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WO2004007771A1 - Test de pre-absorption d'acide nucleique - Google Patents

Test de pre-absorption d'acide nucleique Download PDF

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Publication number
WO2004007771A1
WO2004007771A1 PCT/US2002/021660 US0221660W WO2004007771A1 WO 2004007771 A1 WO2004007771 A1 WO 2004007771A1 US 0221660 W US0221660 W US 0221660W WO 2004007771 A1 WO2004007771 A1 WO 2004007771A1
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WIPO (PCT)
Prior art keywords
nucleic acid
probe
target
hybridization
sequences
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PCT/US2002/021660
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English (en)
Inventor
David L. Scott, Jr.
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D-Squared Biotechnologies, Inc.
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Priority to AU2002322426A priority Critical patent/AU2002322426A1/en
Publication of WO2004007771A1 publication Critical patent/WO2004007771A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical

Definitions

  • the present invention relates to methods and compositions for identifying nucleic acids based upon contacting a probe nucleic acid sequence complimentary to a target nucleic acid sequence under conditions suitable for hybridization between the two sequences.
  • Molecular diagnostic assays such as nucleic acid hybridization techniques and polymerase chain reaction (PCR) techniques are able to overcome the sensitivity and specificity issues associated with immunological-based diagnostic assays.
  • the establishment of the polymerase chain reaction as a robust amplification system and the explosion of techniques and variations of PCR have turned molecular diagnostics into a new entity of molecular biology.
  • PCR-based diagnostics have been successfully implemented for the detection of various genetic, acquired, and infectious diseases.
  • sensitive molecular assays, such as PCR are not amenable to high throughput, large scale testing and require that nucleic acid samples be relative free of contaminants that may inhibit Taq polymerase.
  • RNA amenable to molecular diagnostics is not always a trivial matter.
  • hybridization-based diagnostic assays are inherently less sensitive than target amplification techniques, they are insensitive to most contaminants that affect the functionality and reliability of enzymatic-driven molecular diagnostic assay such as PCR.
  • signal amplification techniques have been described that rival the sensitivity of PCR. These techniques take advantage of the high-throughput capabilities and robustness of hybridization technique to provide diagnostics assay that are more reliable than PCR when dealing with some biological samples.
  • the major drawbacks to many of these signal amplifications techniques is that they can be time consuming and may required a certain skill level to effectively utilize the technology.
  • telomere sequences are based on the principle of hybridization.
  • methods for detecting the presence of particular target nucleic acid sequences involve employing a complementary sequence termed a probe, generally detectably labeled, and incubating this labeled probe sequence with the test sample thought to contain the target nucleic acid sequence.
  • probe-target hybridization complexes hybridize to one another under suitable conditions to form probe-target hybridization complexes that can be identified through the presence of the detectable label.
  • Homogeneous phase hybridization assays permit detection of the hybridized probe-target complex without removal of excess unhybridized probe sequences present in the assay solution. See Tyagi and Kramer, 1996, Nature Biotechnology 14,303-308; Arnold, Waldrop, and Hammond, 1990, U.S. Pat. No. 4,950,613.
  • very sensitive assays require separation of the hybridized complex from the unhybridized probe prior to the detection step. Many different methods have been employed to accomplish this separation, including several that rely on differences in physical characteristics between the two products.
  • Other methods employ the use of a second nucleic acid sequence, described as a "capture" probe, for the purpose of separating the probe-target complex from the unhybridized probe.
  • Capture probes are generally immobilized on a solid support and are selected to hybridize to a different portion of the target nucleic acid sequence than does the labeled probe.
  • a tripartite capture-probe:target:labeled-probe complex forms which is bound to the solid support, while the unhybridized labeled probe remains unbound in solution, allowing the two products to be readily separated.
  • Such assays are referred to as "sandwich” hybridizations. See for example, Engelhardt and Rabbani, 1994, U.S. Pat. No. 5,288,609. Although widely used, these sandwich assays require a number of steps to perform and can be quite time consuming.
