WO2009032779A2

WO2009032779A2 - Methods and compositions for the size-specific seperation of nucleic acid from a sample

Info

Publication number: WO2009032779A2
Application number: PCT/US2008/074689
Authority: WO
Inventors: Min Seob Lee
Original assignee: Sequenom, Inc.
Priority date: 2007-08-29
Filing date: 2008-08-28
Publication date: 2009-03-12
Also published as: WO2009032779A3

Abstract

Provided herein are methods and kits for the separation, extraction and enrichment of short- stranded nucleic acid in the presence of a high background of long-stranded genomic material (e.g., host or maternal nucleic acids). The methods rely on primers that selectively bind to the long- stranded nucleic acid, whereby the primers may be used to subsequently separate the long and short nucleic acid.

Description

METHODS AND COMPOSITIONS FOR THE SIZE-SPECIFIC SEPARATION OF NUCLEIC ACID

FROM A SAMPLE

RELATED APPLICATION This patent application claims the benefit of U.S. Provisional Patent Application No.

60/968,876 filed on August 29, 2007, entitled METHODS AND COMPOSITIONS FOR THE SIZE- SPECIFIC SEPARATION OF NUCLEIC ACID FROM A SAMPLE, naming Min Seob Lee as an inventor, and designated by attorney docket no. SEQ-6013-PV. The entire content of the foregoing provisional patent application is incorporated herein by reference in jurisdictions permitting such incorporation.

FIELD OF THE INVENTION

The invention relates in part to products and processes for the separation and detection of nucleic acids from a sample.

BACKGROUND

The isolation and subsequent analysis of nucleic acids play a central role in molecular biology. Isolated nucleic acids may be used, inter alia, as a starting material for diagnosis and prognosis of diseases and disorders. The recent discovery of trace amounts of short, fragmented nucleic acid in a range of biological samples, including plasma and serum, presents a new opportunity for improved, non-invasive tests. Previously, the recovery of fragmented nucleic acid from biological samples was considered unimportant, and extraction methods were designed to isolate large, undegraded nucleic acid. However, it is short base pair nucleic acid (e.g., highly degraded RNA and DNA) that offers a new source of highly informative genetic material for a wide range of applications, including prenatal diagnostics, early cancer detection and the study of apoptotic DNA from host and non-host sources.

SUMMARY OF THE INVENTION

Provided are improved methods directed to enriching or isolating short, fragmented nucleic acid in the presence of more abundant, longer nucleic acid. These methods are simple, cost- effective and automatable for use in research and clinical environments.

Thus, in one aspect, 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. In the case of cell-free fetal nucleic acid circulating in maternal plasma, 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"). This leads to a relatively enriched fraction of nucleic acid that has a higher concentration of smaller 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. In certain embodiments, 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 separation.

In some embodiments, provided are methods for enriching 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. In certain embodiments, the pair of non-target binding primers preferentially anneal to non-target nucleic acid, but not target nucleic acid. In some embodiments, either the target or non-target nucleic acid may be subsequently analyzed after enrichment. In some embodiments, 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 nucleic acid, thereby yielding a supernatant enriched for the target nucleic acid. In some embodiments, provided are methods for extracting 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. In certain embodiments, the method further comprises analyzing either the target nucleic acid or non- target nucleic acid, or both nucleic acids. In some embodiments, methods of the invention may be repeated or combined with other methods until the desired concentration of nucleic acid is present. In certain embodiments, 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. In some embodiments, 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. In certain embodiments, 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.

Certain embodiments pertain to separating, enriching or extracting 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. In some embodiments, the methods may be particularly useful for discriminating between RNA of varying length. In certain embodiments, the nucleic acid is cell-free nucleic acid. In some embodiments, the target nucleic acid is derived from apoptotic cells. In some embodiments, the target nucleic acid is of fetal origin, and the non-target nucleic acid is of maternal origin.

In some embodiments, 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.

In certain embodiments, 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. In certain embodiments, 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.

