WO2009032779A2 - Procédés et compositions utilisés dans la séparation, en fonction de sa taille, de l'acide nucléique d'un échantillon - Google Patents

Procédés et compositions utilisés dans la séparation, en fonction de sa taille, de l'acide nucléique d'un échantillon Download PDF

Info

Publication number
WO2009032779A2
WO2009032779A2 PCT/US2008/074689 US2008074689W WO2009032779A2 WO 2009032779 A2 WO2009032779 A2 WO 2009032779A2 US 2008074689 W US2008074689 W US 2008074689W WO 2009032779 A2 WO2009032779 A2 WO 2009032779A2
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
target nucleic
sample
target
dna
Prior art date
Application number
PCT/US2008/074689
Other languages
English (en)
Other versions
WO2009032779A3 (fr
Inventor
Min Seob Lee
Original Assignee
Sequenom, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sequenom, Inc. filed Critical Sequenom, Inc.
Publication of WO2009032779A2 publication Critical patent/WO2009032779A2/fr
Publication of WO2009032779A3 publication Critical patent/WO2009032779A3/fr

Links

Classifications

    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification

Definitions

  • the invention relates in part to products and processes for the separation and detection of nucleic acids from a sample.
  • isolated nucleic acids may be used, inter alia, as a starting material for diagnosis and prognosis of diseases and disorders.
  • the recovery of fragmented nucleic acid from biological samples was considered unimportant, and extraction methods were designed to isolate large, undegraded nucleic acid.
  • RNA and DNA short base pair nucleic acid
  • the invention in part relates to the enrichment, separation and analysis of nucleic acids based on their size.
  • Studies have shown that the majority of cell-free nucleic acid resulting from neoplasms, allograft rejection, autoimmune reactions, fetal tissue, and the like, has a relatively small size of approximately 1 ,200 base pairs or less, whereas the majority of cell-free nucleic acid arising in the host from non-programmed cell death-associated events has a size greater than approximately 1 ,200 base pairs.
  • the majority of fetal DNA is relatively small (approximately 500 base pairs or less), whereas the majority of circulatory, extracellular maternal DNA in maternal plasma is greater than approximately 500 base pairs. Further, in certain instances the circulatory DNA material, which is smaller than approximately 500 base pairs, appears to be almost entirely fetal.
  • the present invention therefore, in part provides products and processes for the separation, based on size discrimination, of relatively short nucleic acid (herein referred to as "target nucleic acid”) from a high background of, for example, genomic nucleic acid (herein referred to as “non-target nucleic acid”).
  • target nucleic acid relatively short nucleic acid
  • non-target nucleic acid genomic nucleic acid
  • the methods of the present invention in part, lead to improved methods for detecting low copy number nucleic acid.
  • the present invention in part provides methods for separating nucleic acid from a sample containing a mixture of nucleic acids, where the nucleic acid is separated based on size, comprising the following steps: introducing a pair of forward and reverse primers to the sample, where the primers anneal to nucleic acid at least about 500 base pairs apart; heating and cooling the sample one or more times, where the pair of primers are more likely to anneal to nucleic acid greater than 500 base pairs in length, and less likely to bind to nucleic acid less than about 500 base pairs, thereby yielding primer-bound nucleic acid and non-primer-bound nucleic acid; separating the primer-bound nucleic acid from the non-primer-bound nucleic acid from the previous step, where the primers are used to separate the nucleic acids.
  • the primer-bound nucleic acid is removed from the sample and analyzed. In some embodiments, either species of nucleic acid (primer-bound or non-primer-bound) may be subsequently analyzed after
  • a target nucleic acid in a sample containing a mixture of target and non-target nucleic acid based on the size of the nucleic acid, where the target nucleic acid size is less than the size of the non-target nucleic acid in the mixture comprising the following steps: introducing a pair of forward and reverse non-target binding primers to the sample; heating and cooling the sample one or more times, where the non-target binding primers anneal to non-target nucleic acid, but not target nucleic acid, based on size; and removing non-target nucleic acid from the sample, where the primers annealed to the non-target nucleic acid are used to remove non-target nucleic acid from the sample, whereby the target nucleic acid has been relatively enriched in the sample compared to the non-target nucleic acid.
  • the pair of non-target binding primers preferentially anneal to non-target nucleic acid, but not target nucleic acid.
  • either the target or non-target nucleic acid may be subsequently analyzed after enrichment.
  • the present invention in part provides methods for enriching for a target nucleic acid in a sample containing a mixture of target and non-target nucleic acid based on the size of the nucleic acid, where the target nucleic acid size is less than the size of the non-target nucleic acid in the mixture, comprising the following steps: introducing a pair of forward and reverse non-target binding primers to the sample; heating and cooling the sample one or more times, where the non-target binding primers anneal to non-target nucleic acid, but not target nucleic acid, based on size; and binding the non-target nucleic acid to a solid support in the sample, where the primers annealed to the non-target nucleic acid are used to bind non-target
  • a target nucleic acid from a sample containing a mixture of target and non-target nucleic acid based on the size of the nucleic acid, where the target nucleic acid size is less than the size of the non-target nucleic acid in the mixture comprising the following steps: introducing a pair of forward and reverse non-target binding primers to the sample; heating and cooling the sample one or more times, where the non-target binding primers anneal to non-target nucleic acid, but not target nucleic acid, based on size; binding the non-target nucleic acid to a solid support in the sample, where the primers annealed to the non- target nucleic acid are used to bind non-target nucleic acid, thereby yielding a supernatant enriched for the target nucleic acid; and removing the supernatant that is enriched for target nucleic acid.
  • the method further comprises analyzing either the target nucleic acid or non- target nucleic acid, or both nucleic acids.
  • methods of the invention may be repeated or combined with other methods until the desired concentration of nucleic acid is present.
  • methods of the present invention allow for the selective enrichment of any nucleic acid less than a given size based on the placement of the forward and reverse primers. For example, primers designed to anneal to non-target nucleic acid 500 base pairs apart will allow for the separation, enrichment or extraction of target nucleic acid less than 500 base pairs.
  • certain methods of the invention are used to separate, enrich or extract nucleic acid within the range of about 25 bases to about 10,000 bases from a sample comprising a background of longer nucleic acid.
  • the target nucleic acid is at least about 75 base pairs, but less than about 1200 base pairs. In some embodiments, the target nucleic acid is less than 500 base pairs. In certain embodiments, the target nucleic acid is about 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525 or 550 base pairs in length.
  • nucleic acids such as DNA, RNA, mRNA, miRNA, siRNA, oligonucleosomal, mitochondrial, epigenetically modified, single-stranded, double-stranded, genomic, circular, plasmid, cosmid, yeast artificial chromosomes, artificial or man-made DNA, including unique DNA sequences, and DNA that has been reverse transcribed from an RNA sample, such as cDNA, and combinations thereof.
  • the methods may be particularly useful for discriminating between RNA of varying length.
  • the nucleic acid is cell-free nucleic acid.
  • the target nucleic acid is derived from apoptotic cells.
  • the target nucleic acid is of fetal origin, and the non-target nucleic acid is of maternal origin.
  • the target nucleic acid comprises one or more polymorphic sites. In certain embodiments, the method further comprises determining the identity of at least one allele within the one or more polymorphic sites. In another related embodiment, the non-target nucleic acid also comprises the same one or more polymorphic sites, and the method further comprises determining the identity of at least one allele within the one or more polymorphic sites on the non- target nucleic acid.
  • the present invention relates to separating, enriching or extracting nucleic acid from a sample such as whole blood, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), biopsy material (e.g., from a pre-implantation embryo), fetal nucleated cells or fetal cellular remnants isolated from maternal blood, washings of the female reproductive tract, or aspirated from a pregnant female's reproductive tract (e.g., cervix or vagina), and a sample obtained by celocentesis, urine, feces, sputum, saliva, nasal mucous, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, embryonic cells and fetal cells, or combinations thereof.
  • a sample such as whole blood, serum, plasma, umbilical cord blood, chorionic
  • the biological sample is plasma. In certain embodiments, the biological sample is cell-free or substantially cell-free. In certain embodiments, the biological sample is a sample of previously extracted, isolated or enriched nucleic acids. In some embodiments, the sample is procured by non-invasive means (e.g., maternal blood draw). In some embodiments, the sample is procured from a subject selected from the group consisting of a pregnant female, a subject suspected of suffering from or at high risk for a neoplasm, and a subject who has undergone an organ or tissue transplant or blood transfusion.
  • non-invasive means e.g., maternal blood draw
  • the biological sample is from an animal, most preferably a human.
  • the biological sample is from a pregnant human.
  • the biological sample is collected from a pregnant human at 1-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32, 32-36, 36-40, or 40-44 weeks of fetal gestation, and preferably between 5-28 weeks of fetal gestation.
  • the pregnant human has a relatively elevated concentration of free fetal nucleic acid in her blood, plasma or amniotic fluid.
  • the pregnant human has a relatively decreased concentration of apoptotic nucleic acid in her blood, plasma or amniotic fluid.
  • Methods of the present invention may be performed in conjunction with any known method to elevate fetal nucleic acid in maternal blood, plasma or amniotic fluid, in certain embodiments.
  • methods of the present invention may be performed in conjunction with any known method to decrease apoptotic nucleic acid in maternal blood, plasma or amniotic fluid, in certain embodiments.
  • methods of the present invention may be used to separate, enrich or extract RNA that is expressed by the fetus.
  • the non-target binding primers anneal to the non- target nucleic acid at least about 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 base pairs apart.
  • the non-target binding primers are introduced to the sample at a concentration greater than the concentration of non-target nucleic acid.
  • the primers may be modified to facilitate their capture.
  • modifications include, but are not limited to, inclusion of capture mechanisms, compomers, tags, linkers and adapter molecules.
  • compomers are described in US Patent Application Publication No. 20050287533, filed June 23, 2004.
  • adapters are described in US Patent Application Publication No. 2003021 1489, filed June 20, 2002.
  • capture mechanisms include, but are not limited to, one or more members of one or more binding pairs.
  • Any suitable binding pair can be utilized to effect a non-covalent linkage, including, but not limited to, antibody/antigen, antibody/antibody, antibody/antibody fragment, antibody/antibody receptor, antibody/protein A or protein G, hapten/anti-hapten, biotin/avidin, biotin/streptavidin, folic acid/folate binding protein, FK506/FK506 binding protein, glutathione/glutathione binding protein, vitamin B12/intrinsic factor, nucleic acid/complementary nucleic acid (e.g., hybridization or capture probes; DNA, RNA, PNA).
  • hapten/anti-hapten biotin/avidin
  • biotin/streptavidin biotin/streptavidin
  • folic acid/folate binding protein FK506/FK506 binding protein
  • glutathione/glutathione binding protein glutathione/glutathione binding protein
  • Covalent linkages also can be effected by a binding pair, such as a chemical reactive group/complementary chemical reactive group (e.g., sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative, amine/isotriocyanate, amine/succinimidyl ester, and amine/sulfonyl halides).
  • a member of the binding pair is linked to a solid support in certain embodiments, and methods and conditions for attaching such binding pairs to reagents and effecting binding are known to the person of ordinary skill in the art.
  • the primer contains a universal binding oligonucleotide capable of hybridizing to a capture probe.
  • a solid support is introduced to the sample or the sample is stored in a solid support capable of binding nucleic acid.
  • the solid support is adapted to bind nucleic acids.
  • the solid support may be selected from, for example, the following: paramagnetic microparticles, silica gel, silica particles, controlled pore glass, magnetic beads, biomagnetic separation beads, microspheres, divinylbenzene (DVB) resin, cellulose beads, capillaries, filter membranes, columns, nitrocellulose paper, flat supports, arrays, glass surfaces, fiber optic arrays, metal surfaces, plastic materials, polycarbonate materials, multiwell plates or membranes, wafers, combs, pins and needles, or combination thereof (for example, wells filled with beads).
  • the solid support is a hydroxyl donor (e.g., silica or glass) or contains a functional group that serves as a hydroxyl donor and is attached to a solid support.
  • the solid support is a silica gel membrane.
  • the solid support has a functional group-coated surface.
  • the functional group-coated surface is silica-coated, hydroxyl coated, amine-coated, carboxyl-coated or encapsulated carboxyl group-coated, for example.
  • a bead may be silica-coated or a membrane may contain silica gel in certain embodiments.
  • the solid support is removed from the sample using a method selected from the group consisting of applying a magnetic field, applying vacuum filtration and centrifugation.
  • paramagnetic beads are separated from the sample using magnets or magnetic devices.
  • the primer contains a label.
  • Primers may be labeled with any type of chemical group or moiety that allows for detection including but not limited to radioactive molecules, fluorescent molecules, antibodies, antibody fragments, haptens, carbohydrates, biotin, derivatives of biotin, phosphorescent moieties, luminescent moieties, electrochemiluminescent moieties, chromatic moieties, and moieties having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity, or any combination of labels thereof.
