Patent Description:
The isolation and subsequent amplification of nucleic acids play a central role in molecular biology. For instance, <CIT> relates to a process, a combination of means for isolating nucleic acid from a nucleic acid-containing starting material and a testkit in order to amplify the nucleic acid obtained by said process. Isolated, purified nucleic acids may be used, inter alia, as a starting material for diagnosis and prognosis of diseases or disorders. Therefore, the isolation of nucleic acids, particularly by non-invasive means, is of particular importance for use in genetic analyses.

Current methods for the extraction of nucleic acids include the use of organic-based methods (e.g., phenol/chloroform/isoamyl alcohol), or capitalize upon ion interaction of nucleic acids in an aqueous solution (e.g., salting out in combination with alcohol, solution pH and temperature) alone or in combination with anion exchange chromatography or cation exchange chromatography. Organic-based methods employ the use of phenol/chloroform/isoamyl alcohol or variations thereof for isolating DNA, but have serious disadvantages, namely the processes are very time-consuming, require considerable experimental effort, and are associated with an acute risk of exposure to toxic substances to those carrying out the isolation. Chromatography-based methods increase flexibility and automation since these methods can be used in combination with multiple matrices (e.g., membranes, latex, magnetic beads, micro-titer plate, etc.) and in the presence or absence of ligands (e.g., DEAE, silica, acrylamide, etc.). However, these methods are better suited to extract larger strands of nucleic acids to ensure greater success in downstream analysis.

Previously, the recovery of smaller, fragmented nucleic acids from biological samples was considered unimportant, and extraction methods were designed to isolate large, undegraded nucleic acid molecules. Recently, however, it is shorter base pair nucleic acids (e.g., highly degraded RNA or mRNA and apoptotic DNA) that have been shown to be highly informative for a wide range of applications, including prenatal diagnostics and the study of apoptotic DNA from host or non-host sources. Methods to capture and protect RNA during extraction are now common; however the ability to successfully analyze short, fragmented DNA in the presence of more abundant, longer DNA has remained elusive.

There is a need for improved extraction methods capable of capturing small nucleic acid molecules. At the same time, these methods need to be simple, cost-effective and automatable in order to prove useful in the research and clinical environments. Thus, the invention relates to a kit for extracting target nucleic acid comprising: a) an aqueous solution of functional group-coated paramagnetic microparticles; b) a first binding buffer formulated to comprise a suitable salt, wherein the salt is present at a concentration appropriate for binding a non-target nucleic acid of <NUM> or more base pairs to the paramagnetic microparticles; and c) a second binding buffer formulated to comprise a suitable salt, wherein the salt is present at a higher concentration than that of the first binding buffer, appropriate for binding a target nucleic acid less than <NUM> base pairs, to the paramagnetic microparticles, wherein the suitable salt in the first binding buffer and in the second binding buffer is at least one compound selected from the group consisting of guanidine salt, sodium iodide, sodium perchlorate, potassium iodide, sodium thiocyanate, urea, sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride, cesium chloride, ammonium acetate, sodium acetate and ammonium perchlorate.

According to a preferred embodiment the kit of the invention comprises reagents for the formulation of a wash buffer and an elution buffer, wherein the wash buffer dissolves impurities, but not the nucleic acid bound to paramagnetic microparticles and the elution buffer is a non-salt buffered solution with a pH range between about <NUM> to about <NUM>.

According to a further preferred embodiment of kit of the invention the functional group-coated paramagnetic microparticles are silica-coated, hydroxyl-coated, amine-coated, carboxyl-coated or encapsulated carboxyl group-coated.

According to a yet further preferred embodiment of kit of the invention the salt is guanidine (iso)thiocyanate or sodium perchlorate.

The presence of cell-free nucleic acid in peripheral blood is a well established phenomenon. While cell-free nucleic acid may originate from several sources, it has been demonstrated that one source of circulating extracellular nucleic acid originates from programmed cell death, also known as apoptosis. The source of nucleic acid that arise 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 and allograft rejection.

The present disclosure includes methods, compositions and kits to extract and relatively enrich by physical separation or amplification 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 disclosure provides compositions, methods and kits for the selective extraction and relative enrichment, based on size discrimination, of nucleic acid of approximately <NUM>,<NUM> base pairs or less (herein referred to as "target nucleic acid") in a high background of genomic nucleic acids (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 acids.