Methods of the present invention can be used for separating, enriching or extracting fetal nucleic acid from maternal plasma. In certain embodiments, the biological sample is from an animal, most preferably a human. In certain embodiments, the biological sample is from a pregnant human. In certain embodiments, 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. In some embodiments, the pregnant human has a relatively elevated concentration of free fetal nucleic acid in her blood, plasma or amniotic fluid. In some embodiments, 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. Likewise, 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. In some embodiments, methods of the present invention may be used to separate, enrich or extract RNA that is expressed by the fetus. In some embodiments of the invention, 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. In some embodiments, the non-target binding primers are introduced to the sample at a concentration greater than the concentration of non-target nucleic acid.

In some embodiments of the invention, the primers may be modified to facilitate their capture. For example, modifications include, but are not limited to, inclusion of capture mechanisms, compomers, tags, linkers and adapter molecules. Examples of compomers are described in US Patent Application Publication No. 20050287533, filed June 23, 2004. Examples of adapters are described in US Patent Application Publication No. 2003021 1489, filed June 20, 2002. Examples of 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). 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). For example, 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. In some embodiments, the primer contains a universal binding oligonucleotide capable of hybridizing to a capture probe.

In some embodiments, a solid support is introduced to the sample or the sample is stored in a solid support capable of binding nucleic acid. In one embodiment, 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). In certain embodiments, 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. In certain embodiments, the solid support is a silica gel membrane.

In certain embodiments, the solid support has a functional group-coated surface. In certain embodiments, 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.

In some 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. In certain embodiments, paramagnetic beads are separated from the sample using magnets or magnetic devices. In some embodiments, 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. In some embodiments, the label is only detectable when the primer is bound to nucleic acid.

In some embodiments, provided are 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. Examples of other 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. For example, 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. In some embodiments, 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. In certain embodiments, the salt is a guanidine salt, most preferably guanidine (iso)thiocyanate, or is a sodium salt, most preferably sodium perchlorate. In the methods provided herein, the chaotropic agent often is introduced at a concentration sufficient to bind non-target nucleic acid to a solid support.

In some embodiments, 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. In some embodiments, the solid support-nucleic acid complex is further washed successively with a wash buffer and one or more alcohol-water solutions, and subsequently dried. In certain embodiments, 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.

In some embodiments, provided are methods which further comprise amplifying target nucleic acid, for example, after the non-target nucleic acid is removed. In some embodiments, the target nucleic acid is amplified by a target specific amplification method such as allele-specific PCR. In some embodiments, all of the remaining nucleic acid (both target and non-target 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. Thus, in some embodiments of the invention, provided are methods that further comprise analyzing the non-target nucleic acid, the target nucleic acid or both the non-target and target nucleic acid. Examples of nucleic acid analysis include, but are not limited to, genotype analysis, sequencing analysis, methylation analysis, quantitative analysis and qualitative analysis.

In some embodiments, provided are methods that may be used in multiplexed reactions, where multiple target nucleic acids are extracted, enriched or separated in a single, multiplexed reaction. In certain embodiments, 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.

In some embodiments, 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,

1:801-1:900, 1:901-1:1000, 1:1001-1:2000, 1:2001-1:3000, 1:3001-1:4000, 1:4001-1:5000, 1:5001- 1:6000, 1:6001-1:7000, 1:7001-1:8000, 1:8001-1:9000, 1:9001-1:10,000; 1:10,001-1:20,000, 1:20,001:1:30,000, 1:30,001-1:40,000, 1:40,001-1:50,000, and greater than 1:50,000.

In some embodiments, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

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. As the Figure illustrates, 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.

DETAILED DESCRIPTION OF THE INVENTION

The presence of short, fragmented cell-free nucleic acid in peripheral blood is a well established phenomenon. Cell-free nucleic acid may originate from a number of sources, including programmed cell death, which 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 applications for the detection, extraction and relative enrichment of extracellular nucleic acid from peripheral blood or other body fluids are widespread and may include inter alia, non-invasive prenatal diagnosis, cancer diagnostics, pathogen detection, auto-immune response detection and detection of allograft rejection. In a particular embodiment of the invention, 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. Am J Hum Genet (1999) 64:218-224). Fetal nucleic acid is present in maternal plasma in a much higherfractional concentration than fetal nucleic acid in the cellular fraction of maternal blood (Lo et al. Am J Hum Genet (1998) 62:768-775). Thus, in some embodiments, the target nucleic acid is of fetal origin, the non-target nucleic acid is of maternal origin and 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. In some embodiments, 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,

20030124556, 20030175696, 20030175695, 20030175697, and 20030190605 and U.S. patent application Ser. Nos. 10/326,047, 10/660,997, 10/660,122 and 10/660,996, all of which are herein incorporated by reference in their entirety.