  • the primers can be labeled with one or more chemical groups or moieties. Each primer can be labeled with the same chemical group or moiety, in certain embodiments. Alternatively, each different primer can be labeled with a different chemical group or moiety, in some embodiments.
  • the labeled primers can be dNTPs, ddNTPs, or a mixture of both dNTPs and ddNTPs.
  • the unlabeled primers can be dNTPs, ddNTPs or a mixture of both dNTPs and ddNTPs.
  • the label is only detectable when the primer is bound to nucleic acid.
  • methods for separating, enriching or extracting a nucleic acid which may be performed prior to, subsequent to, or simultaneously with one or more other methods for selectively separating, enriching or extracting nucleic acid.
  • methods for separating, enriching or extracting nucleic acid include, but are not limited to, electrophoresis, liquid chromatography, size exclusion, microdialysis, electrodialysis, centrifugation, centrifugal membrane exclusion, restriction enzyme-based methods, organic or inorganic extraction, affinity chromatography, PCR, genome-wide PCR, sequence-specific PCR, methylation- specific PCR, restriction endonuclease enhanced polymorphic sequence detection, introducing a silica membrane or molecular sieve, nanopore-based methods, fragment selective amplification, or combinations thereof.
  • Examples of separating, enriching or extracting methods are also provided in PCT Patent Application Publication No. PCT/US07/69991 , filed May 30, 2007.
  • Methods provided herein may also be modified to introduce additional steps, for example, in order to improve the extraction of nucleic acid or improve analysis of target nucleic acid following extraction.
  • the sample may be first lysed in the presence of a lysis buffer, which may comprise a chaotropic agent (e.g., salt), a proteinase, a protease or a detergent, or combinations thereof, for example.
  • Chaotropic agents may be added to the sample to improve the binding of the non-target nucleic acid to the nucleic acid-binding solid support, where the longer, non-target nucleic acid is more likely to bind to the solid support than the shorter, target nucleic acid.
  • the chaotropic agent is selected from the group consisting of guanidine salt, sodium iodide, potassium iodide, sodium thiocyanate, urea, sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride, cesium chloride, ammonium acetate, sodium acetate, ammonium perchlorate and sodium perchlorate.
  • the salt is a guanidine salt, most preferably guanidine (iso)thiocyanate, or is a sodium salt, most preferably sodium perchlorate.
  • the chaotropic agent often is introduced at a concentration sufficient to bind non-target nucleic acid to a solid support.
  • methods that comprise target nucleic acid binding to a solid support may further include adding a washing step or steps to remove non-nucleic acid from the solid- support-target nucleic acid complex.
  • the solid support-nucleic acid complex is further washed successively with a wash buffer and one or more alcohol-water solutions, and subsequently dried.
  • the wash buffer comprises a chaotropic agent (e.g., salt), and optionally, a carrier such as LPA, RNA, tRNA, dextran blue, glycogen or polyA RNA, for example.
  • the target nucleic acid is amplified by a target specific amplification method such as allele-specific PCR.
  • all of the remaining nucleic acid are amplified with a common set of PCR primers.
  • Methods of the invention can permit the analysis of fetal genetic traits including those involved in chromosomal aberrations (e.g. aneuploidies or chromosomal aberrations associated with Down's syndrome) or hereditary Mendelian genetic disorders and, respectively, genetic markers associated therewith (e.g. single gene disorders such as cystic fibrosis or the hemoglobinopathies). Size separation of extracellular fetal DNA in the maternal circulation thus facilitates the non-invasive detection of fetal genetic traits, including paternally inherited polymorphisms.
  • methods that further comprise analyzing the non-target nucleic acid, the target nucleic acid or both the non-target and target nucleic acid.
  • nucleic acid analysis include, but are not limited to, genotype analysis, sequencing analysis, methylation analysis, quantitative analysis and qualitative analysis.
  • the multiple reactions are performed under identical reaction conditions. Multiplexing embodiments are particularly important when multiple regions of a target genome need to be analyzed. In one embodiment, greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 100, 200, 300 or 500 target nucleic acids are enriched, separated or extracted.
  • the processes of the present invention are extremely sensitive and allow the detection of low copy number target nucleic acid that are in various ratios (relative to non- target nucleic acid) including but not limited to about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6-1:10, 1:11-1:20, 1:21-1:30, 1:31-1:40, 1:41-1:50, 1:51-1:60, 1:61-1:70, 1:71-1:80, 1:81-1:90, 1:91:1:100, 1:101- 1:200, 1:250, 1:251-1:300, 1:301-1:400, 1:401-1:500, 1:501-1:600, 1:601-1:700, 1:701-1:800,
  • methods of the present invention result in a final relative percentage of target nucleic acid to non-target nucleic acid of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.
  • the present invention also further relates in part to a kit for enriching, separating or extracting nucleic acid from a sample.
  • the kit may comprise primers of the invention, including, but not limited to, modified primers for selectively enriching for target nucleic acid, and instructions for performing the target nucleic acid enrichment.
  • Figure 1 shows an exemplary embodiment of the invention where size specific depletion of nucleic acid is achieved using biotin labeled primers which bind to streptavidin.
  • Figure 2 is a series of mass spectrograms that shows the successful enrichment of the low copy number, small nucleic acid (200 base pair) in the presence of the high copy number amplicon (800 base pair).
  • Figure 3 is a series of mass spectrograms that shows the successful separation of large fragments from heterogeneous mixtures of nucleic acid at different concentrations.
  • the different concentrations (1:1, 1:10 and 1 :50) represent different ratios of small to large nucleic acid fragments.
  • the low concentration small fragments are either hard to detect (1 :10 ratio) or not detectable (1 :50) before the large fragments are separated. However, after the large fragments are selectively removed using the methods of the present invention, the small fragments are detectable.
  • Figure 4 provides sequences of PCR primers, biotin-labeled probes and a genomic sequence that comprises the target and non-target sequences, demonstrated in the Examples.
  • Cell-free nucleic acid may originate from a number of sources, including programmed cell death, which also is known as apoptosis.
  • programmed cell death also is known as apoptosis.
  • the source of nucleic acid that arises as a result of apoptosis may be found in many body fluids and originate from several sources, including, but not limited to, normal programmed cell death in the host, induced programmed cell death in the case of an autoimmune disease, septic shock, neoplasms (malignant or non- malignant), or non-host sources such as an allograft (transplanted tissue), or the fetus or placenta of a pregnant woman.
  • the methods of the invention may be used to enrich for nucleic acid of fetal origin in a maternal sample. It is well established that fetal nucleic acid is present in maternal plasma from the first trimester onwards, with concentrations that increase with progressing gestational age (Lo et al. Am J Hum Genet (1998) 62:768-775). After delivery, fetal nucleic acid is cleared very rapidly from the maternal plasma (Lo et al.
  • the target nucleic acid is of fetal origin
  • the non-target nucleic acid is of maternal origin
  • the sample is maternal plasma.
  • the present invention includes products and processes to extract and relatively enrich by physical separation short base pair nucleic acid in the presence of a high background of genomic material (e.g., host or maternal nucleic acids). More specifically, the present invention in part provides products and processes for the relative enrichment, based on size discrimination, of target nucleic acid in a high background of genomic nucleic acid (herein referred to as "non-target nucleic acid") where the target nucleic acid size is less than the size of the non-target nucleic acid in the mixture.
  • the target nucleic acid may be, for example, approximately 500 base pairs or less.
  • the methods of the present invention may be used to improve pathogen detection by selectively enriching for pathogen nucleic acid. Methods for rapid identification of unknown bioagents using a combination of nucleic acid amplification and determination of base composition of informative amplicons by molecular mass analysis are disclosed and claimed in published U.S. Patent applications 20030027135, 20030082539,
  • extraction refers to the partial or complete separation, and optionally isolation, of a nucleic acid from a biological or non-biological sample comprising other nucleic acids.
  • selective and selective refer to the ability to extract a particular species of nucleic acid molecule, on the basis of molecular size, from a sample that comprises a mixture of nucleic acid molecules.
  • nucleic acid and “nucleic acid molecule” as used herein may be used interchangeably throughout the disclosure.
  • the terms refer to oligonucleotides, oligos, polynucleotides, deoxyribonucleotide (DNA), genomic DNA, mitochondrial DNA (mtDNA), complementary DNA (cDNA), bacterial DNA, viral DNA, viral RNA, RNA, micro RNA (miRNA), message RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), siRNA, catalytic RNA, clones, plasmids, M13, P1 , cosmid, bacteria artificial chromosome (BAC), yeast artificial chromosome (YAC), amplified nucleic acid, amplicon, PCR product and other types of amplified nucleic acid, RNA/DNA hybrids and polyamide nucleic acids (PNAs), all of which can be in either single- or double-stranded form, and unless otherwise limited, would encompass known analog
  • nucleotides refers to both naturally- occurring and modified/non-naturally-occurring nucleotides, including nucleoside tri, di, and monophosphates as well as monophosphate monomers present within polynucleic acid or oligonucleotide.
  • a nucleotide may also be a ribo; 2'-deoxy; 2', 3'-deoxy as well as a vast array of other nucleotide mimics that are well-known in the art.
  • Mimics include chain-terminating nucleotides, such as 3'-O-methyl, halogenated base or sugar substitutions; alternative sugar structures including nonsugar, alkyl ring structures; alternative bases including inosine; deaza- modified; chi, and psi, linker-modified; mass label-modified; phosphodiester modifications or replacements including phosphorothioate, methylphosphonate, boranophosphate, amide, ester, ether; and a basic or complete internucleotide replacements, including cleavage linkages such a photocleavable nitrophenyl moieties.
  • nucleotides such as 3'-O-methyl, halogenated base or sugar substitutions
  • alternative sugar structures including nonsugar, alkyl ring structures
  • alternative bases including inosine
  • deaza- modified chi, and psi, linker-modified
  • mass label-modified mass label-modified
  • phosphodiester modifications or replacements including
  • target nucleic acid refers to the nucleic acid of interest that is extracted or separated based on its molecular size.
  • the target nucleic acid has a molecular size smaller than the non-target nucleic acid present in the biological sample, for example, smaller than about 500 base pairs.
  • the target nucleic acid is fetal DNA, oncogenic DNA, or any non-host DNA.
  • the target nucleic acid is cell-free nucleic acid.
  • the target nucleic acid is oligonucleosomal nucleic acid generated during programmed cell death.
  • non-target nucleic acid refers to the relatively high amount of background nucleic acid present in a sample.
  • non-target nucleic acid has a molecular size larger than target nucleic acid, for example, greater than about 500 base pairs.
  • non-target nucleic acid is from a host or host cell.
  • non-target nucleic acid is of maternal origin.
  • the non-target nucleic acid is separated or extracted from the sample, thereby yielding a relatively enriched target nucleic acid sample.
  • molecular size refers to the size of a nucleic acid molecule, which may be measured in terms of a nucleic acid molecule's mass or length (bases or base pairs).
  • sample includes a specimen or culture (e.g., microbiological cultures) that includes nucleic acids.
  • a sample may include a specimen of synthetic origin.
  • Biological samples include whole blood, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), biopsy material from a pre-implantation embryo, fetal nucleated cells or fetal cellular remnants isolated from maternal blood, urine, feces, sputum, saliva, nasal mucous, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, washings of the female reproductive tract and a sample obtained by celocentesis, cervical mucosa, embryonic cells and fetal cells.
  • the sample comprises a mixture of nucleic acids.
  • the mixture may comprise nucleic acid from different species or from different individuals.
  • the biological sample contains cellular elements or cellular remnants in maternal blood.
  • the sample is from a pregnant female.
  • the sample is procured through non-invasive means (e.g., a blood draw).
  • non-invasive refers a method for collecting a sample that poses minimal risk to an individual (e.g., the mother, fetus, victim, and the like).
  • An example of a non-invasive method is a blood draw; whereas examples of invasive methods include amniocentesis and chorionic villus sampling, both of which constitute a finite risk to the fetus.
  • the sample is cervical mucosa, which is obtained, for example, by an aspiration catheter.
  • the biological sample is blood, and more preferably plasma.
  • blood encompasses whole blood or any fractions of blood, such as serum and plasma as conventionally defined.
  • Blood plasma refers to a fraction of whole blood, which may result from centrifugation of blood treated with anticoagulants.
  • Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated.
  • the biological sample is cell-free or substantially cell-free.
  • the biological sample is a sample containing previously extracted, isolated or purified nucleic acids.
  • One way of targeting target nucleic acid is to use the non-cellular fraction of a biological sample; thus limiting the amount of intact cellular material (e.g., large strand genomic DNA) from contaminating the sample.
  • a cell-free sample (such as pre-cleared plasma, urine, and the like) is first treated to inactivate intracellular nucleases through the addition of an enzyme, a chaotropic substance, a detergent or any combination thereof.
  • the biological sample is first treated to remove substantially all cells from the sample by any of the methods known in the art, for example, centrifugation, filtration, affinity chromatography, and the like.
  • a cell lysis inhibitor is introduced to the sample. In some embodiments, lysis may be blocked.
  • the sample may be mixed with an agent that inhibits cell lysis to inhibit the lysis of cells, if cells are present, where the agent is a membrane stabilizer, a cross-linker, or a cell lysis inhibitor.