The methods of the present disclosure may be used to improve pathogen detection. 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 in published <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, <CIT>, and <CIT> and <CIT>, <CIT>, <CIT>and <CIT>.

The term "host cell" as used herein is any cell into which exogenous nucleic acid can be introduced, producing a host cell which contains exogenous nucleic acid, in addition to host cell nucleic acid. As used herein the terms "host cell nucleic acid" and "endogenous nucleic acid" refer to nucleic acid species (e.g., genomic or chromosomal nucleic acid) that are present in a host cell as the cell is obtained. As used herein, the term "exogenous" refers to nucleic acid other than host cell nucleic acid; exogenous nucleic acid can be present into a host cell as a result of being introduced in the host cell or being introduced into an ancestor of the host cell. Thus, for example, a nucleic acid species which is exogenous to a particular host cell is a nucleic acid species which is non-endogenous (not present in the host cell as it was obtained or an ancestor of the host cell). Appropriate host cells include, but are not limited to, bacterial cells, yeast cells, plant cells and mammalian cells.

The term "extraction" as used herein refers to the partial or complete separation and 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 combination which includes or is a mixture of species of nucleic acid molecules.

The terms "nucleic acid" and "nucleic acid molecule" may be used interchangeably throughout the disclosure. The terms refer to a deoxyribonucleotide (DNA), ribonucleotide polymer (RNA), RNA/DNA hybrids and polyamide nucleic acids (PNAs) 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.

The term "target nucleic acid" as used herein refers to the nucleic acid of interest that is extracted based on its molecular size, preferably in a second extraction step, and further isolated for downstream analysis. The target nucleic acid may have a molecular size smaller than the non-target nucleic acid present in the biological sample, for example, smaller than <NUM> base pairs. The target nucleic acid may be from apoptotic DNA, fetal DNA, oncogenic DNA, or any non-host DNA. The target nucleic acid may be cell-free nucleic acid. The target nucleic acid may be oligonucleosomal nucleic acid generated during programmed cell death.

The term "non-target nucleic acid" as used herein refers to the relatively high amount of non-desired background nucleic acid present in a biological sample, which is extracted, preferably, in a first extraction step. Non-target nucleic acid may have a molecular size larger than target nucleic acid, for example, greater than <NUM> base pairs. Non-target nucleic acid may be from a host or host cell. Non-target nucleic acid may be of maternal origin.

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).

Fetal nucleic acid is present in maternal plasma from the first trimester onwards, with concentrations that increase with progressing gestational age (<NPL>). After delivery, fetal nucleic acid is cleared very rapidly from the maternal plasma (<NPL>). Fetal nucleic acid is present in maternal plasma in a much higher fractional concentration than fetal nucleic acid in the cellular fraction of maternal blood (<NPL>). Thus, the target nucleic acid may be of fetal origin, and the non-target nucleic acid may be of maternal origin.

The present disclosure relates to extracting nucleic acid from a biological sample such as whole blood, serum, plasma, umbilical cord blood, chorionic villi, amniotic fluid, cerbrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, athroscopic), 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. The biological sample may be 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 the fraction of whole blood resulting from centrifugation of blood treated with anticoagulants. Blood serum refers to the watery portion of fluid remaining after a blood sample has coagulated. 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.

The biological sample may be cell-free or substantially cell-free. The biological sample may be 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, etc. may be first treated to inactivate intracellular nucleases through the addition of an enzyme, a chaotropic substance, a detergent or any combination thereof. The biological sample may be 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, etc..

The term "concentration sufficient to selectively bind" as used herein refers to an amount sufficient to cause at least <NUM>%, more preferably <NUM>%, even more preferably <NUM>% or more of the target nucleic acid to bind to an adsorptive surface. Suitable solid phase carriers include, but are not limited to, other particles, fibers, beads and or supports which have an affinity for nucleic acids or may be modified (e.g., the addition of a functional group or groups) to bind nucleic acids, and which can embody a variety of shapes, that are either regular or irregular in form, provided that the shape maximizes the surface area of the solid phase, and embodies a carrier which is amenable to microscale manipulations. Silica-coated magnetic beads may be used. The solid support may be modified to reversibly bind nucleic acid. The solid support may have a functional group-coated surface. The functional group-coated surface may be silica-coated, hydroxyl-coated, amine-coated, carboxyl-coated and encapsulated carboxyl group-coated.