The term "extraction" as used herein 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. The terms "selective" and "selectively" as used herein 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.

The terms "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 analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides and combinations and/or mixtures thereof. Thus, the term "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. The term "target nucleic acid" as used herein refers to the nucleic acid of interest that is extracted or separated based on its molecular size. In certain embodiments, 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. In certain embodiments, the target nucleic acid is fetal DNA, oncogenic DNA, or any non-host DNA. In another related embodiment, the target nucleic acid is cell-free nucleic acid. In another related embodiment, the target nucleic acid is oligonucleosomal nucleic acid generated during programmed cell death.

The term "non-target nucleic acid" as used herein refers to the relatively high amount of background nucleic acid present in a sample. In certain embodiments, non-target nucleic acid has a molecular size larger than target nucleic acid, for example, greater than about 500 base pairs. In certain embodiments, non-target nucleic acid is from a host or host cell. In certain embodiments, non-target nucleic acid is of maternal origin. In some embodiments, the non-target nucleic acid is separated or extracted from the sample, thereby yielding a relatively enriched target nucleic acid sample. The term "molecular size" as used herein 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). The term "sample" as used herein 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. In certain embodiments, the sample comprises a mixture of nucleic acids. For example, the mixture may comprise nucleic acid from different species or from different individuals. In a further embodiment, the biological sample contains cellular elements or cellular remnants in maternal blood.

In one embodiment, the sample is from a pregnant female. In certain embodiments, the sample is procured through non-invasive means (e.g., a blood draw). The term "non-invasive" as used herein 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. In another related embodiment, the sample is cervical mucosa, which is obtained, for example, by an aspiration catheter. In certain embodiments, the biological sample is blood, and more preferably plasma. As used herein, the term "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. These examples are not to be construed as limiting the sample types applicable to the present invention.

In a preferred method, blood handling protocols are followed to ensure minimal degradation of nucleic acid in the sample and to minimize the creation of apoptotic nucleic acid in the sample. Blood handling methods are well known in the art. In certain embodiments, the biological sample is cell-free or substantially cell-free. In certain embodiments, 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. In an embodiment of the invention, 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. In some embodiments, 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. In some embodiments, a cell lysis inhibitor is introduced to the sample. In some embodiments, lysis may be blocked. In these embodiments, 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. In some of these embodiments, 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. Other suitable methods are provided in 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. Examples of 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. 5,679,524 and 5,952,174, and WO 01/27326), mismatch sequence determination methods (e.g., U.S. Pat. Nos. 5,851 ,770; 5,958,692; 6,1 10,684; and 6,183,958), allele specific oligonucleotide (ASO) analysis, methylation-specific PCR (MSPCR), pyrosequencing analysis, acycloprime analysis, Reverse dot blot, Dynamic allele-specific hybridization (DASH), Peptide nucleic acid (PNA) and locked nucleic acids (LNA) probes, 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-directed incorporation (TDI), fluorescence polarization, Colorimetric oligonucleotide ligation assay (OLA), Sequence-coded OLA, Microarray ligation, Ligase chain reaction, Padlock probes, and Invader assay, microarray sequence determination methods, restriction fragment length polymorphism (RFLP) procedures, single primer linear nucleic acid amplification, as described in U.S. Pat. No. 6,251 ,639, PCR-based assays (e.g., TAQMAN® PCR System (Applied Biosystems)), nucleotide sequencing methods, hybridization methods, conventional dot blot analyses, single strand conformational polymorphism analysis (SSCP, e.g., U.S. Patent Nos. 5,891 ,625 and 6,013,499; Orita et al., Proc. Natl. Acad. Sci. U.S.A 86: 27776-2770 (1989)), BeadArray, Invader assay, denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and techniques described in Sheffield et al., Proc. Natl. Acad. Sci. USA 49: 699-706 (1991 ), White et al., Genomics 12: 301-306 (1992), Grompe et al., Proc. Natl. Acad. Sci. USA 86: 5855-5892 (1989), and Grompe, Nature

Genetics 5: 1 11-1 17 (1993), detection by mass spectrometry, for example Sequenom Inc.'s primer extension method (e.g., iPLEX™) 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.