  • the agent is a cell lysis inhibitor such as glutaraldehyde, derivatives of glutaraldehyde, formaldehyde, formalin, or derivatives of formaldehyde. See U.S. patent application 20040137470, which is hereby incorporated by reference, for examples of methods relating to the use of cell lysis inhibitors.
  • Known methods for nucleic acid isolation or extraction from blood, plasma, or serum can be performed prior to, after, or in combination with the methods of the present invention.
  • Any standard DNA or RNA isolation technique can be used to isolate nucleic acid including, but not limited to, QIAamp DNA Blood Midi Kit supplied by QIAGEN.
  • Other standard methods of DNA isolation are described, for example, in (Sambrook et al., Molecular Biology: A Laboratory Approach, Cold Spring Harbor, N. Y. 1989; Ausubel, et al., Current protocols in Molecular Biology, Greene Publishing, Y, 1995).
  • a preferred method for isolation of plasma DNA is described in Chiu et al., 2001 , Clin. Chem. 47: 1607-1613, which is herein incorporated by reference in its entirety.
  • Example 2 of PCT International Application Publication Number 2007/028155, filed on September 1 , 2006; PCT International Application Number PCT/US07/69991 , filed May 31 , 2007; US Provisional Application No. 60/805,073, filed June 16, 2006; and US Provisional Application No. 60/908,167, filed March 26, 2007.
  • the methods of the present invention may further comprise analyzing the non-target nucleic acid, the target nucleic acid, or both the non-target and target nucleic acid prior to, after, or in combination with the separation, extraction or enrichment methods of the present invention.
  • analyzing a nucleic acid may include, but are not limited to, genotyping, sequencing, quantitative analysis and qualitative analysis.
  • Nucleic acid analysis methods known in the art include, for example, PCR, allele specific PCR, gel electrophoresis, ELISA, mass spectrometry, MALDI-TOF mass spectrometry hybridization, primer extension or microsequencing methods, ligase sequence determination methods (e.g., U.S. Pat. Nos.
  • mismatch sequence determination methods e.g., U.S. Pat. Nos. 5,851 ,770; 5,958,692; 6,1 10,684; and 6,183,958
  • ASO allele specific oligonucleotide
  • MSPCR methylation-specific PCR
  • pyrosequencing analysis acycloprime analysis
  • Reverse dot blot Dynamic allele-specific hybridization
  • DASH Dynamic allele-specific hybridization
  • PNA Peptide nucleic acid
  • LNA locked nucleic acids
  • Molecular Beacons Intercalating dye, fluorescence detection, fluorescence resonance energy transfer (FRET), FRET primers, AlphaScreen, SNPstream, genetic bit analysis (GBA), Multiplex minisequencing, SNaPshot, GOOD assay, Microarray miniseq, arrayed primer extension (APEX), Microarray primer extension, Tag arrays, Coded microspheres, Template-
  • PCR-based assays e.g., TAQMAN® PCR System (Applied Biosystems)
  • nucleotide sequencing methods e.g., nucleotide sequencing methods
  • hybridization methods conventional dot blot analyses
  • SSCP single strand conformational polymorphism analysis
  • BeadArray Invader assay
  • DGGE denaturing gradient gel electrophoresis
  • heteroduplex analysis mismatch cleavage detection, and techniques described in Sheffield et al., Proc.
  • Sequenom Inc. detection by mass spectrometry, for example Sequenom Inc.'s primer extension method (e.g., iPLEXTM) or MassCLEAVE® assay (information regarding these and other Sequenom assays may be found at, for example, the sequenom.com website on the World Wide Web) real time-PCR (e.g., US Patent Nos. US 5,210,015, US 5,487,972), or hybridization with a suitable nucleic acid primer specific for the sequence to be detected.
  • Suitable nucleic acid primers can be provided in a format such as a gene chip, bead, or any combination thereof.
  • the target nucleic acid can be analyzed by a variety of methods including but not limited to fluorescence detection, DNA sequencing gel, capillary electrophoresis on an automated DNA sequencing machine, microchannel electrophoresis, and other methods of sequencing, mass spectrometry, time of flight mass spectrometry, quadrupole mass spectrometry, magnetic sector mass spectrometry, electric sector mass spectrometry infrared spectrometry, ultraviolet spectrometry, palentiostatic amperometry or by DNA hybridization techniques including Southern Blots, Slot Blots, Dot Blots, and DNA microarrays, where DNA fragments would be useful as both "probes" and "targets," ELISA, fluorimetry, Fluorescence Resonance Energy Transfer (FRET), SNP-IT, GeneChips, HuSNP, BeadArray, TaqMan assay, Invader assay, MassExtend, or MassCleave T (hMC) method.
  • FRET Fluorescence Resonance Energy Transfer
  • the term “genotype” refers to the identity of alleles or non-homologous variants present in an individual or sample.
  • the term “genotyping a sample” or “genotyping an individual” refers to determining a specific allele or specific nucleotide(s) or polymorphism(s) in a sample or carried by an individual at particular region(s).
  • an "allele” is one of several alternate forms of a gene or non-coding regions of nucleic acid that occupy the same position on a chromosome.
  • the term “allele” can be used to describe nucleic acid from any organism including but not limited to bacteria, viruses, fungi, protozoa, molds, yeasts, plants, humans, non-humans, animals, and archeabacteria.
  • Alleles can have the identical sequence or can vary by a single nucleotide (SNP) or more than one nucleotide.
  • SNP single nucleotide
  • organisms that have two copies of each chromosome if both chromosomes have the same allele, the condition is referred to as homozygous. If the alleles at the two chromosomes are different, the condition is referred to as heterozygous.
  • the locus of interest is SNP X on chromosome 1
  • the maternal chromosome contains an adenine at SNP X (A allele) and the paternal chromosome contains a guanine at SNP X (G allele)
  • the individual is heterozygous at SNP X.
  • polymorphism refers to an allelic variant.
  • Polymorphisms can include single nucleotide polymorphisms (SNPs) as well as simple sequence length polymorphisms.
  • SNPs single nucleotide polymorphisms
  • a polymorphism can be due to one or more nucleotide substitutions at one allele in comparison to another allele or can be due to an insertion or deletion, duplication, inversion and other alterations known to the art.
  • Other polymorphisms include, but are not limited to, restriction fragment length polymorphisms (RFLPs), insertions/deletions, short tandem repeats, such as di-, tri-or tetra- nucleotide repeats (STRs), copy number variations, and the like.
  • RFLPs restriction fragment length polymorphisms
  • STRs tetra- nucleotide repeats
  • polymorphism may include epigenetic variants, as long as cleavage by non-epigenetic
  • amplification reaction refers to any in vitro means for multiplying the copies of nucleic acid.
  • “Amplifying” refers to a step of submitting a sample to conditions sufficient to allow for amplification.
  • Components of an amplification reaction may include, but are not limited to, for example, primers, a polynucleotide template, polymerase, nucleotides, dNTPs and the like.
  • the term “amplifying” typically refers to an "exponential" increase in target nucleic acid. However, “amplifying” as used herein can also refer to linear increases in the numbers of a select target sequence of nucleic acid, but is different than a one-time, single primer extension step.
  • PCR Polymerase chain reaction
  • Oligomers as used herein refers to linear oligomers of natural or modified nucleosidic monomers linked by phosphodiester bonds or analogs thereof.
  • Oligonucleotides include deoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptide nucleic acids (PNAs), and the like, capable of specifically binding to a target nucleic acid.
  • PNAs peptide nucleic acids
  • monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., 3-4, to several tens of monomeric units, e.g., 40-60.
  • oligonucleotide is represented by a sequence of letters, such as "ATGCCTG,” it will be understood that the nucleotides are in 5'-3' order from left to right and that "A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, “T” denotes deoxythymidine, and “U” denotes the ribonucleoside, uridine, unless otherwise noted.
  • oligonucleotides of the invention comprise the four natural deoxynucleotides; however, they may also comprise ribonucleosides or non-natural nucleotide analogs.
  • an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity, e.g., single stranded DNA, RNA/DNA duplex, or the like
  • selection of appropriate composition for the oligonucleotide or polynucleotide substrates is well within the knowledge of one of ordinary skill.
  • the terms "primer”, "oligonucleotide primer” and “probe” are interchangeable when used to discuss an oligonucleotide that anneals to a nucleic acid.
  • an oligonucleotide primer serves as a point of initiation of nucleic acid synthesis.
  • oligonucleotide primers of the invention may not be used solely for nucleic acid synthesis. Rather, they may be used as probes to selectively bind to non-target nucleic acid and to "fish out” or otherwise isolate the nucleic acid to which it is annealed.
  • Primers of the invention can be a variety of lengths and are often less than 50 nucleotides in length, for example 12-25 nucleotides in length. The length and sequences of primers for use in the invention can be designed based on principles known to those of skill in the art.
  • flanking means the sequences of the primers are such that at least a portion of the 3' region of one primer is complementary to the antisense strand of the template DNA and upstream from the locus of interest site (forward primer), and at least a portion of the 3' region of the other primer is complementary to the sense strand of the template DNA and downstream of the locus of interest (reverse primer).
  • a "primer pair” refers to a forward primer and a reverse primer. Primers can be prepared by a variety of methods including but not limited to cloning of appropriate sequences and direct chemical synthesis using methods well known in the art (Narang et al., Methods Enzymol.
  • Primers can also be obtained from commercial sources such as Operon Technologies, Amersham Pharmacia Biotech, Sigma, and Life Technologies.
  • the primers can have an identical melting temperature.
  • the lengths of the primers can be extended or shortened at the 5' end or the 3' end to produce primers with desired melting temperatures.
  • the annealing position of each primer pair can be designed such that the sequence and, length of the primer pairs yield the desired melting temperature.
  • Computer programs can also be used to design primers, including but not limited to Array Designer Software (Arrayit Inc.), Oligonucleotide Probe Sequence Design Software for Genetic Analysis (Olympus Optical Co.), NetPrimer, and DNAsis from Hitachi Software Engineering.
  • Array Designer Software Arrayit Inc.
  • Oligonucleotide Probe Sequence Design Software for Genetic Analysis Oligonucleotide Probe Sequence Design Software for Genetic Analysis
  • NetPrimer NetPrimer
  • DNAsis from Hitachi Software Engineering.
  • the TM (melting or annealing temperature) of each primer can be easily calculated using methods well known in the art.
  • the primers can be modified with a tag that facilitates isolation and/or extraction of the nucleic acid.
  • the primers are modified with a tag that facilitates isolation and/or extraction of the nucleic acids. The modification is preferably the same for all primers.
  • the tag can be any chemical moiety including but not limited to a radioisotope, fluorescent reporter molecule, chemiluminescent reporter molecule, antibody, antibody fragment, hapten, biotin, derivative of biotin, photobiotin, iminobiotin, digoxigenin, avidin, enzyme, acridinium, sugar, enzyme, apoenzyme, homopolymeric oligonucleotide, hormone, ferromagnetic moiety, paramagnetic moiety, diamagnetic moiety, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity, or combinations thereof.
  • a primer is labeled with biotin which may bind to immobilized streptavidin (Kandpal et al., Nucleic Acids Res. 18:1789-1795 (1990); Kaneoka et al., Biotechniques 10:30-34 (1991 ); Green et al., Nucleic Acids Res. 18:6163-6164 (1990)).
  • biotin provides an affinity tag that can be used to separate the target nucleic acid from the non-target nucleic acid.
  • Biotinylated molecules can be purified using a streptavidin coated substrate, including but not limited to Streptawell, transparent, High-Bind plates from Roche Molecular Biochemicals (catalog number 1 645 692, as listed in Roche Molecular Biochemicals, 2001 Biochemicals Catalog).
  • the term "functional group-coated surface” as used herein refers to a surface which is coated with moieties which bind nucleic acids.
  • One example is a surface which is coated with moieties which each have a free functional group which is bound to the amino group of the amino silane or the solid support; as a result, the surfaces of the solid support are coated with the functional group containing moieties.
  • the functional group is a carboxylic acid.
  • a suitable moiety with a free carboxylic acid functional group is a succinic acid moiety in which one of the carboxylic acid groups is bonded to the amine of amino silanes through an amide bond and the second carboxylic acid is unbonded, resulting in a free carboxylic acid group attached or tethered to the surface of the paramagnetic microparticle.
  • Suitable solid phase carriers having a functional group coated surface that reversibly binds nucleic acid molecules are for example, magnetically responsive solid phase carriers having a functional group-coated surface, such as, but not limited to, silica-coated, hydroxyl-coated, amino-coated, carboxyl-coated and encapsulated carboxyl group-coated magnetic beads.
  • phosphorus dendrimer linkers are introduced to the solid support to capture nucleic acid.
  • Archer et al. describes a magnetic bead-based method for capturing target nucleic acid with a probe (Anal Biochem. 2006 Aug 15;355(2):285-97).
  • certain methods of the present invention are combined with the improved magnetic bead-based capture methods described by Archer et al.
  • the methods include adding a washing step or steps to remove non- nucleic acid molecules, for example salts, from the solid-support-target nucleic acid complex or surrounding solution.
  • Non-nucleic acid molecules are then removed with an alcohol-based wash and the target nucleic acid is eluted under low- or no-salt conditions (TE buffer or water) in small volumes, ready for immediate use without further concentration.
  • extraction is improved by the introduction of a carrier such as tRNA, glycogen, polyA RNA, dextran blue, linear poly acrylamide (LPA), or any material that increases the recovery of nucleic acid.
  • a carrier such as tRNA, glycogen, polyA RNA, dextran blue, linear poly acrylamide (LPA), or any material that increases the recovery of nucleic acid.
  • the final relative percentage of target nucleic acid to non-target nucleic acid is at least about 5-6% target DNA, about 7-8% target DNA, about 9-10% target DNA, about 1 1-12% target DNA, about 13-14% target DNA. about 15-16% target DNA, about 16-17% target DNA, about 17-18% target DNA, about 18-19% target DNA, about 19-20% target DNA, about 20-21% target DNA, about 21-22% target DNA, about 22-23% target DNA, about 23-24% target DNA, about 24-25% target DNA, about 25-35% target DNA, about 35-45% target DNA, about 45- 55% target DNA, about 55-65% target DNA, about 65-75% target DNA, about 75-85% target DNA, about 85-90% target DNA, about 90-91 % target DNA, about 91-92% target DNA, about 92-93% target DNA, about 93-94% target DNA, about 94-95% target DNA, about 95-96% target DNA, about 96-97% target DNA, about 97-98% target DNA, about 98-99% target DNA, or about 99-