The term "functional group-coated surface" as used herein refers to a surface which is coated with moieties which reversibly 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 may be 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 another example, an oligonucleotide (e.g., an adapter or primer) is labeled with biotin which may bind to immobilized streptavidin.

The extraction of nucleic acid from biological material requires cell lysis, inactivation of cellular nucleases and separation of the desired nucleic acid from cellular debris. Common lysis procedures include mechanical disruption (e.g., grinding, hypotonic lysis), chemical treatment (e.g., detergent lysis, chaotropic agents, thiol reduction), and enzymatic digestion (e.g., proteinase K). The biological sample may be first lysed in the presence of a lysis buffer, chaotropic agent (e.g., salt) and proteinase or protease. Cell membrane disruption and inactivation of intracellular nucleases may be combined. For instance, a single solution may contain detergents to solubilise cell membranes and strong chaotropic salts to inactivate intracellular enzymes. After cell lysis and nuclease inactivation, cellular debris may easily be removed by filtration or precipitation.

Lysis may be blocked. In these cases, 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 may be a cell lysis inhibitor, and may be glutaraldehyde, derivatives of glutaraldehyde, formaldehyde, formalin, or derivatives of formaldehyde.

The method may 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 may be 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. The carriers may be added to the second binding solution or washing buffer.

The final relative percentage of target nucleic acid to non-target nucleic acid may be at least about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA. about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, about <NUM>-<NUM>% fetal DNA, or about <NUM>-<NUM>% fetal DNA.

The methods provided herein may also be modified to combine steps, for example, in order to improve automation.

In another example, the methods of the present disclosure may be used in conjunction with any known technique suitable for the extraction, isolation or purification of nucleic acids, including, but not limited to, cesium chloride gradients, gradients, sucrose gradients, glucose gradients, centrifugation protocols, boiling, Microcon <NUM> filter, Chemagen viral DNA/RNA <NUM> kit, Chemagen blood kit, Qiagen purification systems, Qiagen MinElute kits, QIA DNA blood purification kit, HiSpeed Plasmid Maxi Kit, QlAfilter 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.

The first extraction method may be any known or modified technique suitable for the extraction, isolation or purification of non-target nucleic acids (i.e., larger than target nucleic acids), including, but not limited to, cesium chloride gradients, gradients, sucrose gradients, glucose gradients, centrifugation protocols, boiling, Microcon <NUM> filter, Chemagen viral DNA/RNA <NUM> kit, Chemagen blood kit, Qiagen purification systems, Qiagen MinElute kits, QIA DNA blood purification kit, HiSpeed Plasmid Maxi Kit, QlAfilter 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. One or more of the above methods may be modified to selectively extract larger non-target nucleic acids while not extracting smaller target nucleic acids. For example, the temperature, pH or reagent concentrations of one or more of the above methods may be modified.

The second extraction method may be any known or modified technique suitable for the extraction, isolation or purification of target nucleic acids (i.e., smaller than non-target nucleic acids), including, but not limited to, cesium chloride gradients, gradients, sucrose gradients, glucose gradients, centrifugation protocols, boiling, Microcon <NUM> filter, Chemagen viral DNA/RNA <NUM> kit, Chemagen blood kit, Qiagen purification systems, Qiagen MinElute kits, QIA DNA blood purification kit, HiSpeed Plasmid Maxi Kit, QlAfilter 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. One or more of the above methods may be modified to selectively extract smaller nucleic acids, for example, present in a supernatant from a previously extracted sample. For example, the temperature, pH or reagent concentrations of one or more of the above methods may be modified.

The present specification also further relates to kits for practicing the disclosed methods.

Programmed cell death or apoptosis is an essential mechanism in morphogenesis, development, differentiation, and homeostasis in all multicellular organisms. Typically, apoptosis is distinguished from necrosis by activation of specific pathways that result in characteristic morphological features including DNA fragmentation, chromatin condensation, cytoplasmic and nuclear breakdown, and the formation of apoptotic bodies.

Caspase-activated DNase (CAD), alternatively called DNA fragmentation factor (DFF or DFF40), has been shown to generate double-stranded DNA breaks in the internucleosomal linker regions of chromatin leading to nucleosomal ladders consisting of DNA oligomers of approximately <NUM> base pairs or multiples thereof. The majority of the ladder fragments (up to <NUM>%) occur as nucleosomal monomers of 180bp. All fragments carry <NUM>'- phosphorylated ends and the majority of them are blunt-ended (<NPL>). Since non-apoptotic DNA is lacking this feature, any method that can select for DNA fragments with blunt, <NUM>'- phosphorylated ends, is suitable to select for specific features (such as size, sequence and DNA base methylation differences) of the apoptotic DNA in a given biological sample. See for example, <CIT>, <CIT>, <CIT>, or <CIT>.