As used herein, 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).

As used herein, 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. With regard to 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. For example, if the locus of interest is SNP X on chromosome 1 , and 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.

The term "polymorphism" as used herein refers to an allelic variant. Polymorphisms can include single nucleotide polymorphisms (SNPs) as well as simple sequence length 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. As used herein, polymorphism may include epigenetic variants, as long as cleavage by non-epigenetic-specific cleavage agents is used.

The term "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.

"Polymerase chain reaction" or "PCR" refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression. PCR is well known to those of skill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, lnnis et al., eds, 1990. "Oligonucleotide" 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. Usually 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. Whenever an 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. Usually oligonucleotides of the invention comprise the four natural deoxynucleotides; however, they may also comprise ribonucleosides or non-natural nucleotide analogs. Where an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity, e.g., single stranded DNA, RNA/DNA duplex, or the like, then selection of appropriate composition for the oligonucleotide or polynucleotide substrates is well within the knowledge of one of ordinary skill. As used herein, the terms "primer", "oligonucleotide primer" and "probe" are interchangeable when used to discuss an oligonucleotide that anneals to a nucleic acid. In amplification embodiments of the invention, an oligonucleotide primer serves as a point of initiation of nucleic acid synthesis. Alternatively, 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.

As used herein, the term "flanking" a locus of interest 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. 68:90 (1979); Brown et al., Methods Enzymol. 68:109 (1979)). 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. Also, 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. The simplest equation for determining the melting temperature of primers smaller than 25 base pairs is the Wallace Rule (Td=2(A+T)+4(G+C)). 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. The TM (melting or annealing temperature) of each primer can be easily calculated using methods well known in the art.

When isolation and extraction of the primer bound nucleic acid is preferred, the primers can be modified with a tag that facilitates isolation and/or extraction of the nucleic acid. In certain embodiments, 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. In some embodiments of the invention, 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)). The 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. In one embodiment, 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.

In some embodiments, phosphorus dendrimer linkers are introduced to the solid support to capture nucleic acid. For example, 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). In an embodiment of the invention, certain methods of the present invention are combined with the improved magnetic bead-based capture methods described by Archer et al.

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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 99-99.7% target DNA. The methods provided herein may also be modified to combine steps, for example, in order to improve automation.

In another example, 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.

Diagnostic applications

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. In certain embodiments, the amount, concentration or characteristic of enriched nucleic acid may be compared to a control (e.g., nucleic acid from a healthy individual).

The phrase "evaluating a disease condition" refers to assessing the disease condition of a patient. For example, 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.

Cancer

Methods provided herein may be used to separate, enrich or extract oncogenic nucleic acid, which may be further used for the detection, diagnosis or prognosis of a cancer-related disorder. In plasma from cancer patients, nucleic acids, including DNA and RNA, 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. 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 (Jahr S, et al. Cancer Res (2001 ) 61 ,1659-1665).

If a cancer shows specific viral DNA sequences or tumor suppressor and/or oncogene mutant sequences, PCR-specific strategies can be developed. However, for most cancers (and most Mendelian disorders), clinical application awaits optimization of methods to isolate, quantify and characterize the tumor-specific DNA compared to the patient's normal DNA, which is also present in plasma. Therefore, understanding the molecular structure and dynamics of DNA in plasma of normal individuals is necessary to achieve further advancement in this field. Thus, 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. Specifically, 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. In particular, 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.

Prenatal Diagnostics

Since 1997, it is known that free fetal DNA can be detected in the blood circulation of pregnant women. In the absence of pregnancy-associated complications, 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.