  • certain methods of the present invention may be used together with any known technique suitable for the extraction, isolation or enrichment of nucleic acids, including, but not limited to, cesium chloride gradients, gradients, sucrose gradients, glucose gradients, centrifugation protocols, boiling, Microcon 100 filter, Chemagen viral DNA/RNA 1 k kit, Chemagen blood kit, Qiagen purification systems, Qiagen MinElute kits, QIA DNA blood purification kit, HiSpeed Plasmid Maxi Kit, QIAfilter plasmid kit, Promega DNA purification systems, MangeSil Paramagnetic Particle based systems, Wizard SV technology, Wizard Genomic DNA purification kit, Amersham purification systems, GFX Genomic Blood DNA purification kit, Invitrogen Life Technologies Purification Systems, CONCERT purification system, Mo Bio Laboratories purification systems, UltraClean BloodSpin Kits, and UlraClean Blood DNA Kit.
  • Circulating nucleic acids in the plasma and serum of patients can be used to diagnose or prognose certain diseases and conditions (See, Lo YMD et al., N Eng J Med 1998;339:1734-8; Chen XQ, et al., Nat Med 1996;2:1033-5, Nawroz H et al., Nat Med 1996;2:1035-7; Lo YMD et al., Lancet 1998;351 :1329-30; Lo YMD, et al., Clin Chem 2000;46:319-23).
  • the characteristics and biological origin of circulating nucleic acids are not completely understood. However, it is likely that cell death, including apoptosis, is one major factor (Fournie et al., Gerontology 1993;39:215-21 ; Fournie et al., Cancer Lett 1995;91 :221-7). Without being bound by theory, as cells undergoing apoptosis dispose nucleic acids into apoptotic bodies, it is possible that at least part of the circulating nucleic acids in the plasma or serum of human subjects is short, fragmented DNA that takes the form of particle-associated nucleosomes.
  • the present invention in part provides methods for extracting the short, fragmented circulating nucleic acids present in biological samples, thereby enriching the short, predictive nucleic acids relative to the background genomic DNA.
  • the present invention in part provides methods of evaluating a disease condition in a patient suspected of suffering or known to suffer from the disease condition.
  • Some embodiments of the invention include obtaining a biological sample from the patient suspected of suffering or known to suffer from a disease condition, selectively extracting or enriching extracellular nucleic acid in the sample based on its size using the methods provided herein, and evaluating the disease condition by determining the amount, concentration or characteristic of enriched nucleic acid.
  • the amount, concentration or characteristic of enriched nucleic acid may be compared to a control (e.g., nucleic acid from a healthy individual).
  • evaluating a disease condition refers to assessing the disease condition of a patient.
  • evaluating the condition of a patient can include detecting the presence or absence of the disease in the patient. Once the presence of disease in the patient is detected, evaluating the disease condition of the patient may include determining the severity of disease in the patient. It may further include using that determination to make a disease prognosis, e.g. a prognosis or treatment plan. Evaluating the condition of a patient may also include determining if a patient has a disease or has suffered from a disease condition in the past. Evaluating the disease condition in that instant might also include determining the probability of reoccurrence of the disease condition or monitoring the reoccurrence in a patient.
  • Evaluating the disease condition might also include monitoring a patient for signs of disease. Evaluating a disease condition therefore includes detecting, diagnosing, or monitoring a disease condition in a patient as well as determining a patient prognosis or treatment plan. The method of evaluating a disease condition aids in risk stratification.
  • nucleic acids including DNA and RNA
  • RNA RNA
  • RNA molecules are known to be present (Lo KW, et al. CHn Chem (1999) 45,1292-1294). These molecules are likely packaged in apoptotic bodies and, hence, rendered more stable compared to 'free RNA' (Anker P and Stroun M, CHn Chem (2002) 48, 1210-1211 ; Ng EK, et al. Proc Natl Acad Sci USA (2003) 100, 4748-4753).
  • Nucleic acid derived from cancer patients display tumor-specific characteristics, including decreased strand stability, Ras and p53 mutations, mircrosatellite alterations, abnormal promoter hypermethylation, mitochondrial DNA mutations and tumor-related viral DNA (Stroun M, et al. Oncology (1989) 46,318-322; Chen XQ, et al. Nat Med (1996) 2,1033-1035; Anker P, et al. Cancer Metastasis Rev (1999) 18,65-73; Chan KC and Lo YM, Histol Histopathol (2002) 17,937-943).
  • Tumor-specific DNA for a wide range of malignancies has been found: haematological, colorectal, pancreatic, skin, head-and-neck, lung, breast, kidney, ovarian, nasopharyngeal, liver, bladder, gastric, prostate and cervix.
  • haematological, colorectal, pancreatic, skin, head-and-neck, lung, breast, kidney, ovarian, nasopharyngeal, liver, bladder, gastric, prostate and cervix In aggregate, the above data show that tumor-derived DNA in plasma is ubiquitous in affected patients, and likely the result of a common biological process such as apoptosis. Investigations into the size of these plasma DNA fragments from cancer patients have revealed that the majority show lengths in multiples of nucleosomal DNA, a characteristic of apoptotic DNA fragmentation (Gri S, et al. Cancer Res (2001 ) 61 ,1659-1665).
  • the present invention in part relates to detection of specific extracellular nucleic acid in plasma or serum fractions of human or animal blood associated with neoplastic, pre-malignant or proliferative disease.
  • the invention in part relates to detection of nucleic acid derived from mutant oncogenes or other tumor-associated DNA, and to those methods of detecting and monitoring extracellular mutant oncogenes or tumor-associated DNA found in the plasma or serum fraction of blood by using DNA extraction with enrichment for mutant DNA as provided herein.
  • the invention in part relates to the detection, identification, or monitoring of the existence, progression or clinical status of benign, premalignant, or malignant neoplasms in humans or other animals that contain a mutation that is associated with the neoplasm through the size selective enrichment methods provided herein, and subsequent detection of the mutated nucleic acid of the neoplasm in the enriched DNA.
  • the present invention in part features methods for identifying DNA originating from a tumor in a biological sample. These methods may be used to differentiate or detect tumor-derived DNA in the form of apoptotic bodies or nucleosomes in a biological sample. In certain embodiments embodiments, the non-cancerous DNA and tumor-derived DNA are differentiated by observing nucleic acid size differences, where low base pair DNA is associated with cancer.
  • the total concentration of circulating DNA is in the range of about 10 to about 100ng or 1 ,000 to 10,000 genome equivalents/ml plasma (Bischoff et al., Hum Reprod Update. 2005 Jan-Feb;11 (1 ):59-67 and references cited therein) while the concentrations of the fetal DNA fraction increases from about 20 copies/ml in the first trimester to greater than 250 copies/ml in the third trimester.
  • the present invention in part features methods for separating DNA species originating from different individuals in a biological sample. These methods may be used to enrich and thereby detect fetal DNA in a maternal sample. The separation of maternal and fetal DNA may be performed with or without quantifying the concentration of fetal DNA in the sample. In embodiments where the fetal DNA is quantified, the measured concentration may be used to predict, monitor or diagnose or prognosticate a pregnancy-associated disorder.
  • the first marker that was developed for fetal DNA detection in maternal plasma was the Y chromosome (Lo et al. Am J Hum Genet (1998) 62:768-775).
  • the robustness of Y chromosomal markers has been reproduced by many researchers in the field (Costa JM, et al. Prenat Diagn 21 :1070-1074).
  • This approach constitutes a highly accurate method for the determination of fetal gender, which is useful for the prenatal investigation of sex-linked diseases (Costa JM, Ernault P (2002) Clin Chem 48:679-680).
  • Maternal plasma DNA analysis is also useful for the noninvasive prenatal determination of fetal RhD blood group status in RhD-negative pregnant women (Lo et al.
  • the present invention in part features methods of detecting abnormalities in a fetus by detecting fetal DNA in a biological sample obtained from a mother.
  • certain methods according to the present invention provide for detecting fetal DNA in a maternal sample by separating the fetal DNA from the maternal DNA based on DNA characteristics (e.g., size). See Chan et al. CHn Chem.
  • fetal DNA that is predictive of a genetic anomaly or genetic-based disease may be detected and analyzed, thereby providing improved methods for prenatal diagnosis.
  • These methods are applicable to any and all pregnancy-associated conditions for which nucleic acid changes, mutations or other characteristics (e.g., methylation state) are associated with a disease state.
  • Exemplary diseases that may be diagnosed include, for example, preeclampsia, preterm labor, hyperemesis gravidarum, ectopic pregnancy, fetal chromosomal aneuploidy (such as trisomy 18, 21 , or 13), intrauterine growth retardation, achondroplasia, myotonic dystrophy, cystic fibrosis, Huntington disease and congenital adrenal hyperplasia. It is expected that the spectrum of such applications will increase with enrichment methods herein.
  • products and processes of the present invention allow for the detection of chromosomal aberrations (e.g. aneuploidies or chromosomal aberrations associated with Down's syndrome) and hereditary Mendelian genetic disorders, including genetic markers associated therewith (e.g. single gene disorders such as cystic fibrosis or the hemoglobinopathies). Therefore, the size-based separation and enrichment of extracellular fetal DNA as described herein facilitates the non-invasive detection of fetal genetic traits, including paternally inherited alleles.
  • pregnancy-associated disorder refers to any condition or disease that may affect a pregnant woman, the fetus the woman is carrying, or both the woman and the fetus.
  • Such a condition or disease may manifest its symptoms during a limited time period, e.g., during pregnancy or delivery, or may last the entire life span of the fetus following its birth.
  • a pregnancy-associated disorder include ectopic pregnancy, preeclampsia, preterm labor, sex-linked disorders, and fetal chromosomal abnormalities such as trisomy 13, 18, or 21.
  • chromosomal abnormality refers to a deviation between the structure of the subject chromosome and a normal homologous chromosome.
  • normal refers to the predominate karyotype or banding pattern found in healthy individuals of a particular species.
  • a chromosomal abnormality can be numerical or structural, and includes, but is not limited to, aneuploidy, polyploidy, inversion, a trisomy, a monosomy, duplication, deletion, deletion of a part of a chromosome, addition, addition of a part of chromosome, insertion, a fragment of a chromosome, a region of a chromosome, chromosomal rearrangement, and translocation.
  • a chromosomal abnormality can also be correlated with presence of a pathological condition or with a predisposition to develop a pathological condition.
  • certain products and processes of the invention may be used in conjunction with other non-invasive and invasive techniques available for detecting pregnancy- associated disorders, including ultrasonography, nuchal translucency, amniocentesis, chorionic villi sampling (CVS), fetal blood cells in maternal blood, maternal serum alpha-fetoprotein, maternal serum beta-HCG, maternal serum estriol, and other prenatal diagnostic techniques described, for example, in the following U.S. Patents and Applications: U.S. Patent Application No. 09/380,696, which issued July 10, 2001 as U.S. Patent No. 6,258,540; U.S. Patent Application No. 10/759,783, which published October 14, 2004 as Application Publication No. 20040203037; U.S. Patent Application No.
  • apoptotic or nucleosomal nucleic acid that may be detected by the methods provided herein.
  • Other diseases and disorders believed to produce apoptotic nucleic acid include diabetes, heart disease, stroke, trauma, rheumatoid arthritis and lupus erythematosus (SLE) (Rumore and Steinman J Clin Invest. 1990 Jul;86(1 ):69-74). Rumore et al.
  • the present invention provides methods of evaluating the disease condition of a patient suspected of having suffered from a trauma or known to have suffered from a trauma.
  • the methods include obtaining a sample of plasma or serum from the patient suspected of having suffered from a trauma or known to have had suffered from a trauma, and detecting the quantity or concentration of mitochondrial nucleic acid in the sample.
  • biotin labeled forward and reverse primer mix was added as described in Table 3.
  • the biotin labeled forward and reverse primer mix consists of the probes designed to anneal to the non-target nucleic acid, where the biotin can be used to "fish out” or separate the non- target nucleic acid from the target nucleic acid.
  • the primer sequences and DNA fragment sequences are provided in Figure 4.
  • the total final volume for each well in this plate was 36ul.
  • 2OuI of DNA and Biotin-Primer Mix were used in the subsequent binding step.
  • the 96 well plate with 2OuI of DNA & Biotin-Primer mix was denatured by heating to 95°C for 5 minutes, then cooled down to room temperature for 30 minutes. After the plates cooled to room temperature, the following 6OuI streptavidin magnetic bead master mix was added to each well: 2ul beads, 4OuI binding buffer and 18ul water. The plates were vortexed at mid speed of 4 with a vortexor for 10 minutes at room temperature to keep the beads dispersed. The plates were then spun down and placed in a magnetic bed. About 60 ul of supernatant were transferred to a new plate for MassARRAY® analysis.
  • the remaining DNA was subjected to PCR amplification using the reagents provided in Table 4.
  • the MassARRAY® primers are provided in Figure 4.
  • PCR cycling was performed for 45 cycles, where each cycle is 94° C for 15 minutes, 94° C for 20 seconds, 56° C for 30 seconds, 72° C for 1 minute, 72° C for 3 minutes. Then the products were maintained at 4° C thereafter.
  • each cycle includes 94° C for 30 seconds, 94° C for 5 seconds, 52° C for 5 seconds, 80° C for 5 seconds and 72° C for 3 minutes, and then the products were maintained at 4° C thereafter.
  • reference to “a subset” includes a plurality of such subsets
  • reference to “a nucleic acid” includes one or more nucleic acids and equivalents thereof known to those skilled in the art, and so forth.
  • the term “or” is not meant to be exclusive to one or the terms it designates.
  • a or B may denote A alone, B alone, or both A and B.
  • all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and systems similar or equivalent to those described herein can be used in the practice or testing of the present invention, the methods, devices, and materials are now described.

