold. Enhanced resolution (5000 FWHM) and mass measurement accuracy of better than 5 ppm imply that differences between nucleosides as small as 9 amu (Daltons) can be accurately measured.
  • the TOF mass analyzer performs very high frequency sampling (10 spectra/sec) of all ions simultaneously across the full mass range of interest.
  • the duty cycle of the LC-TOF-MS allows high sensitivity spectra to be recorded in quick succession making the instrument compatible with more efficient separations techniques such as narrow bore LC, capillary chromatography (CE) and capillary electrochromatography (CEC).
  • CE capillary chromatography
  • CEC capillary electrochromatography
  • the ES or APCI aerosol spray is directed perpendicularly past the sampling cone, which is displaced from the central axis of the instrument. Ions are extracted orthogonally from the spray into the sampling cone aperture leaving large droplets, involatile materials, particulates and other unwanted components to collect in the vent port that is protected with an exchangeable liner.
  • the second orthogonal step enables the volume of gas (and ions) sampled from atmosphere to be increased compared with conventional API sources. Gas at atmospheric pressure sampled through an aperture into a partial vacuum forms a freely expanding jet, which represents a region of high performance compared to the surrounding vacuum. When this jet is directed into the second aperture of a conventional API interface it increases the flow of gas through the second aperture.
  • Maintaining a suitable vacuum in the MS-TOF therefore places a restriction on the maximum diameter of the apertures in such an LC interface. Ions in the partial vacuum of the ion block are extracted electrostatically into the hexapole ion bridge that efficiently transports ions to the analyzer.
  • the coupling of the TOF mass analyzers with MUX-technology allows the connection of up to 8 HPLC columns in parallel to a single LC-TOF- MS. (Micromass, Manchester UK).
  • a multiplexed electrospray (ESI) interface is used for on-line LC-MS utilizing an indexed stepper motor to sequentially sample from up to 8 HPLC columns or liquid inlets operated in parallel.
  • LC-TOF-MS is sometimes preferred over use of MALDI- TOF because LC-TOF-MS is a quantitative method for analysis of the molecular weight of polymers. LC-TOF-MS does not fragment the polymers and it employs a very gentle ionization process compared to matrix-assisted- lazer-desorption-ionization (MALDI). Because every MALDI blast is different, the ionization is not quantitative. LC-TOF-MS does, however, produce different m/z values for polymers.
  • MALDI matrix-assisted- lazer-desorption-ionization
  • HPLC High-Performance Liquid Chromatography
  • HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Compounds are separated by injecting an aliquot of the sample mixture onto the column. The different components in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase.
  • the pumps provide a steady high performance with no pulsating, and can be programmed to vary the composition of the solvent during the course of the separation.
  • Exemplary detectors useful within the methods of present invention include UV-VIS absorption, or fluorescence after excitation with a suitable wavelength, mass spectrometers and IR spectrometers.
  • IP- RO-HPLC on non-porous PS/DVB particles with chemically bonded alkyl chains have been shown to be rapid alternatives to capillary electrophoresis in the analysis of both single and double-strand nucleic acids providing similar degrees of resolution.
  • IP-RP-HPLC In contrast to ion-exchange chromatography, which does not always retain double-strand DNA as a function of strand length (since AT base pairs interact with the positively charged stationary phase, more strongly than GC base- pairs), IP-RP-HPLC enables a strictly size-dependent separation.
  • Denaturing HPLC is an ion-pair reversed-phase high performance liquid chromatography methodology (IP-RP-HPLC) that uses a non-porous C-18 column as the stationary phase.
  • the column is comprised of a polystyrene-divinylbenzene copolymer.
  • the mobile phase is comprised of an ion-pairing agent of triethylammonium acetate (TEAA), which mediates binding of DNA to the stationary phase, and acetonitrile (ACN) as an organic agent to achieve subsequent separation of the DNA from the column.