  • displacement assays Alternative assay methods, termed displacement assays, were developed in an attempt to simplify the method of identifying nucleic acids. See Diamond, S. E., et al., 1988, U.S. Pat. No. 4,766,062; Williams, J. I., et al. 1988, U.S. Pat. No. 4,766,064; Vary, 1987, Nucleic Acids Res. 15, 6883-6897; Vary et al., 1986, Clinical Chemistry 32, 1696-1701.
  • a tether nucleic acid sequence complementary to the target nucleic acid sequence, is hybridized to a shorter, detectably labeled signal nucleic acid sequence, complementary to a specific subsequence of the tether sequence.
  • the signal nucleic acid is fully base-paired with the tether nucleic acid in this signal-tether complex, but the longer tether nucleic acid retains a single-stranded region.
  • the target hybridizes to the single-stranded portion of the tether component. Since the target is homologous to the entire length of the tether, a homologous strand exchange reaction with the signal nucleic acid is initiated, and the target displaces the signal from the tether. This strand exchange reaction proceeds rapidly in the direction of signal nucleic acid displacement because the target is longer and forms a more stable duplex with the tether. See Green, C.
  • the amount of displaced labeled signal nucleic acid is measured to determine the amount of target nucleic acid in the sample.
  • the tether component of the probe complex can also be linked to a solid support, so that separation of the solid and solution phases results in isolation of the signal nucleic acid.
  • standard displacement assays do have certain drawbacks. For example, when target nucleic acid hybridizes to a tether not hybridized to a signal nucleic acid, or when a displaced signal nucleic acid hybridizes to a tether nucleic acid, which was not previously hybridized to a signal nucleic acid, a decrease in the detection signal produced per unit of target hybridized results. Moreover, if the hybridized complex is not stable, an undesirable background signal can be introduced, which complicates interpretation of the assay results and reduces the sensitivity of the assay.
  • the present invention overcomes the limitations of the aforementioned hybridization assays by contacting a pre-determined amount of a probe nucleic acid sequence complementary to a target nucleic acid sequence under conditions suitable for hybridization between the two sequences.
  • the unhybridized probe is separated from the probe-target complex and its concentration is determined. The reduction of in probe concentration is indicative of the presence of target nucleic acid sequences.
  • the "nucleic acid pre-absorption assay"[NAPA] is not limited by unstable hybridization complexes as described above.
  • the combination with a random oligonucleotide library is useful in identifying unique sequences in target nucleic acids SUMMARY OF THE INVENTION
  • the present invention is based upon contacting a probe nucleic acid sequence complementary to a target nucleic acid sequence under conditions suitable for hybridization between the two sequences.
  • the probe-target complex is separated from unhybridized probe nucleic acid by size exclusion.
  • the unhybridized probe can be contacted to a second target nucleic acid sequence.
  • the unhybridized probe recovered separately from the probe-target complex can be used as probe nucleic acid to distinguish the first sequence from the second.
  • the unhybridized probe is quantified by 1 ) directly immobilizing the probe nucleic acid to a solid support; 2) contacting the probe nucleic acid sequence to a complementary nucleic acid sequence immobilized to a solid support under conditions suitable for hybridization between the two sequences
  • One aspect of the present invention pertains to the detection of the unhybridized probe nucleic acid without the separation of the probe-target complex.
  • the quantity of unhybridized probe is determined by contacting the solution to a complementary nucleic acid sequence immobilized to a solid support under conditions suitable for hybridization between the two sequences.
  • the probe nucleic acid contains a detectable label.
  • Another aspect of the present invention pertains to contacting multiple probe nucleic acids having different base sequences complementary to multiple target nucleic acid sequences under conditions suitable for hybridization between probe and target nucleic acid sequences.
  • the probe-target complexes are separated from unhybridized probe nucleic acid sequences by size exclusion or alternatively, by contacting the solution to an immobilized complementary nucleic acid sequence under conditions suitable for hybridization between the two sequences.
  • the probe nucleic acid contains a detectable label.
  • the present invention pertains to a kit for detecting the presence of a target nucleic acid sequence in a test sample.