Abstract

L'invention concerne des procédés et des trousses utilisés dans la séparation, l'extraction et l'enrichissement de l'acide nucléique à courte chaîne en présence d'un fond important d'un matériau génomique à longue chaîne (par exemple, des acides nucléiques hôtes ou maternels). Ces procédés reposent sur des amorces qui se lient sélectivement à l'acide nucléique à longue chaîne, ces amorces pouvant être utilisées pour séparer par la suite l'acide nucléique à longue chaîne et à courte chaîne.
PCT/US2008/074689 2007-08-29 2008-08-28 Procédés et compositions utilisés dans la séparation, en fonction de sa taille, de l'acide nucléique d'un échantillon WO2009032779A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US96887607P 2007-08-29 2007-08-29
US60/968,876 2007-08-29

Publications (2)

Publication Number Publication Date
WO2009032779A2 true WO2009032779A2 (fr) 2009-03-12
WO2009032779A3 WO2009032779A3 (fr) 2009-05-07

Family

ID=40429663

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/074689 WO2009032779A2 (fr) 2007-08-29 2008-08-28 Procédés et compositions utilisés dans la séparation, en fonction de sa taille, de l'acide nucléique d'un échantillon

Country Status (1)

Country Link
WO (1) WO2009032779A2 (fr)