Very short, single base <NUM>' and <NUM>'-overhangs have also been detected but represent a minority of the DNA species in apoptotic ladders (<NPL>; Widlak et al, <NUM>). Hence, methods that are selective for both, blunt, and <NUM>'-phosphorylated blunt ends, are only slightly less sensitive but retain very high specificity for DNA of apoptotic origin.

For enrichment and detection of apoptotic DNA ladders in mammalian tissues, a method has been described that takes advantage of the presence of blunt, <NUM>'-phosphorylated ends in apoptotic DNA by ligation of synthetic, blunt-ended linkers to both ends of linear apoptotic DNA fragments with T4 ligase which is able to form a covalent bond between the <NUM>'-hydroxy ends of the synthetic linker and the <NUM>'-phosphorylated ends of the DNA fragments (<NPL>). The method can only be used as a generic tool to characterize the size distribution of apoptotic ladders in specific tissues in general, and is not site or sequence specific.

A variation of the method, that employs biotinylated hairpin probes stained with fluorescence dye streptavidin conjugates had been introduced described patent (Didenko et al <NUM>; <CIT> and <CIT>) to selectively detect terminal apoptotic activities in tissue sections.

Recently, the concept of blunt-end ligation-mediated whole genome amplification of apoptotic and necrotic plasma DNA has been introduced (<NPL>) for the analysis of allelic imbalance in tumor-specific DNA biomarkers. In this approach, isolated plasma DNA is first treated with T4 DNA polymerase to convert DNA fragments to blunt-ends before the blunt, <NUM>'-phophorylated DNA termini are self-ligated or cross ligated. The self-ligated, circular fragments are then amplified approximately <NUM>,<NUM> fold via random primer-initiated multiple displacement amplification. However, since this approach amplifies all apoptotic DNA sequences present in the sample, at least 1ng (which represents about <NUM> genome equivalents of human DNA) is required to maintain equal genomic representation and gene-dosage and allelic ratios present before amplification.

Thus, there is an increasing need to characterize known mutations and epimutations of specific DNA fragments from specific cells or tissues or present as extracellular fragments in biological fluids in a target-specific manner in the presence of high background of wild-type DNA (e.g. somatic mutations of DNA from cells responding to a xenobiotic of drug treatment; from inflamed, malignant or otherwise diseased tissues; from transplants or from differences of fetal and maternal DNA during pregnancy).

The present specification, therefore, also provides a method for selectively amplifying short, fragmented nucleic acid by adapter mediated ligation and other related methods. The method capitalizes on the blunt end and <NUM>'-phosphorylated nature of the target nucleic acid as a means to attach a non-genome specific adapter to the blunt ends using a ligation process. While the nature of the termini of all cell-free nucleic acid is unknown, coupling this method with short extension times during amplification will favor the amplification of the oligonucleosome monomer and short multimers. Since the target nucleic acid is shorter than the non-targeted nucleic acid, the target nucleic acid can be enriched over the non-target nucleic acid. This method can be further coupled with specific amplification of a nucleic acid region of interest for further analysis. The <NUM>' and <NUM>' dephosphorylated adapters are complementary and form a double-stranded blunt end adapter complex. The <NUM>' adapter of the adapter complex ligates to the <NUM>' phosphorylated strand of the target nucleic acid, and heat is introduced to release the shorter, unligated <NUM>' adapter. Next, the <NUM>' protruding ends of the ligated complex are filled in by a thermostable DNA polymerase. The <NUM>' adapter is reintroduced and serves as a PCR primer for whole genome amplification.

The method is particularly useful for detecting oligonucleosomes. Oligonucleosomes are the repeating structural units of chromatin, each consisting of approximately <NUM> base pairs of DNA wound around a histone core that partially protects the DNA from nuclease digestion in vitro and in vivo. These units can be found as monomers or multimers and produce what is commonly referred to as an apoptotic DNA ladder. The units are formed by nuclease digestion of the flanking DNA not bound to histone resulting in the majority of oligonucleosomes being blunt ended and <NUM>'-phorsphorylated. In biological systems in which only a small percentage of cells are apoptotic, or in which apoptosis is occurring asynchronously, oligonucleosomes are hard to detect and harder to isolate; however, they can serve as predictors for disease and other conditions (see <CIT>).