  • TEAA triethylammonium acetate
  • ACN acetonitrile
  • a linear gradient of acetonitrile allows separation DHPLC identifies mutations and polymorphisms based on detection of heteroduplex formation between mismatched nucleotides in double stranded PCR amplified DNA. Sequence variation creates a mixed population of heteroduplexes and homoduplexes during reannealling of wild type and mutant DNA of fragments based on size and/or presence of heteroduplexes (this is the traditional use of the DHPLC technology). When this mixed population is analyzed by HPLC under partially denaturing temperatures, the heteroduplexes elute from the column earlier than the homoduplexes because of their reduced melting temperature. Analysis can be performed on individual samples to determine heterozygosity, or on mixed samples to identify sequence variation between individuals.
  • the various nicking and amplification reactions described above may also be readout by detectors that measure real-time fluorescence, such as the MJ Opticon from MJ Research (Boston, MA), the ABI Prism 7000 instrument (Foster City, CA), and endpoint plate readers, such as the Ultramark from Biorad (Hercules, CA).
  • Real time monitoring is a very useful method as it enables parameters such as initial rates to be determined with accuracy and ease.
  • the use of double-strand specific fluorescent dyes such as SYBR ® green from Molecular Probes (Eugene OR) is especially useful when used during the amplification reactions described above. Dyes that bind to single strand nucleic acids can also be used, perhaps at times with slightly less efficacy than double-strand specific dyes.
  • intercalating dyes such as SYBR ®
  • dual labeled probes FRET (fluorescent energy transfer) probes
  • Molecular Beacons exemplary fluorescent intercalating agents include, without limitation, those disclosed in U.S. Pat. Nos.
  • Fluorescence produced by fluorescent intercalating agents may be detected by various detectors, including PMTs, CCD cameras, fluorescent-based microscopes, fluorescent-based scanners, fluorescent-based microplate readers, fluorescent- based capillary readers.
  • the nicking agent in the reaction mixture may be inactivated (e.g., by heat) and a fresh DNA polymerase be added.
  • a fresh DNA polymerase e.g., by heat
  • the presence of the active DNA polymerase, but not the nicking agent, allows any partial duplexes to be extended to completely double-stranded nucleic acid fragments.
  • the generation of such completely double-stranded nucleic acid fragments allow the binding of a greater number of fluorescent intercalating agents, which in turn increase the signal that may be detected.
  • the present invention provides a composition or kit comprising polynucleotide, a nicking agent and a polymerase.
  • inventive compositions have unique properties that render them particularly useful in idenfying the source organism of a nucleic acid sample.
  • a source organism may be a bacterium, fungus, virus, plant, non-human animal or human.
  • Such a composition or kit generally comprises the template oligonucleotide(s) useful for amplifying initiating oligonucleotides described above. It may also further comprise at least one, two, several, or each of the following components: (1) a nicking agent (e.g., a NE or a RE) that recognizes the nicking agent recognition sequence of which one strand is present in the template oligonucleotide(s); (2) a suitable buffer for nicking agent (1); (3) a RE that functions in combination with a nicking agent (with may be identical to or different from nicking agent (1); (4) a suitable buffer for RE (3); (5) a DNA polymerase; (6) a suitable buffer for the DNA polymerase (5); (7) dNTPs; (8) a modified dNTP; (9) a strand displacement facilitator (e.g., 1 M trehalose); and (10) a fluorescent intercalating agent.
  • a nicking agent e.g., a
  • the present invention alleviates and overcomes many drawbacks of the present state of the art through the discovery of novel methods and kits for rapidly fingerprinting DNA to identify prokaryotic and eukaryotic species, subspecies, and especially strains or individuals of the subspecies.
  • the present invention is especially suited for identifying different bacterial strains involved in, for example, nosocomial infections, since the methods and kits are to be sensitive enough to detect differences between, for example, bacterial isolates of the same species.
  • the present invention contemplates identifying, for instance, species, subspecies, and the differences between the individuals of the subspecies, such as pedigrees.
  • the method can be used for: (1) diagnosis of bacterial disease, in plants animals and humans; (2) monitoring for bacterial content and/or contamination in the environment; (3) monitoring food for bacterial contamination; (4) monitoring manufacturing processes for bacterial contamination; (5) monitoring quality assurance/quality control of laboratory tests involving microbiological assays; (6) tracing bacterial contamination and/or outbreaks of bacterial infections; (7) genome mapping; (8) monitoring bioremediation sites; and (9) monitoring agricultural sites for test crops, bacteria and recombinant molecules.