Cited By (89)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8206926B2 (en) 2008-03-26 2012-06-26 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection
EP2569453A2 (fr) * 2010-05-14 2013-03-20 Fluidigm Corporation Méthodes d'isolement de l'acide nucléique
WO2013052907A2 (fr) 2011-10-06 2013-04-11 Sequenom, Inc. Méthodes et procédés pour évaluation non invasive de variations génétiques
WO2013052913A2 (fr) 2011-10-06 2013-04-11 Sequenom, Inc. Procédés et processus d'évaluation non invasive de variations génétiques
WO2013055817A1 (fr) 2011-10-11 2013-04-18 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
WO2013177086A1 (fr) 2012-05-21 2013-11-28 Sequenom, Inc. Procédés et méthodes d'évaluation non invasive de variations génétiques
WO2013192562A1 (fr) 2012-06-22 2013-12-27 Sequenom, Inc. Procédés et processus pour l'évaluation non invasive de variations génétiques
WO2014011928A1 (fr) 2012-07-13 2014-01-16 Sequenom, Inc. Procédés et compositions pour l'enrichissement basé sur la méthylation d'un échantillon maternel en acide nucléique fœtal, utiles pour les diagnostics prénatals non invasifs
US8652780B2 (en) 2007-03-26 2014-02-18 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection
US8688388B2 (en) 2011-10-11 2014-04-01 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
WO2014055774A1 (fr) 2012-10-04 2014-04-10 Sequenom, Inc. Procédés et processus d'estimation non invasive de variations génétiques
WO2014055790A2 (fr) 2012-10-04 2014-04-10 Sequenom, Inc. Procédés et processus d'évaluation non invasive de variations génétiques
WO2014116598A2 (fr) 2013-01-25 2014-07-31 Sequenom, Inc. Procédés et processus d'évaluation non invasive de variations génétiques
WO2014164838A1 (fr) 2013-03-12 2014-10-09 Sequenom, Inc. Quantification de marqueurs d'acides nucléiques spécifiques de cellules présentant un état de méthylation particulier
WO2014165596A1 (fr) 2013-04-03 2014-10-09 Sequenom, Inc. Procédés et systèmes d'évaluation non invasive de variations génétiques
WO2014168711A1 (fr) 2013-03-13 2014-10-16 Sequenom, Inc. Amorces pour analyse de la méthylation de l'adn
US8949036B2 (en) 2010-05-18 2015-02-03 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US8962247B2 (en) 2008-09-16 2015-02-24 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non invasive prenatal diagnoses
WO2015051163A2 (fr) 2013-10-04 2015-04-09 Sequenom, Inc. Méthodes et processus d'évaluation non invasive de variations génétiques
WO2015054080A1 (fr) 2013-10-07 2015-04-16 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de modifications chromosomiques
US9051608B2 (en) 2006-12-05 2015-06-09 Agena Bioscience, Inc. Detection and quantification of biomolecules using mass spectrometry
US9163282B2 (en) 2010-05-18 2015-10-20 Natera, Inc. Methods for non-invasive prenatal ploidy calling
WO2015159979A1 (fr) * 2014-04-18 2015-10-22 凸版印刷株式会社 Procédé de récupération d'acides nucléiques à courte chaîne
WO2015183872A1 (fr) 2014-05-30 2015-12-03 Sequenom, Inc. Déterminations de représentation de chromosomes
US9228234B2 (en) 2009-09-30 2016-01-05 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US9305756B2 (en) 2013-03-13 2016-04-05 Agena Bioscience, Inc. Preparation enhancements and methods of use for MALDI mass spectrometry
US9310378B2 (en) 2008-01-15 2016-04-12 Agena Bioscience, Inc. Compositions and processes for improved mass spectrometry analysis
WO2016057901A1 (fr) 2014-10-10 2016-04-14 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US9424392B2 (en) 2005-11-26 2016-08-23 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US9453257B2 (en) 2006-05-31 2016-09-27 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US9499870B2 (en) 2013-09-27 2016-11-22 Natera, Inc. Cell free DNA diagnostic testing standards
US9605313B2 (en) 2012-03-02 2017-03-28 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US9639657B2 (en) 2008-08-04 2017-05-02 Natera, Inc. Methods for allele calling and ploidy calling
US9677118B2 (en) 2014-04-21 2017-06-13 Natera, Inc. Methods for simultaneous amplification of target loci
WO2017205826A1 (fr) 2016-05-27 2017-11-30 Sequenom, Inc. Méthodes de détection de variations génétiques
US9850480B2 (en) 2009-04-03 2017-12-26 Sequenom, Inc. Nucleic acid preparation compositions and methods
WO2018022906A1 (fr) 2016-07-27 2018-02-01 Sequenom, Inc. Procédés d'évaluation non invasive de l'instabilité génomique
WO2018022890A1 (fr) 2016-07-27 2018-02-01 Sequenom, Inc. Classifications de modifications du nombre de copies génétiques
US9920361B2 (en) 2012-05-21 2018-03-20 Sequenom, Inc. Methods and compositions for analyzing nucleic acid
US9926593B2 (en) 2009-12-22 2018-03-27 Sequenom, Inc. Processes and kits for identifying aneuploidy
US9984198B2 (en) 2011-10-06 2018-05-29 Sequenom, Inc. Reducing sequence read count error in assessment of complex genetic variations
US10011870B2 (en) 2016-12-07 2018-07-03 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
WO2018136888A1 (fr) 2017-01-20 2018-07-26 Sequenom, Inc. Procédés d'évaluation non invasive d'alterations genetique
WO2018136882A1 (fr) 2017-01-20 2018-07-26 Sequenom, Inc. Procédés d'évaluation non invasive de variations du nombre de copies
WO2018136881A1 (fr) 2017-01-20 2018-07-26 Sequenom, Inc. Fabrication et utilisation d'adaptateur de séquençage
WO2018140521A1 (fr) 2017-01-24 2018-08-02 Sequenom, Inc. Méthodes et procédés d'évaluation de variations génétiques
WO2018170511A1 (fr) 2017-03-17 2018-09-20 Sequenom, Inc. Procédés et techniques d'évaluation d'un mosaïcisme génétique
US10083273B2 (en) 2005-07-29 2018-09-25 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10081839B2 (en) 2005-07-29 2018-09-25 Natera, Inc System and method for cleaning noisy genetic data and determining chromosome copy number
US10113196B2 (en) 2010-05-18 2018-10-30 Natera, Inc. Prenatal paternity testing using maternal blood, free floating fetal DNA and SNP genotyping
JP2018183189A (ja) * 2012-07-24 2018-11-22 ナテラ， インコーポレイテッド 高度多重ｐｃｒ法および組成物
US10179937B2 (en) 2014-04-21 2019-01-15 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10196681B2 (en) 2011-10-06 2019-02-05 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US10262755B2 (en) 2014-04-21 2019-04-16 Natera, Inc. Detecting cancer mutations and aneuploidy in chromosomal segments
US10316362B2 (en) 2010-05-18 2019-06-11 Natera, Inc. Methods for simultaneous amplification of target loci
US10323268B2 (en) 2011-10-06 2019-06-18 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
WO2019140201A1 (fr) 2018-01-12 2019-07-18 Claret Bioscience, Llc Procédés et compositions d'analyse d'acide nucléique
EP3540076A1 (fr) 2013-06-21 2019-09-18 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US10424394B2 (en) 2011-10-06 2019-09-24 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US10504613B2 (en) 2012-12-20 2019-12-10 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
EP3578670A1 (fr) 2013-05-24 2019-12-11 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US10526658B2 (en) 2010-05-18 2020-01-07 Natera, Inc. Methods for simultaneous amplification of target loci
US10577655B2 (en) 2013-09-27 2020-03-03 Natera, Inc. Cell free DNA diagnostic testing standards
US10738358B2 (en) 2008-09-16 2020-08-11 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
WO2020206143A1 (fr) 2019-04-05 2020-10-08 Claret Bioscience, Llc Procédés et compositions d'analyse d'acide nucléique
EP3760739A1 (fr) 2014-07-30 2021-01-06 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US10894976B2 (en) 2017-02-21 2021-01-19 Natera, Inc. Compositions, methods, and kits for isolating nucleic acids
WO2021072037A1 (fr) 2019-10-09 2021-04-15 Claret Bioscience, Llc Procédés et compositions d'analyse d'acide nucléique
WO2021087491A1 (fr) 2019-10-31 2021-05-06 Sequenom, Inc. Application d'un rapport du mosaïcisme dans des gestations multifœtales et évaluation de risque personnalisé
US11004537B2 (en) 2011-06-24 2021-05-11 Sequenom, Inc. Methods and processes for non invasive assessment of a genetic variation
US11111544B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US11111543B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
WO2021262805A1 (fr) 2020-06-24 2021-12-30 Claret Bioscience, Llc Procédés et compositions pour analyse d'acide nucléique
US11299780B2 (en) 2016-07-15 2022-04-12 The Regents Of The University Of California Methods of producing nucleic acid libraries
WO2022076574A1 (fr) 2020-10-08 2022-04-14 Claret Bioscience, Llc Procédés et compositions d'analyse d'acide nucléique
US11322224B2 (en) 2010-05-18 2022-05-03 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11326208B2 (en) 2010-05-18 2022-05-10 Natera, Inc. Methods for nested PCR amplification of cell-free DNA
US11332785B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11332793B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for simultaneous amplification of target loci
US11339429B2 (en) 2010-05-18 2022-05-24 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11365447B2 (en) 2014-03-13 2022-06-21 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US11408031B2 (en) 2010-05-18 2022-08-09 Natera, Inc. Methods for non-invasive prenatal paternity testing
EP3973081A4 (fr) * 2019-05-23 2022-09-07 American Molecular Laboratories, Inc. Procédés de détection de séquences d'adn rares dans des échantillons fécaux
US11479812B2 (en) 2015-05-11 2022-10-25 Natera, Inc. Methods and compositions for determining ploidy
US11485996B2 (en) 2016-10-04 2022-11-01 Natera, Inc. Methods for characterizing copy number variation using proximity-litigation sequencing
US11525159B2 (en) 2018-07-03 2022-12-13 Natera, Inc. Methods for detection of donor-derived cell-free DNA
US11629345B2 (en) 2018-06-06 2023-04-18 The Regents Of The University Of California Methods of producing nucleic acid libraries and compositions and kits for practicing same
US11697849B2 (en) 2012-01-20 2023-07-11 Sequenom, Inc. Methods for non-invasive assessment of fetal genetic variations that factor experimental conditions
US11939634B2 (en) 2010-05-18 2024-03-26 Natera, Inc. Methods for simultaneous amplification of target loci

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9404150B2 (en) 2007-08-29 2016-08-02 Sequenom, Inc. Methods and compositions for universal size-specific PCR

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5387505A (en) * 1990-05-04 1995-02-07 Eastman Kodak Company Preparation and isolation of single-stranded biotinylated nucleic acids by heat avidin-biotin cleavage
US5484701A (en) * 1990-01-26 1996-01-16 E. I. Du Pont De Nemours And Company Method for sequencing DNA using biotin-strepavidin conjugates to facilitate the purification of primer extension products
US20010031467A1 (en) * 1999-12-10 2001-10-18 Johannes Dapprich Method for selectively isolating a nucleic acid
US20020022224A1 (en) * 2000-04-28 2002-02-21 Hornby David P. Method for isolating single-stranded DNA

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5484701A (en) * 1990-01-26 1996-01-16 E. I. Du Pont De Nemours And Company Method for sequencing DNA using biotin-strepavidin conjugates to facilitate the purification of primer extension products
US5387505A (en) * 1990-05-04 1995-02-07 Eastman Kodak Company Preparation and isolation of single-stranded biotinylated nucleic acids by heat avidin-biotin cleavage
US20010031467A1 (en) * 1999-12-10 2001-10-18 Johannes Dapprich Method for selectively isolating a nucleic acid
US20020022224A1 (en) * 2000-04-28 2002-02-21 Hornby David P. Method for isolating single-stranded DNA