  • nucleic acid or “polynucleotide sequence” and their respective plurals are used interchangeably herein and are intended to include deoxyribonucleic acid [hereinafter "DNA”] and ribonucleic acid [hereinafter "RNA”]. Both single stranded and double stranded nucleic acids are embraced by this invention. Higher ordered structures of nucleic acids, for example, RNA that has folded upon its linear strand forming a secondary loop structure, are also within the scope of the present invention. Nucleic acid sequences encompassed by the present invention can be from about 3 to about 10,000 nucleotides in length. There is no absolute minimum or maximum length requirement for target nucleic acid sequences, however, a preferred range is from about 10 to about 2,000, preferably about 30 to about 1 ,000, most preferably about 30 to about 100.
  • hybridize or “hybridization” is intended to include admixing of at least two nucleic acid sequences or nucleic acid analog sequences, under conditions such that when at least two complementary nucleic acid sequences are present, they will form a double-stranded structure through base-pairing.
  • label is intended to include radioactive isotopes, fluorescent moieties, chemiluminescent moieties, and direct enzyme conjugates, as well as indirect labels, such as affinity labels for use with secondary or tertiary labeled molecules, such as in fluorescent energy transfer assays.
  • nucleic acid sequences that can form a double-stranded structure through base- pairing. Mismatched base pairing is intended to fall within the scope of this invention.
  • Nucleotide mismatch can affect the affinity between nucleic acid sequences. The greater the mismatch between nucleic acid sequences, generally, the lower the affinity between them as compared to perfectly matched nucleic acid sequences. Generally, the greater the mismatch between nucleic acid sequences, the more readily the hybridization that exists between them can be disrupted. When mismatches between base pairs are present, they generally account for no more than 5% of the region of base-pairing.
  • the degree of complementality between hybridization partners is from about 100% to about 95%, most preferably about 100%.
  • base-pairing is intended to include those reactions that occur in an antiparallel manner as well as those which occur in a parallel fashion.
  • Base-pairing itself is understood to essentially follow a complementary pattern wherein a purine pairs with a pyrimidine via hydrogen bonds. More particularly, it is understood that complementary base-pairing of individual base pairs generally follows Chargaff s Rule wherein an adenine pairs with a thymine (or uracil) and guanine pairs with cytosine.
  • Chargaff s Rule wherein an adenine pairs with a thymine (or uracil) and guanine pairs with cytosine.
  • there are modified bases that account for unconventional base-pairing and these are also considered to be within the scope of the instant invention.
  • modified nucleic acid is intended to include a DNA or RNA nucleic acid molecule that contains chemically modified nucleotides.
  • nucleic acid analog is intended to include non-nucleic acid molecules that can engage in base-pairing interactions with conventional nucleic acids such as phosphorothioates and phosphoroamidates. These modified bases and nucleic acid analogs are considered to be within the scope of the instant invention. It is apparent to those skilled in the art which nucleic acid analogues are useful in strategies for developing probe nucleic acid sequences. Kumas, A. et al., J. Med Chem., 33(9): 2368-2735 (1990); McGuigan, C.
  • a "probe” is a general term for a piece of DNA or RNA corresponding to a gene or sequence of interest, that has been labeled either radioactively, or with some other detectable molecule, such as biotin, digoxygenin or fluorescein. As stretches of DNA or RNA with complementary sequences will hybridize, a probe will label viral plaques, bacterial colonies or bands on a gel that contain the gene of interest.
  • isolated refers to, e.g., a peptide, DNA, or RNA separated from other peptides, DNAs, or RNAs, respectively, and being found in the presence of (if anything) only a solvent, buffer, ion or other component normally present in a biochemical solution of the same. "Isolated” does not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure substances or as solutions.
  • Ligands are defined as molecules that bind to other molecules.
  • a ligand is typically a soluble molecule, such as a hormone or neurotransmitter that binds to a receptor.
  • Nucleic acid aptamers as used herein are artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and other molecules. Aptamers can be selected against many kinds of targets, including proteins, small organic molecules, and carbohydrates. See Tuerk and Gold, Science 1990 249 5050-510; Joyce, Gene 1989, 82 83-87; Ellington and Szostak, Nature 1990, 346, 818-822]; Klug and Famulok, Molecular Biology Reports 1994, 20, 97-107.