The term "<NUM>' dephosphorylated adapter" as used herein refers to a nucleic acid which comprises about <NUM> to <NUM> base pairs that is complementary to a short dephosphorylated adapter and capable of hybridizing thereto to form a double-stranded, blunt end adapter complex capable of ligating to target nucleic acid. Specifically, the <NUM>'adapter ligates to the <NUM>'phosphorylated base of the target nucleic acid.

The term "<NUM>' dephosphorylated adapter" refers to a nucleic acid which comprises about <NUM> to <NUM> base pairs that is complementary to the <NUM>' adapter at the <NUM>' end, thus capable of creating a double-stranded blunted end necessary for ligation. The <NUM>' adapter does not bind or ligate to the oligonucleosomal DNA.

The term "<NUM>' adapter primer" as used herein refers to the same oligonucleotide sequence as the <NUM>' dephosphorylated adapter, but is later reintroduced to the ligated sample to facilitate the whole genome amplification.

The term "adapter complex" as used herein refers to the hybridized, double-stranded <NUM>' adapter and <NUM>' adapter molecule.

The method is semi quantitative. By comparing the numbers of PCR cycles needed to detect target nucleic acid in two samples, the relative amount of target nucleic acid occurring in each sample can be estimated.

The <NUM>' adapters may be bound to a solid support for increased enrichment of the target nucleic acid. In this case, non-ligated, non-target nucleic acid is substantially removed from the solution, and amplification can proceed using only the targeted material that has ligated to the <NUM>' adapters. This improves the enrichment of the target nucleic acid by removing genomic non-target nucleic acid that may compete with the target nucleic acid in the target-specific amplification step. For example, in a maternal sample, if the target sequence is present in both the mother and the fetus, and the maternal sample is very abundant (><NUM>%), under normal circumstances the fetal nucleic acid would not be detectable as it would be out-competed by the maternal nucleic acid in the first cycles of amplification. If the fetal nucleic acid is part or wholly oligonucleosomal in nature, and the majority of the ligated sample is fetal in nature, maternal nucleic acid is still present in the sample, which can compete with the fetal nucleic acid in the target-specific amplification step. Separation of non-target nucleic acid from the target nucleic acid (i.e., ligated sample) increases the detection of fetal nucleic acid in cases where there is an abundance of maternal nucleic acid in the initial biological sample, there is sample degradation, or there is a maternal condition (e.g., autoimmune disease, transplant rejection, cancer) that increases the amount of maternal oligonucleosomes. Spacer arms may be introduced between the solid support and <NUM>' adapter to improve ligation of the target nucleic acid to the adapter molecule. The ligated sample bound to a solid support may be combined with a ligated sample that does not have a solid support prior to the amplification step.

The term "spacer arms" as used herein refers to any molecule that can be used in single or multiples to create space between the solid support and an oligonucleotide (e.g., target nucleic acid). One or more hexathylene (HEG) spacer units may be inserted between the aminohexyl groups and the <NUM>' end of the <NUM>' adapter. The aminohexyl group is used for covalent coupling to the solid support. Other examples of spacer arms that may be used herein include multiple dTTP's (up to <NUM>), spacer <NUM> (an <NUM> atom hexa-ethylene glycol spacer), spacer <NUM> (a triethylene glycol spacer), or photocleavable spacers known in the art.

An alternative method disclosed herein selectively detects and amplifies target nucleic acid using a combination of the following <NUM> steps:.

Any combination of these <NUM> steps will selectively enrich the double-stranded, blunt-ended <NUM>'-phosphorylated DNA such as DNA from apoptotic ladders by several orders of magnitude over the DNA fragments present in the biological sample that cannot by circularized due to lack of blunt ends and/or missing <NUM>'-terminal phosphate groups and allow a comparison of its sequence with the wild-type sequence of the same organism or the host organism in case of a transplant or a maternal sequence in case of a pregnancy, for instance.

In a variation of the method, before step <NUM>, an aliquot of the total DNA is either treated with methylation-sensitive or methylation-resistant enzymes or with chemicals that convert methylated bases into different bases so that methylated bases in the apoptotic DNA fragments can be characterized after step <NUM> and <NUM>.