It has been demonstrated that the circulating DNA molecules are significantly larger in size in pregnant women than in non-pregnant women. Chan et al. demonstrated that the median percentages of total plasma DNA of greater than 201 base pairs were 57% and 14% for pregnant and non-pregnant women, respectively, while the median percentages of fetal-derived DNA with sizes greater than 193 base pairs and greater than 313 base pairs were only 20% and 0%, respectively (Chan et al, Clin Chem. 2004 Jan;50(1 ):88-92). These findings were independently confirmed by Li et al. (Clin Chem. 2004 Jun;50(6):1002-11 ; and Patent application US200516424). They showed that a greater than 5 fold relative enrichment of fetal DNA from ca. 5% to greater than 28% of total circulating plasma DNA is possible by means of size exclusion chromatography via preparative agarose gel electrophoresis and elution of the less than 300bp size fraction. However, this method of enrichment can be impractical for research or clinical use because it is difficult to automate. Also, DNA material may be lost when recovered from the relevant Agarose gel section. Thus, 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.

Pregnancy-Associated Disorders

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. (1998) N Engl J Med 339:1734-1738). This approach has been shown by many groups to be accurate, and has been introduced as a routine service by the British National Blood Service since 2001 (Finning KM, et al. (2002) Transfusion 42:1079-1085). Maternal plasma DNA analysis has been addressed in the following studies, for example: Chiu RWK, et al. (2002) Lancet 360:998-1000; Fucharoen G, et al. (2003) Prenat Diagn 23:393-396); Saito H, et al. (2000) Lancet 356:1 170); Amicucci P, et al. (2000) CHn Chem 46:301-302); Gonzalez-Gonzalez MC, et al. (2002) Prenat Diagn 22:946-948); Gonzalez-Gonzalez MC, et al. (2003) Prenat Diagn 23:232-234) and (Rijnders RJ, et al. (2001 ) Obstet Gynecol 98:374-378). Thus 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. In some embodiments, 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. 2004 Jan;50(1 ):88-92; and Li et al. Clin Chem. 2004 Jun;50(6):1002-11. Employing such methods, 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.

In certain embodiments, 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. The term "pregnancy-associated disorder," as used herein, 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. Some examples of a pregnancy-associated disorder include ectopic pregnancy, preeclampsia, preterm labor, sex-linked disorders, and fetal chromosomal abnormalities such as trisomy 13, 18, or 21. The term "chromosomal abnormality" refers to a deviation between the structure of the subject chromosome and a normal homologous chromosome. The term "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. In certain embodiments, 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. 11/378,598, which published November 9, 2006 as Application Publication No. 20060252068; U.S. Patent Application No. 11/384,128, which published November 9, 2006 as Application Publication No. 20060252071 ; U.S. Patent Application No. 10/661 ,165, which published July 15, 2004 as Application Publication No. 20040137470; U.S. Patent No. 6,927,028, which issued August 9, 2005; U.S. Patent Application No. 10/346,514, which published November 13, 2003 as Application Publication No. 2003021 1522; U.S. Patent Application No. 09/944,951 , which issued August 9, 2005 as U.S. Patent No. 6,927,028; U.S. Patent Application No. 11/144,951 , which published January 26, 2006 as Application Publication No. 20060019278; U.S. Patent Application No. 10/575,119, which published March 15, 2007 as Application Publication No. 20070059707; U.S. Patent Application No. 10/852,943, which published February 17, 2005 as Application Publication No. 20050037388; and U.S. Patent Application No. 1 1/232,335, which published May 4, 2006 as Application Publication No. 20060094039.

Other diseases

In addition to cancer and pregnancy, many other diseases, disorders and conditions (e.g., tissue or organ rejection) produce 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. noted that DNA purified from SLE plasma formed discrete bands, corresponding to sizes of about 150-200, 400, 600, and 800 base pairs, closely resembling the characteristic 200 bp "ladder" found with oligonucleosomal DNA. In certain embodiments, 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.

Each document cited throughout the specification, and each document cited therein, is hereby expressly incorporated by reference in its entirety.