  • Target nucleic acid libraries often contain sequences of interest and their identification is disclosed herein. Identification of said unique sequences using oligonucleotide aptamers, and hence using the apatamers as ligands offer a simple and flexible mechanism for obtaining a probe nucleic acid sequence against many target nucleic acid sequences.
  • an oligonucleotide comprising a variable region adjacent to one constant region, or adjacent to two constant regions on either side of the variable region is a preferred embodiment for the probe sequences of this invention.
  • the variable region of the oligonucleotide comprises randomized sequences while the constant region[s] contains primer binding sites for amplification and/or sequencing of the target nucleic acid.
  • the constant region may also contain restriction sites for cloning, while the variable region is designed to contain molecular diversity from which specific ligands can be selected.
  • an aptamer library [1 pmol-10 pmol] is added to appropriate hybridization buffer containing target nucleic acid sequences.
  • the hybridization mixture is incubated at a desirable temperature range to stimulate the formation of the probe-target complex(es).
  • the formation of the probe-target complex(es) is from about 15 C to about 90 C, depending upon the variables of other properties of the solution, such as ionic strength, pH, nucleic acid sequence concentration, degree of complementality of the sequences, as well as other components of the aqueous medium that may affect melting temperatures.
  • the hybridization reaction is allowed to proceed over a period of 0.5 h - 6 h.
  • the unbound aptamers are separated from aptamer-target complexes] by contacting the hybridization reaction to a filter with a specified molecular weight cut off.
  • a filter with a specified molecular weight cut off. For example, a 30 micron filter will retain nucleic acid sequences greater than 100 bases, while allowing nucleic acid sequences less than 100 bp to be collected in the filtrate.
  • the retentate containing the aptamer-target complex[es] are used as substrate for PCR.
  • the resulting amplicons are purified and used as probe nucleic acids.
  • the probe nucleic acids are added to appropriate hybridization buffer containing target nucleic acid sequences.
  • the hybridization mixture is incubated at a desirable temperature range to stimulate the formation of the probe-target complex[es].
  • the formation of the probe-target complex[es] is from about 15 C. to about 90 C, depending upon the variables of other properties of the solution, such as ionic strength, pH, nucleic acid sequence concentration, degree of complementality of the sequences, as well as other components of the aqueous medium that may affect melting temperatures.
  • the hybridization reaction is allowed to proceed over a period of 0.5 h - 6 h.
  • the unbound aptamers are separated from aptamer-target complexes] by contacting the hybridization reaction to a filter with a specified MW limitation.
  • a micron 30 filter will retain nucleic acid sequences greater than 100 bases, while allowing nucleic acid sequences less than 100 bp to be collected in the filtrate.
  • the filtrate is used as substrate for DNA sequencing.
  • the resulting sequences are unique to the primary target nucleic acid sequence.
  • the invention described herein also pertains to compositions and methods useful for analyzing nucleic acids in a variety of test samples.
  • the methods described herein comprise steps involving the formation of hybridization complexes and the detection of unhybridized probe nucleic acid sequences.
  • the recovery of nucleic acids from biological samples which may be used in molecular diagnostics requires cell lysis, nuclease inactivation, and separation from contaminating cellular components.
  • nucleic acids are preferably recovered from a test sample using an extraction buffer that simultaneously releases nucleic acids from test samples and neutralize nucleases.
  • the extraction buffer comprises 4-6 M Guanidine Hydrochloride; 1% -5% Sarkosyl; 10 mM-50 mM EDTA; 0.5%-1.5% Thiourea; 1 mM- 10 mM DTT; and 1mM-100 mM Autocrintricarboxylic acid.
  • the "test sample” can be any sample containing proteins, nucleic acids or any other charged molecules and includes samples of biological origin such as blood, urine, other bodily fluids, cells [both plant and animal], cell extract, tissues and tissue extract as well as samples of non-biologic origin.
  • solid test samples preferably between about 10 mg- 250 mg of sample, can be ground to a fine powder in liquid nitrogen using a mortar and pestle, and suspended in the extraction buffer Alternatively, homogenized tissues in a hand-held glass-Teflon homogenizer (a loose- fitting homogenizer with a tolerance greater than about 0.1-0.15 mm) can be used.