Cited By (192)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10083273B2 (en) 2005-07-29 2018-09-25 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10266893B2 (en) 2005-07-29 2019-04-23 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US11111544B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US11111543B2 (en) 2005-07-29 2021-09-07 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10260096B2 (en) 2005-07-29 2019-04-16 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10392664B2 (en) 2005-07-29 2019-08-27 Natera, Inc. System and method for cleaning noisy genetic data and determining chromosome copy number
US10227652B2 (en) 2005-07-29 2019-03-12 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US10081839B2 (en) 2005-07-29 2018-09-25 Natera, Inc System and method for cleaning noisy genetic data and determining chromosome copy number
US10711309B2 (en) 2005-11-26 2020-07-14 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US10597724B2 (en) 2005-11-26 2020-03-24 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US10240202B2 (en) 2005-11-26 2019-03-26 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US9424392B2 (en) 2005-11-26 2016-08-23 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US11306359B2 (en) 2005-11-26 2022-04-19 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US9695477B2 (en) 2005-11-26 2017-07-04 Natera, Inc. System and method for cleaning noisy genetic data from target individuals using genetic data from genetically related individuals
US11952569B2 (en) 2006-05-31 2024-04-09 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US9453257B2 (en) 2006-05-31 2016-09-27 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US10662421B2 (en) 2006-05-31 2020-05-26 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US9051608B2 (en) 2006-12-05 2015-06-09 Agena Bioscience, Inc. Detection and quantification of biomolecules using mass spectrometry
US8652780B2 (en) 2007-03-26 2014-02-18 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection
US9310378B2 (en) 2008-01-15 2016-04-12 Agena Bioscience, Inc. Compositions and processes for improved mass spectrometry analysis
US9873912B2 (en) 2008-01-15 2018-01-23 Agena Bioscience, Inc. Compositions and processes for improved mass spectrometry analysis
US10329612B2 (en) 2008-01-15 2019-06-25 Agena Bioscience, Inc. Compositions and processes for improved mass spectrometry analysis
US8206926B2 (en) 2008-03-26 2012-06-26 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection
US9639657B2 (en) 2008-08-04 2017-05-02 Natera, Inc. Methods for allele calling and ploidy calling
US8962247B2 (en) 2008-09-16 2015-02-24 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non invasive prenatal diagnoses
US10612086B2 (en) 2008-09-16 2020-04-07 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
US10738358B2 (en) 2008-09-16 2020-08-11 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
US10858645B2 (en) 2009-04-03 2020-12-08 Sequenom, Inc. Nucleic acid preparation compositions and methods
US10053685B2 (en) 2009-04-03 2018-08-21 Sequenom, Inc. Nucleic acid preparation compositions and methods
US9850480B2 (en) 2009-04-03 2017-12-26 Sequenom, Inc. Nucleic acid preparation compositions and methods
US10061889B2 (en) 2009-09-30 2018-08-28 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10216896B2 (en) 2009-09-30 2019-02-26 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10522242B2 (en) 2009-09-30 2019-12-31 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10061890B2 (en) 2009-09-30 2018-08-28 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US9228234B2 (en) 2009-09-30 2016-01-05 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11180799B2 (en) 2009-12-22 2021-11-23 Sequenom, Inc. Processes and kits for identifying aneuploidy
US9926593B2 (en) 2009-12-22 2018-03-27 Sequenom, Inc. Processes and kits for identifying aneuploidy
EP2569453A4 (fr) * 2010-05-14 2013-11-20 Fluidigm Corp Méthodes d'isolement de l'acide nucléique
EP2569453A2 (fr) * 2010-05-14 2013-03-20 Fluidigm Corporation Méthodes d'isolement de l'acide nucléique
US10017812B2 (en) 2010-05-18 2018-07-10 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10316362B2 (en) 2010-05-18 2019-06-11 Natera, Inc. Methods for simultaneous amplification of target loci
US11322224B2 (en) 2010-05-18 2022-05-03 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10557172B2 (en) 2010-05-18 2020-02-11 Natera, Inc. Methods for simultaneous amplification of target loci
US11326208B2 (en) 2010-05-18 2022-05-10 Natera, Inc. Methods for nested PCR amplification of cell-free DNA
US10590482B2 (en) 2010-05-18 2020-03-17 Natera, Inc. Amplification of cell-free DNA using nested PCR
US10538814B2 (en) 2010-05-18 2020-01-21 Natera, Inc. Methods for simultaneous amplification of target loci
US11525162B2 (en) 2010-05-18 2022-12-13 Natera, Inc. Methods for simultaneous amplification of target loci
US10731220B2 (en) 2010-05-18 2020-08-04 Natera, Inc. Methods for simultaneous amplification of target loci
US11519035B2 (en) 2010-05-18 2022-12-06 Natera, Inc. Methods for simultaneous amplification of target loci
US11332785B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11482300B2 (en) 2010-05-18 2022-10-25 Natera, Inc. Methods for preparing a DNA fraction from a biological sample for analyzing genotypes of cell-free DNA
US10526658B2 (en) 2010-05-18 2020-01-07 Natera, Inc. Methods for simultaneous amplification of target loci
US11312996B2 (en) 2010-05-18 2022-04-26 Natera, Inc. Methods for simultaneous amplification of target loci
US8949036B2 (en) 2010-05-18 2015-02-03 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10774380B2 (en) 2010-05-18 2020-09-15 Natera, Inc. Methods for multiplex PCR amplification of target loci in a nucleic acid sample
US10597723B2 (en) 2010-05-18 2020-03-24 Natera, Inc. Methods for simultaneous amplification of target loci
US10793912B2 (en) 2010-05-18 2020-10-06 Natera, Inc. Methods for simultaneous amplification of target loci
US11306357B2 (en) 2010-05-18 2022-04-19 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10113196B2 (en) 2010-05-18 2018-10-30 Natera, Inc. Prenatal paternity testing using maternal blood, free floating fetal DNA and SNP genotyping
US11939634B2 (en) 2010-05-18 2024-03-26 Natera, Inc. Methods for simultaneous amplification of target loci
US11332793B2 (en) 2010-05-18 2022-05-17 Natera, Inc. Methods for simultaneous amplification of target loci
US10174369B2 (en) 2010-05-18 2019-01-08 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11408031B2 (en) 2010-05-18 2022-08-09 Natera, Inc. Methods for non-invasive prenatal paternity testing
US11111545B2 (en) 2010-05-18 2021-09-07 Natera, Inc. Methods for simultaneous amplification of target loci
US11286530B2 (en) 2010-05-18 2022-03-29 Natera, Inc. Methods for simultaneous amplification of target loci
US9163282B2 (en) 2010-05-18 2015-10-20 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US9334541B2 (en) 2010-05-18 2016-05-10 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US11746376B2 (en) 2010-05-18 2023-09-05 Natera, Inc. Methods for amplification of cell-free DNA using ligated adaptors and universal and inner target-specific primers for multiplexed nested PCR
US11339429B2 (en) 2010-05-18 2022-05-24 Natera, Inc. Methods for non-invasive prenatal ploidy calling
US10655180B2 (en) 2010-05-18 2020-05-19 Natera, Inc. Methods for simultaneous amplification of target loci
US11004537B2 (en) 2011-06-24 2021-05-11 Sequenom, Inc. Methods and processes for non invasive assessment of a genetic variation
US10196681B2 (en) 2011-10-06 2019-02-05 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US11560586B2 (en) 2011-10-06 2023-01-24 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
WO2013052907A2 (fr) 2011-10-06 2013-04-11 Sequenom, Inc. Méthodes et procédés pour évaluation non invasive de variations génétiques
US10323268B2 (en) 2011-10-06 2019-06-18 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
WO2013052913A2 (fr) 2011-10-06 2013-04-11 Sequenom, Inc. Procédés et processus d'évaluation non invasive de variations génétiques
US9984198B2 (en) 2011-10-06 2018-05-29 Sequenom, Inc. Reducing sequence read count error in assessment of complex genetic variations
US11001884B2 (en) 2011-10-06 2021-05-11 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US11492659B2 (en) 2011-10-06 2022-11-08 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
EP3922731A2 (fr) 2011-10-06 2021-12-15 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US10424394B2 (en) 2011-10-06 2019-09-24 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US11437121B2 (en) 2011-10-06 2022-09-06 Sequenom, Inc. Methods and processes for non-invasive detection of a microduplication or a microdeletion with reduced sequence read count error
US8688388B2 (en) 2011-10-11 2014-04-01 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
EP3243908A1 (fr) 2011-10-11 2017-11-15 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
WO2013055817A1 (fr) 2011-10-11 2013-04-18 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US11697849B2 (en) 2012-01-20 2023-07-11 Sequenom, Inc. Methods for non-invasive assessment of fetal genetic variations that factor experimental conditions
US10738359B2 (en) 2012-03-02 2020-08-11 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US9605313B2 (en) 2012-03-02 2017-03-28 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
EP3757210A1 (fr) 2012-03-02 2020-12-30 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US11312997B2 (en) 2012-03-02 2022-04-26 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
EP3401399A1 (fr) 2012-03-02 2018-11-14 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
EP4155401A1 (fr) 2012-03-02 2023-03-29 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US11306354B2 (en) 2012-05-21 2022-04-19 Sequenom, Inc. Methods and compositions for analyzing nucleic acid
EP4276194A2 (fr) 2012-05-21 2023-11-15 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
WO2013177086A1 (fr) 2012-05-21 2013-11-28 Sequenom, Inc. Procédés et méthodes d'évaluation non invasive de variations génétiques
EP3978621A1 (fr) 2012-05-21 2022-04-06 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US9920361B2 (en) 2012-05-21 2018-03-20 Sequenom, Inc. Methods and compositions for analyzing nucleic acid
EP3663409A1 (fr) 2012-05-21 2020-06-10 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
EP4137579A1 (fr) 2012-06-22 2023-02-22 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
WO2013192562A1 (fr) 2012-06-22 2013-12-27 Sequenom, Inc. Procédés et processus pour l'évaluation non invasive de variations génétiques
EP3473731A1 (fr) 2012-06-22 2019-04-24 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US10497461B2 (en) 2012-06-22 2019-12-03 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
WO2014011928A1 (fr) 2012-07-13 2014-01-16 Sequenom, Inc. Procédés et compositions pour l'enrichissement basé sur la méthylation d'un échantillon maternel en acide nucléique fœtal, utiles pour les diagnostics prénatals non invasifs
US11332791B2 (en) 2012-07-13 2022-05-17 Sequenom, Inc. Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
JP2018183189A (ja) * 2012-07-24 2018-11-22 ナテラ， インコーポレイテッド 高度多重ｐｃｒ法および組成物