Circulating nucleic acids in the plasma and serum of patients are associated with certain diseases and conditions (See,<NPL>; <NPL>, <NPL>;<NPL>; <NPL>). Further, the method of nucleic acid isolation may affect the ability to detect these disease-associated nucleic acids circulating in the blood (<NPL>).

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 (<NPL>; <NPL>). 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 particle-associated nucleosomes. The present disclosure provides methods for extracting the short, fragmented circulating nucleic acids present in the plasma or serum of subjects, thereby enriching the short, predictive nucleic acids relative to the background genomic DNA.

The present specification provides methods of evaluating a disease condition in a patient suspected of suffering or known to suffer from the disease condition. The specification describes obtaining a biological sample from the patient suspected of suffering or known to suffer from a disease condition, selectively extracting and 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 or concentration or characteristic of enriched extracellular nucleic acid and comparing the amount or concentration or characteristic of enriched extracellular nucleic acid to a control (e.g., background genomic DNA from biological sample).

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.

The methods provided herein may be used to 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 (<NPL>). These molecules are likely packaged in apoptotic bodies and, hence, rendered more stable compared to 'free RNA' (<NPL>; <NPL>).

In the late <NUM> and <NUM> several groups demonstrated that plasma DNA derived from cancer patients displayed tumor-specific characteristics, including decreased strand stability, Ras and p53 mutations, mircrosatellite alterations, abnormal promoter hypermethylation of selected genes, mitochondrial DNA mutations and tumor-related viral DNA (<NPL>; <NPL>; <NPL>; <NPL>). 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 has revealed that the majority show lengths in multiples of nucleosomal DNA, a characteristic of apoptotic DNA fragmentation (<NPL>).

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 specification describes detection of specific extracellular nucleic acid in plasma or serum fractions of human or animal blood associated with neoplastic, premalignant or proliferative disease. Specifically, the specification 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 specification 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 specification 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 preferred cases, the non-cancerous DNA and tumor-derived DNA are differentiated by observing nucleic acid size differences, wherein low base pair DNA is associated with cancer.

Since <NUM>, it is known that free fetal DNA can be detected in the blood circulation of pregnant women. In absence of pregnancy-associated complications, the total concentration of circulating DNA is in the range of <NUM>-100ng or <NUM>,<NUM> to <NUM>,<NUM> genome equivalents/ml plasma (<NPL> and references cited therein) while the concentrations of the fetal DNA fraction increases from ca. <NUM> copies/ml in the first trimester to ><NUM> copies/ml in the third trimester. After electron microscopic investigation and ultrafiltration enrichment experiments, the authors conclude that apoptotic bodies carrying fragmented nucleosomal DNA of placental origin are the source of fetal DNA in maternal plasma.

It has been demonstrated that the circulating DNA molecules are significantly larger in size in pregnant women than in non-pregnant women with median percentages of total plasma DNA of ><NUM> bp at <NUM>% and <NUM>% for pregnant and non-pregnant women, respectively while the median percentages of fetal-derived DNA with sizes ><NUM> bp and ><NUM> bp were only <NUM>% and <NUM>%, respectively (<NPL>).

These findings have been independently confirmed (<NPL>); Patent application <CIT>) who showed as a proof of concept, that a >5fold relative enrichment of fetal DNA from ca. <NUM>% to ><NUM>% of total circulating plasma DNA is possible be means of size exclusion chromatography via preparative agarose gel electrophoresis and elution of the <300bp size fraction. Unfortunately, the method is not very practical for reliable routine use because it is difficult to automate and due to possible loss of DNA material and the low concentration of the DNA recovered from the relevant Agarose gel section.

Thus, the present specification features methods for differentiating DNA species originating from different individuals in a biological sample. These methods may be used to differentiate or detect fetal DNA in a maternal sample. The DNA species can be differentiated by observing nucleic acid size differences.

The differentiation between maternal and fetal DNA may be performed with or without quantifying the concentration of fetal DNA in maternal plasma or serum. Where the fetal DNA is quantified, the measured concentration may be used to predict, monitor or diagnose or prognosticate a pregnancy-associated disorder.