  • a hand-held glass-Teflon homogenizer a loose- fitting homogenizer with a tolerance greater than about 0.1-0.15 mm
  • about 10 mg-500 mg solid samples can be homogenized in 1 ml extraction buffer by applying as few strokes as possible. Typically, 5-10 strokes are required for complete homogenization. 500 ⁇ l of the homogenate is transferred to a 1.5 ml microcentrifuge tube and an equal volume of molecular grade chloroform is added to the lysate ⁇ homogenate.
  • aqueous phase or top phase
  • chloroform extraction step and centrifugation are repeated to recover nucleic acids.
  • a sterile blade can be used to excise plant tissue, preferably about 10 mg -250 mg of sample, which is then immediately ground to a fine powder in liquid nitrogen using a mortar and pestle, and suspended in 500 ⁇ l of extraction buffer.
  • a sample solution is centrifuged at 12,000 x g for 5 min., then the pellet is re-suspended in 500 ⁇ l of extraction buffer and an equal volume of molecular grade chloroform is added to the lysate ⁇ homogenate.
  • the mixture is made homogenous by inversion and then centrifuged at 12,000 x g for 5 min.
  • the aqueous phase, or top phase is transferred to a new tube and the chloroform extraction step and centrifugation are repeated.
  • Nucleic acid can also be recovered from soil samples by mixing 500 ⁇ l of extraction buffer with 10 mg-100 mg soil sample and vortex vigorously.
  • the recovered nucleic acid solution can be used directly or diluted in a buffer suitable for the hybridization of a probe nucleic acid sequence[s] to a complementary target nucleic acid sequence[s].
  • the probe nucleic acid sequence[s] is a synthetic oligonucleotide from about 18 to about 40 nucleotides in length, and most preferably about 20 to about 25.
  • the directionality of the nucleic acids of the current invention may be either 5' to 3' or the reverse, that is, 3' to 5'.
  • the oligonucleotide probe is synthesized by methods common to the art.
  • the probe nucleic acid sequence(s), preferably about 1 Dmol-100 Dmol, are added to the recovered nucleic acids diluted ten-fold in hybridization buffer (4M -6 M Guanidine Hydrochloride;10 mM-50 mM EDTA and incubated at a desirable temperature range to stimulate the formation of the probe-target complex[es].
  • hybridization buffer 4M -6 M Guanidine Hydrochloride;10 mM-50 mM EDTA and incubated at a desirable temperature range to stimulate the formation of the probe-target complex[es].
  • the formation of the probe-target complex[es] is from about 15 oC. to about 90 oC, depending upon the variables of other properties of the solution, such as ionic strength, pH, nucleic acid sequence concentration, degree of complementality of the sequences, as well as other components of the aqueous medium that may affect melting temperatures.
  • the probe-target complex(es) are allowed to form over a period of about 0.5 h - 6 h
  • probe nucleic acid sequences that are not hybridized with complementary target nucleic acid sequences are separated from probe-target complexes by affinity separation, or a second hybridization reaction or by size exclusion.
  • the unhybridized probe nucleic acid sequence(s) are separated from probe-target complex(es) by contacting the hybridization reaction to DEAE or hydroxylapatite columns under conditions in which the support preferentially binds double-strand nucleic acids.
  • nucleic acid sequences that are 100% complementary to the probe nucleic acid sequences are synthesized and immobilized to a solid support. Immobilization of the nucleic acid can be accomplished by direct or indirect attachment to a solid support.
  • the solid support can be a filter membrane or microtiter plate depending on the application being employed.
  • Direct attachment to the polymeric components of the medium can be accomplished by the formation of covalent bonds between the complementary probe nucleic acid ligand and the polymer. Covalent attachment is generally preferred.
  • the free probe nucleic acid sequencefs] hybridize to their complementary nucleic acid sequence[s] under conditions described in above.
  • noncovalent binding between the ligand and polymer substituents can also be used. For instance, strong noncovalent binding provided by the widely-used biotinstreptavidin and digoxigenin-antidigoxigenin systems can be used to attach ligands to appropriately modified polymeric media.