WO2014055790A2 (fr) 2012-10-04 2014-04-10 Sequenom, Inc. Procédés et processus d'évaluation non invasive de variations génétiques
EP4009329A1 (fr) 2012-10-04 2022-06-08 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
EP3591067A1 (fr) 2012-10-04 2020-01-08 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US10482994B2 (en) 2012-10-04 2019-11-19 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
WO2014055774A1 (fr) 2012-10-04 2014-04-10 Sequenom, Inc. Procédés et processus d'estimation non invasive de variations génétiques
US10504613B2 (en) 2012-12-20 2019-12-10 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US10497462B2 (en) 2013-01-25 2019-12-03 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
EP4261828A2 (fr) 2013-01-25 2023-10-18 Sequenom, Inc. Procédés et procédés d'évaluation non invasive de variations génétiques
WO2014116598A2 (fr) 2013-01-25 2014-07-31 Sequenom, Inc. Procédés et processus d'évaluation non invasive de variations génétiques
WO2014164838A1 (fr) 2013-03-12 2014-10-09 Sequenom, Inc. Quantification de marqueurs d'acides nucléiques spécifiques de cellules présentant un état de méthylation particulier
US9305756B2 (en) 2013-03-13 2016-04-05 Agena Bioscience, Inc. Preparation enhancements and methods of use for MALDI mass spectrometry
WO2014168711A1 (fr) 2013-03-13 2014-10-16 Sequenom, Inc. Amorces pour analyse de la méthylation de l'adn
EP3597774A1 (fr) 2013-03-13 2020-01-22 Sequenom, Inc. Amorces pour analyse de la méthylation de l'adn
US11060145B2 (en) 2013-03-13 2021-07-13 Sequenom, Inc. Methods and compositions for identifying presence or absence of hypermethylation or hypomethylation locus
US10204771B2 (en) 2013-03-13 2019-02-12 Agena Bioscience, Inc. Preparation enhancements and methods of use for MALDI mass spectrometry
WO2014165596A1 (fr) 2013-04-03 2014-10-09 Sequenom, Inc. Procédés et systèmes d'évaluation non invasive de variations génétiques
EP4187543A1 (fr) 2013-04-03 2023-05-31 Sequenom, Inc. Procédés et procédés d'évaluation non invasive de variations génétiques
US10930368B2 (en) 2013-04-03 2021-02-23 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US11462298B2 (en) 2013-05-24 2022-10-04 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US10699800B2 (en) 2013-05-24 2020-06-30 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
EP3578670A1 (fr) 2013-05-24 2019-12-11 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
EP3540076A1 (fr) 2013-06-21 2019-09-18 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US10622094B2 (en) 2013-06-21 2020-04-14 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
US10577655B2 (en) 2013-09-27 2020-03-03 Natera, Inc. Cell free DNA diagnostic testing standards
US9499870B2 (en) 2013-09-27 2016-11-22 Natera, Inc. Cell free DNA diagnostic testing standards
EP4258269A2 (fr) 2013-10-04 2023-10-11 Sequenom, Inc. Procédés et procédés d'évaluation non invasive de variations génétiques
WO2015051163A2 (fr) 2013-10-04 2015-04-09 Sequenom, Inc. Méthodes et processus d'évaluation non invasive de variations génétiques
US10964409B2 (en) 2013-10-04 2021-03-30 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
WO2015054080A1 (fr) 2013-10-07 2015-04-16 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de modifications chromosomiques
US11929146B2 (en) 2013-10-07 2024-03-12 Sequenom, Inc. Systems for non-invasive assessment of chromosome alterations using changes in subsequence mappability
EP3851539A1 (fr) 2013-10-07 2021-07-21 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive d'altérations chromosomiques
EP3495496A1 (fr) 2013-10-07 2019-06-12 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive d'altérations chromosomiques
US11365447B2 (en) 2014-03-13 2022-06-21 Sequenom, Inc. Methods and processes for non-invasive assessment of genetic variations
WO2015159979A1 (fr) * 2014-04-18 2015-10-22 凸版印刷株式会社 Procédé de récupération d'acides nucléiques à courte chaîne
US10597709B2 (en) 2014-04-21 2020-03-24 Natera, Inc. Methods for simultaneous amplification of target loci
US11408037B2 (en) 2014-04-21 2022-08-09 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11371100B2 (en) 2014-04-21 2022-06-28 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11319595B2 (en) 2014-04-21 2022-05-03 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10179937B2 (en) 2014-04-21 2019-01-15 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11319596B2 (en) 2014-04-21 2022-05-03 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US11486008B2 (en) 2014-04-21 2022-11-01 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US10597708B2 (en) 2014-04-21 2020-03-24 Natera, Inc. Methods for simultaneous amplifications of target loci
US11390916B2 (en) 2014-04-21 2022-07-19 Natera, Inc. Methods for simultaneous amplification of target loci
US10351906B2 (en) 2014-04-21 2019-07-16 Natera, Inc. Methods for simultaneous amplification of target loci
US11414709B2 (en) 2014-04-21 2022-08-16 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US9677118B2 (en) 2014-04-21 2017-06-13 Natera, Inc. Methods for simultaneous amplification of target loci
US10262755B2 (en) 2014-04-21 2019-04-16 Natera, Inc. Detecting cancer mutations and aneuploidy in chromosomal segments
US11530454B2 (en) 2014-04-21 2022-12-20 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
EP3598452A1 (fr) 2014-05-30 2020-01-22 Sequenom, Inc. Déterminations de représentation de chromosomes
WO2015183872A1 (fr) 2014-05-30 2015-12-03 Sequenom, Inc. Déterminations de représentation de chromosomes
US11783911B2 (en) 2014-07-30 2023-10-10 Sequenom, Inc Methods and processes for non-invasive assessment of genetic variations
EP3760739A1 (fr) 2014-07-30 2021-01-06 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
WO2016057901A1 (fr) 2014-10-10 2016-04-14 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
EP3730629A1 (fr) 2014-10-10 2020-10-28 Sequenom, Inc. Méthodes et procédés d'évaluation non invasive de variations génétiques
US11479812B2 (en) 2015-05-11 2022-10-25 Natera, Inc. Methods and compositions for determining ploidy
US11946101B2 (en) 2015-05-11 2024-04-02 Natera, Inc. Methods and compositions for determining ploidy
EP4043581A1 (fr) 2016-05-27 2022-08-17 Sequenom, Inc. Méthode pour générer un système d'essai de paralogue
WO2017205826A1 (fr) 2016-05-27 2017-11-30 Sequenom, Inc. Méthodes de détection de variations génétiques
US11299780B2 (en) 2016-07-15 2022-04-12 The Regents Of The University Of California Methods of producing nucleic acid libraries
WO2018022890A1 (fr) 2016-07-27 2018-02-01 Sequenom, Inc. Classifications de modifications du nombre de copies génétiques
US11515003B2 (en) 2016-07-27 2022-11-29 Sequenom, Inc. Copy number alteration and reference genome mapping
WO2018022906A1 (fr) 2016-07-27 2018-02-01 Sequenom, Inc. Procédés d'évaluation non invasive de l'instabilité génomique
US11485996B2 (en) 2016-10-04 2022-11-01 Natera, Inc. Methods for characterizing copy number variation using proximity-litigation sequencing
US10577650B2 (en) 2016-12-07 2020-03-03 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US11519028B2 (en) 2016-12-07 2022-12-06 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US10533219B2 (en) 2016-12-07 2020-01-14 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US10011870B2 (en) 2016-12-07 2018-07-03 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
US11530442B2 (en) 2016-12-07 2022-12-20 Natera, Inc. Compositions and methods for identifying nucleic acid molecules
WO2018136888A1 (fr) 2017-01-20 2018-07-26 Sequenom, Inc. Procédés d'évaluation non invasive d'alterations genetique
WO2018136881A1 (fr) 2017-01-20 2018-07-26 Sequenom, Inc. Fabrication et utilisation d'adaptateur de séquençage
EP4235676A2 (fr) 2017-01-20 2023-08-30 Sequenom, Inc. Procédés d'évaluation non invasive d'alterations genetique
WO2018136882A1 (fr) 2017-01-20 2018-07-26 Sequenom, Inc. Procédés d'évaluation non invasive de variations du nombre de copies
WO2018140521A1 (fr) 2017-01-24 2018-08-02 Sequenom, Inc. Méthodes et procédés d'évaluation de variations génétiques
US11694768B2 (en) 2017-01-24 2023-07-04 Sequenom, Inc. Methods and processes for assessment of genetic variations
US10894976B2 (en) 2017-02-21 2021-01-19 Natera, Inc. Compositions, methods, and kits for isolating nucleic acids
WO2018170511A1 (fr) 2017-03-17 2018-09-20 Sequenom, Inc. Procédés et techniques d'évaluation d'un mosaïcisme génétique
EP3998350A1 (fr) 2017-03-17 2022-05-18 Sequenom, Inc. Procédés et techniques d'évaluation d'un mosaïcisme génétique
WO2019140201A1 (fr) 2018-01-12 2019-07-18 Claret Bioscience, Llc Procédés et compositions d'analyse d'acide nucléique
US11584929B2 (en) 2018-01-12 2023-02-21 Claret Bioscience, Llc Methods and compositions for analyzing nucleic acid
US11629345B2 (en) 2018-06-06 2023-04-18 The Regents Of The University Of California Methods of producing nucleic acid libraries and compositions and kits for practicing same
US11525159B2 (en) 2018-07-03 2022-12-13 Natera, Inc. Methods for detection of donor-derived cell-free DNA
WO2020206143A1 (fr) 2019-04-05 2020-10-08 Claret Bioscience, Llc Procédés et compositions d'analyse d'acide nucléique
EP3973081A4 (fr) * 2019-05-23 2022-09-07 American Molecular Laboratories, Inc. Procédés de détection de séquences d'adn rares dans des échantillons fécaux
WO2021072037A1 (fr) 2019-10-09 2021-04-15 Claret Bioscience, Llc Procédés et compositions d'analyse d'acide nucléique
WO2021087491A1 (fr) 2019-10-31 2021-05-06 Sequenom, Inc. Application d'un rapport du mosaïcisme dans des gestations multifœtales et évaluation de risque personnalisé
WO2021262805A1 (fr) 2020-06-24 2021-12-30 Claret Bioscience, Llc Procédés et compositions pour analyse d'acide nucléique
WO2022076574A1 (fr) 2020-10-08 2022-04-14 Claret Bioscience, Llc Procédés et compositions d'analyse d'acide nucléique

Also Published As

Publication number Publication date
WO2009032779A3 (fr) 2009-05-07

Similar Documents

Publication Publication Date Title
US9404150B2 (en) Methods and compositions for universal size-specific PCR
US11952569B2 (en) Methods and compositions for the extraction and amplification of nucleic acid from a sample
WO2009032779A2 (fr) Procédés et compositions utilisés dans la séparation, en fonction de sa taille, de l'acide nucléique d'un échantillon
US20200385810A1 (en) Methods for determining fraction of fetal nucleic acids in maternal samples
US20080096766A1 (en) Methods and compositions for the amplification, detection and quantification of nucleic acid from a sample
EP3378954B1 (fr) Quantification d'une minorité d'espèces d'acide nucléique
AU2010343276B2 (en) Methods for determining fraction of fetal nucleic acid in maternal samples
EP3755813B1 (fr) Détection améliorée de l'instabilité microsatellitaire
EP2531618B1 (fr) Procédé de détection de motifs de séquences répétées dans un acide nucléique
EP2324063A2 (fr) Génotypage rhd f tal non invasif à partir de sang maternel entier
US20240052417A1 (en) Method for early determination of gender by multiplex pcr detecting four genes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08798912

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08798912

Country of ref document: EP

Kind code of ref document: A2