There are a variety of non-invasive and invasive techniques available for prenatal diagnosis including ultrasonography, amniocentesis, chorionic villi sampling (CVS), fetal blood cells in maternal blood, maternal serum alpha-fetoprotein, maternal serum beta-HCG, and maternal serum estriol. However, the techniques that are non-invasive are less specific, and the techniques with high specificity and high sensitivity are highly invasive. Furthermore, most techniques can be applied only during specific time periods during pregnancy for greatest utility
The first marker that was developed for fetal DNA detection in maternal plasma was the Y chromosome, which is present in male fetuses (<NPL>). The robustness of Y chromosomal markers has been reproduced by many workers in the field (<NPL>). This approach constitutes a highly accurate method for the determination of fetal gender, which is useful for the prenatal investigation of sex-linked diseases (<NPL>).

Maternal plasma DNA analysis is also useful for the noninvasive prenatal determination of fetal RhD blood group status in RhD-negative pregnant women (<NPL>). 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 <NUM> (<NPL>).

More recently, maternal plasma DNA analysis has been shown to be useful for the noninvasive prenatal exclusion of fetal β-thalassemia major (<NPL>). A similar approach has also been used for prenatal detection of the HbE gene (<NPL>).

Other genetic applications of fetal DNA in maternal plasma include the detection of achondroplasia (<NPL>), myotonic dystrophy (<NPL>), cystic fibrosis (<NPL>), Huntington disease (<NPL>), and congenital adrenal hyperplasia (<NPL>). It is expected that the spectrum of such applications will increase over the next few years.

Also described herein is that the patient is pregnant and the method of evaluating a disease or physiological condition in the patient or her fetus aids in the detection, monitoring, prognosis or treatment of the patient or her fetus. More specifically, the present specification features methods of detecting abnormalities in a fetus by detecting fetal DNA in a biological sample obtained from a mother. The methods according to the present specification provide for detecting fetal DNA in a maternal sample by differentiating the fetal DNA from the maternal DNA based on DNA characteristics (e.g., size, weight, <NUM>' phosphorylated, blunt end). See <NPL>; and<NPL>. Employing such methods, fetal DNA that is predictive of a genetic anomaly or genetic-based disease may be identified thereby providing 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 <NUM>, <NUM>, or <NUM>), and intrauterine growth retardation.

The compositions, methods and kits of the present disclosure allow for 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-based extraction of extracellular fetal DNA in the maternal circulation thus facilitates the non-invasive detection of fetal genetic traits, including paternally inherited polymorphisms which permit paternity testing.

The term "pregnancy-associated disorder," as used in this application, 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, and fetal chromosomal abnormalities such as trisomy <NUM>, <NUM>, or <NUM>.

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 be correlated with presence of a pathological condition or with a predisposition to develop a pathological condition.

Many diseases, disorders and conditions (e.g., tissue or organ rejection) produce apoptotic or nucleosomal DNA that may be detected by the methods provided herein. Diseases and disorders believed to produce apoptotic DNA include diabetes, heart disease, stroke, trauma and rheumatoid arthritis. Lupus erythematosus (SLE) (<NPL>). Rumore et al. noted that DNA purified from SLE plasma formed discrete bands, corresponding to sizes of about <NUM>-<NUM>, <NUM>, <NUM>, and <NUM> bp, closely resembling the characteristic <NUM> bp "ladder" found with oligonucleosomal DNA.

The present specification also provides a method of evaluating the disease condition of a patient suspected of having suffered from a trauma or known to have suffered from a trauma. The method includes 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.

The examples hereafter illustrate but do not limit the invention.

The example provides a procedure, using a method provided herein, to selectively extract DNA based on its size.

Add a low concentration of chaotropic salt, for example, less than <NUM>% solution (weight per volume) to the sample solution to denature proteins and inactivate nucleases, proteinase K or any protease, for example <NUM> to <NUM>µg) to further inactivate nucleases and break down proteins in solution. Alternatively, detergents, for example SDS or Triton-X <NUM> up to <NUM>% volume per volume, may be used alone or in combination with a salt.

Mix the solution thoroughly, and incubate for <NUM>-<NUM> minutes at <NUM>, or sufficient time and temperature for the enzyme in use.

Add a low concentration of salt, for example, <NUM>-<NUM>% weight per volume, and add the solid support.

Mix the solution thoroughly, and incubate <NUM>-<NUM> minutes at ambient temperature.

Transfer the solid support or solution from the vessel to a new vessel.

Add a high concentration of salt, for example, <NUM>-<NUM>% weight per volume, and add (fresh) solid support.