  • the free probe nucleic acid sequence(s) are separated from probe-target complex(es) by contacting the hybridization reaction to a filter with a specified molecular weight cutoff.
  • a filter with a specified molecular weight cutoff. For example, a 30 micron filter will retain nucleic acid sequences greater than 100 base pairs, while allowing nucleic acid sequences less than 100 bp to be collected in the filtrate. Such filtrates are commonly known in the art.
  • Detection of the target nucleic acid may be accomplished in a variety of ways, but generally through detecting and/or measuring the value or change in value of any chemical or physical property of the product of the assay.
  • physical properties suitable for use in detection and/or measurement include mass or density properties as determined by mass spectrometry or plasmon resonance, optical properties as determined by methods detecting emission, absorption, fluorescence, phosphorescence, luminescence, chemiluminescence, polarization, and refractive index changes, or other known techniques, electrical properties as determined by methods detecting conductivity, absorption or emission of other electromagnetic energy, radioactive properties, as well as induced changes in solution properties such as viscosity, turbidity, and optical rotation.
  • the probe nucleic acid comprises a nucleic acid or analog and a detectable label.
  • the label can be bound ionically, covalently or in any other appropriate manner known in the art.
  • the label is preferably bound to any region of the nucleic acid comprising the probe or tether nucleic acid sequences.
  • the present invention further pertains to a kit used for determining the presence of a target nucleic acid sequence within a test sample.
  • the kit contains a first probe nucleic acid sequence component complementary to the target molecule and a second tether nucleic acid sequence component complementary to at least one subsequence of the probe component.
  • the first and second components are selected such that when admixed, under conditions suitable for hybridization, the first and second components form a probe-tether complex containing at least one double stranded segment and at least one single stranded segment.
  • the components may be provided singly or in combination.
  • the kit may also include additional components.
  • the probe and/or the tether component are detectably labeled.
  • the probe component is detectably labeled.

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Abstract

L'invention concerne des procédés de mise en contact d'un acide nucléique sonde, ou d'une séquence analogue d'acide nucléique complémentaire, avec une séquence d'acide nucléique cible dans des conditions convenant à l'hybridation des deux séquences. L'acide nucléique sonde peut être marqué de façon détectable. La séquence de la sonde ou de la cible peut être immobilisée sur une matrice solide ou sur une surface. La séquence d'acide nucléique sonde non hybridée est séparée du complexe d'acide nucléique cible-sonde et la concentration de la sonde non hybridée est déterminée. L'utilisation de test de pré-absorption d'acide nucléique avec une banque d'oligonucléotides aléatoires est utile afin d'identifier des séquences uniques dans les acides nucléiques cibles.
PCT/US2002/021660 2002-07-10 2002-07-10 Test de pre-absorption d'acide nucleique WO2004007771A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009072812A3 (fr) * 2007-12-04 2009-08-20 Panagene Inc Méthode de marquage sélectif et de détection d'acides nucléiques cibles au moyen de sondes d'acides nucléiques peptidiques immobilisées

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6156502A (en) * 1995-12-21 2000-12-05 Beattie; Kenneth Loren Arbitrary sequence oligonucleotide fingerprinting
US6255456B1 (en) * 1997-12-09 2001-07-03 Incyte Genomics, Inc. Cyclic GMP phosphodiesterase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6156502A (en) * 1995-12-21 2000-12-05 Beattie; Kenneth Loren Arbitrary sequence oligonucleotide fingerprinting
US6255456B1 (en) * 1997-12-09 2001-07-03 Incyte Genomics, Inc. Cyclic GMP phosphodiesterase

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SOUTHERN E.M.: "Detection of specific sequences among DNA fragments separated by gel electrophoresis", JOURNAL OF MOLECULAR BIOLOGY, vol. 98, 1975, pages 503 - 517, XP000564884 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009072812A3 (fr) * 2007-12-04 2009-08-20 Panagene Inc Méthode de marquage sélectif et de détection d'acides nucléiques cibles au moyen de sondes d'acides nucléiques peptidiques immobilisées

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