EXAMPLES

The examples hereafter illustrate but do not limit the invention.

EXAMPLE 1 Selective Enrichment of Short-Stranded DNA Using the Size Specific Depletion Method

The below example provides a procedure, using a method provided herein, to selectively separate, and thereby enrich, DNA based on its size using biotin labeled primers. More specifically, 200bp DNA target is shown to be selectively enriched in a sample containing a background of non- target 800bp DNA. A schematic showing the general features of this method is provided in Figure 1.

1. DNA Dilutions

First, 800bp and 200bp DNA fragments were generated by PCR. Both the long (actual length = 783 bp) and short (actual length = 169 bp) fragments included a single nucleotide polymorphism (SNP) rs6687785 - an A/T polymorphism. Two different samples (Sample 1 and Sample 2) of known sequence at SNP rs6687785 were used to generate the long and short DNA fragments, thereby ensuring the long DNA fragment was homozygote for the A allele, and the short DNA fragment was homozygote for the T allele. See Figure 2.

Next, long and short DNA fragments were diluted according to Table 1 to ensure equal starting concentrations of the 800bp and 200bp products.

TABLE 1 : Equal DNA Starting Concentration

Next, the long and short DNA mixes from Table 1 were combined at different ratios as summarized below in Table 2:

TABLE 2: Test Ratios of Long-to-Short DNA

Quantitative analysis of the long and short DNA mixes was performed as described below in Section 4. This analysis is shown in a spectrograph in Figure 3, where the "Before" spectrographs represent samples comprising varying ratios of target and non-target nucleic acid that have not been enriched for target nucleic acid using certain methods of the invention.

2. DNA and Biotin-Primers Mix

Next, 2OuI of the varying ratios of long and short DNA from Table 2 were added to a 96 well plate. To the 96 well plate, 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. Then, 2OuI of DNA and Biotin-Primer Mix were used in the subsequent binding step.

TABLE 3

3. Capture of Long-Stranded DNA to Magnetic Beads

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.

4. Quantitative Analysis of Long and Short Fragment DNA

Following the capture of the long-stranded DNA using biotin labeled primers, the remaining quantity of long and short-stranded DNA present in the supernatant was determined using Sequenom's MassARRAY® technology.

First, 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.

PCR amplification was followed by SAP cleanup using the reagents in Table 5.

TABLE 5: SAP Reagents

Two microliters of the SAP mix were added to each 5 microliter PCR reaction; then maintained at the following temperatures: 37° C for 20 minutes, 85° C for 5 minutes and 4° C thereafter.

Next, a MassEXTEND® reaction was performed using the reagents provided in Table 6. TABLE 6: MassEXTEND® Reagents

For iPLEX extension, 200 short cycles were carried out, where 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.

The samples were deslated with 6 mg of resin, dispensed to SpectroChip® Bioarrays and analyzed on a Sequenom® MALDI-TOF MS system. The resulting spectrographs are provided in Figure 3. They show the relative enrichment of the target nucleic acid after separation of the target and non-target nucleic acid. The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of the above patents, patent applications, publications and documents is not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, 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. For example, as it is used in a phrase of the structure "A or B" may denote A alone, B alone, or both A and B. Unless defined otherwise, 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. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the processes, systems, and methodologies that are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

Claims

What is claimed is:

1. A method for separating nucleic acid in a sample containing a mixture of non-target and target nucleic acid based on the size of the nucleic acid, wherein the target nucleic acid size is less than the size of the non-target nucleic acid in the mixture, comprising the steps of: a) introducing a pair of forward and reverse non-target binding primers to the sample; b) heating and cooling the sample of step a) one or more times, wherein the non-target binding primers anneal to non-target nucleic acid, but not target nucleic acid, based on size; and c) separating the non-target nucleic acid from target nucleic acid in the sample, wherein the primers annealed to the non-target nucleic acid are used to separate the non-target nucleic acid from the target nucleic acid.

2. The method of claim 1 , wherein the target nucleic acid comprises at least about 75 base pairs, but less than about 1200 base pairs.

3. The method of claim 1 , wherein the non-target binding primers anneal to the non-target nucleic acid at least 300 base pairs apart.