  • a further aspect of the present invention is a machine for automating the identification of bacterial strains, particularly by mass spectrometry.
  • the present invention affords the medical community with a means to not only identify the infectious agents, but also to rapidly characterize the strain or strains involved so that effective measures may be timely employed.
  • Another application of this method is in the manufacturing process.
  • a number of manufacturing processes for instance drugs, microorganism-aided synthesis, food manufacturing, chemical manufacturing and fermentation process all rely either on the presence or absence of bacteria.
  • the method of the present invention can be used. It can monitor bacterial contamination or test that strain purity is being maintained.
  • This method can also be used to test stored blood for bacterial contamination. This would be important in blood banking where bacteria such as Yersinia enterocolitica can cause serious infection and death if it is in transfused blood.
  • the procedure can also be used for quality assurance and quality control in monitoring bacterial contamination in laboratory tests.
  • the Guthrie bacterial inhibition assay uses a specific strain of bacteria to measure phenylalanine in newborn screening. If this strain changes it could affect test results and thus affect the accuracy of the newborn screening program.
  • This method of the present invention can be used to monitor the strain's purity. Any other laboratory test that uses or relies on bacteria in the assay can be monitored. The laboratory or test environment can also be monitored for bacterial contamination by sampling the lab and testing for specific strains of bacteria. This procedure will also be useful in hospitals for tracing the origin and distribution of bacterial infections. It can show whether or not the infection of the patient is a hospital-specific strain.
  • the type of treatment and specific anti-bacterial agent can depend on the source and nature of the bacteria. There are a variety of applications for the fingerprinting technology described here.
  • the fingerprinting technology described herein may be useful to detect polymorphisms in the human genome, in view of the large number of fragments that can be generated.
  • the methods and compositions of the present invention may be used to interrogate a sample for the presence of fragments that uniquely identify all pathogens, and fragments obtained from the human genome that can be used to uniquely identify individuals.
  • oligonucleotides uniquely identify K12, and 15 oligonucleotides uniquely identify O157 (Table 4). These oligonucleotides unique for distinguishing the two E. coli strains are not found in the oligonucleotides that would be generated in the presence of N.BstNB I from chromosome 21 of the human genome (see Table 5).
  • Table 6 lists the relative probability of obtaining an overlapping mass (composition) as a function of the length of the trigger in a background of DNA that is as complex as the human genome (4 billion bases).
  • Table 7 lists the number of oligonucleotides having 6-16 nucleotides that would be generated in the presence of N.BstNB I from 35 bacterial species. The average number of oligonucleotides that would be generated per organism is 19. Greater than 99% of these fragments have a unique mass and sequence, thus virtually every one of the fingerprinting oligonucleotides is unique to the organism from which it is generated.
  • Table 1 E. coli K12 oligonucleotide fragments.
  • Table 2 E. coli O157 oligonucleotide fragments.
  • oligonucleotide fragments from human chromosome 21 Sig. (signature) indicates the number of As-Cs-Gs- Ts (A-C-T-G).
  • the "sequence” includes the sequence of the sense or antisense strand of the recognition sequence(s) of N.BstNB I and/or N.AIw I

Abstract

Selon l'invention, un échantillon d'acide nucléique, caractéristique d'une source particulière, par ex. une bactérie, peut être analysé(e), afin de déterminer si cet échantillon particulier provient d'une source particulière. Le protocole d'analyse soumet l'échantillon à une réaction par coupure de brin, afin de produire une famille de fragments à brin coupé. Dans certains modes de réalisation, la réaction par coupure de brin s'effectue en présence d'une polymérase et de triphosphates de nucléoside, afin d'amplifier les fragments à brin coupé ou des parties desdits fragments. Les fragments à brin coupé ou les parties desdits fragments peuvent être analysé(e)s pour déterminer la source de l'échantillon d'acide nucléique.
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