4. The method of claim 1 , wherein the non-target binding primers are introduced to the sample at a concentration greater than the concentration of non-target nucleic acid.

5. The method of claim 1 , wherein the nucleic acid is a cell-free nucleic acid.

6. The method of claim 1 , wherein the target nucleic acid is an apoptotic product.

7. The method of claim 1 , wherein the target nucleic acid is of fetal origin.

8. The method of claim 1 , wherein the target nucleic acid comprises one or more loci of interest.

9. The method of claim 8, which further comprises determining the identity of at least one allele within the one or more loci of interest.

10. The method of claim 1 , wherein the non-target nucleic acid is of maternal origin.

11. The method of claim 1 , wherein the sample is from a human.

12. The method of claim 1 , wherein the sample is from a pregnant human.

13. The method of claim 12, wherein the sample is collected after the fifth week of gestation.

14. The method of claim 1 , wherein the sample is selected from the group consisting of whole blood, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerbrospinal fluid, spinal fluid, lavage fluid, biopsy sample, urine, feces, sputum, saliva, nasal mucous, prostate fluid, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, embryonic cells and fetal cells.

15. The method of claim 1 , wherein the sample is plasma.

16. The method of claim 1 , wherein the sample is a previously isolated sample of nucleic acids.

17. The method of claim 1 , wherein one or more of the primers further comprise a capture mechanism.

18. The method of claim 17, wherein the capture mechanism is selected from the group consisting of biotin, a mechanism capable of binding to a solid support, and an oligonucleotide complimentary to a capture probe.

19. The method of claim 17, wherein the capture mechanism is capable of binding to a solid support, further wherein the solid support is selected from the group consisting of 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, glass surfaces, metal surfaces, plastic materials, multiwell plates or membranes, wafers, combs, pins and needles.

20. The method of claim 19, wherein the solid support is adapted to bind nucleic acids.

21. The method of claim 19, wherein the solid support is removed from the sample using a method selected from the group of methods consisting of: applying a magnetic field, applying vacuum filtration and centrifugation.

22. The method of claim 1 , wherein the primer contains a label.

23. A method for enriching for a target nucleic acid, wherein the method of claim 1 is performed prior to, subsequent to, or simultaneously with another method for selectively extracting nucleic acid.

24. The method of claim 23, wherein the other method for extracting nucleic acid is selected from the group consisting of electrophoresis, liquid chromatography, size exclusion, microdialysis, electrodialysis, centrifugal membrane exclusion, 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, and fragment selective amplification, or combinations thereof.

25. The method of claim 1 , further comprising quantifying the separated target nucleic acid.

26. The method of claim 1 , wherein multiple target nucleic acid species are separated in a single, multiplexed reaction.

27. A method for separating nucleic acid from a sample containing a mixture of nucleic acids, wherein the nucleic acid is separated based on size, comprising the steps of: a) introducing a pair of forward and reverse primers to the sample, wherein the primers anneal to nucleic acid at least about 500 base pairs apart; b) heating and cooling the sample of step a) one or more times, wherein 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; c) separating the primer-bound nucleic acid and non-primer-bound nucleic acid of step b), wherein the primers are used to separate the nucleic acids.

28. A method for removing 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, wherein the target nucleic acid size is less than the size of the non-target nucleic acid in the mixture, comprising the steps of: a) introducing a pair of forward and reverse non-target binding primers to the sample; b) heating and cooling the sample of step a) one or more times, wherein the non-target binding primers (preferentially) anneal to non-target nucleic acid, but not target nucleic acid, based on size; c) binding the non-target nucleic acid to a solid support in the sample, wherein 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 d) removing the supernatant that is enriched for target nucleic acid.

29. The method of claim 28, which further comprises amplifying target nucleic acid after the non- target nucleic acid is removed.

30. The method of claim 29, wherein the target nucleic acid is amplified by a target specific amplification method.

31. The method of claim 30, wherein the target specific amplification method is allele-specific PCR.

32. The method of claim 28, wherein the final relative percentage of target nucleic acid to non- target nucleic acid is at least about 25%.