Discard the supernatant and proceed to washing the target nucleic acid-bound solid support.

Wash target nucleic acid-bound solid support using an appropriate washing solution comprised of salt, buffer, water and alcohol. (Addition of carrier to wash solution may increase recovery).

Remove washing solution and repeat by gradually increasing alcohol concentration in the wash solution.

Air dry solid support at ambient temperature or by exposing to heat to completely dry and remove any remaining alcohol that would inhibit downstream use of the sample.

Release the target nucleic acid from the solid support by addition of sufficient sterile water or buffered solution (e.g. 1xTE pH <NUM>-<NUM>) at ambient temperature or by exposing to heat.

<FIG> shows the successful extraction of low base pair DNA from a 1kb DNA ladder (Promega™) in the presence of guanidine thiocyanate (GuSCN). The DNA is first bound to silica at various low concentrations of guanidine thiocyanate as shown in <FIG>. The supernatant from the first binding solution is subsequently bound to silica at varying guanidine thiocyanate concentrations, with a finishing high concentration of <NUM>. These steps are followed by wash and elution steps.

The method can be employed to produce size selective separation of a commercially available DNA ladder with DNA strands ranging in mass from 250bp to <NUM>,<NUM> bp from normal human plasma in the presence of guanidine thiocyanate as the chaotropic salt. The following steps are performed:.

<FIG> shows the successful extraction of low base pair DNA from a 1kb DNA ladder (Promega™) in the presence of sodium perchlorate (NaClO<NUM>). The DNA is first bound to silica at various low concentrations of sodium perchlorate as shown in <FIG>. The supernatant from the first binding solution is subsequently bound to silica at varying sodium perchlorate concentrations, with a finishing high concentration of <NUM>. These steps are followed by wash and elution steps.

The method can be employed to produce size selective separation of a commercially available DNA ladder with DNA strands ranging in mass from 250bp to <NUM>,<NUM> bp from normal human plasma in the presence of sodium perchlorate (NaClO<NUM>) as the chaotropic salt. The following steps are performed:.

The below example provides a procedure, using the method provided herein, to selectively amplify target nucleic acid that is blunt-ended and <NUM>'-phosphorylated. The method relies on the ligation of a non-genome specific adapter to the blunt ends, which allows for whole genome amplification followed by target-specific amplification. While the nature of the termini of all cell-free nucleic acid is unknown, coupling this method with short extension times during amplification will favor the amplification of the oligonucleosome monomer and short multimers. Since the target nucleic acid is shorter than the non-targeted nucleic acid, the target nucleic acid can be enriched over the non-target nucleic acid. This method can be further coupled with specific amplification of a nucleic acid region of interest for further analysis. <FIG> shows results of the method.

The <NUM>' adapter is complementary to the <NUM>'-end of <NUM>' adapter to create the blunt-end, double-stranded adapter complex.

Alternatively, the <NUM>' adapter molecule is modified such that the new sequence consists of <NUM> nucleobases with a dideoxy-nucleotide at its <NUM>'-position. This terminator nucleotide does not allow extension of the <NUM>' adapter molecule by any polymerase, thus improving assay efficiency and detection.

The <NUM>' adapter is shorter to reduce the melting temperature and allow for release from the <NUM>' adapter following ligation. Both adapters are non-phosphorylated, and may be made of any sequence that is nonspecific to the nucleic acid to be amplified to prevent non-specific amplification of the genome.

An exemplary procedure is provided below:.

Prepare total or size selective nucleic acid sample in water or buffer. Add ligation buffer, <NUM>' adapter, <NUM>' adapter, and water to reaction volume. Place the reaction into a thermocycler and heat the reaction to <NUM> for <NUM> minutes, then slowly ramp down the temperature to <NUM> over <NUM> hour. Add <NUM>µl T4 ligase (<NUM>-<NUM> U per ul) (or ligation enzyme) and mix well and incubate for <NUM> at <NUM>, then ramp temperature up to <NUM> and incubate for <NUM> minutes to over night (<NUM>-<NUM> hours). Add <NUM>' primer, 10x PCR buffer, MgCl<NUM>, dNTPs, and polymerase. Incubate at <NUM> for <NUM> minutes (displacement of <NUM>' adapter and initial extension of the ligated sample)
<NUM>. Thermocycle for non-template specific amplification.

Add template-specific <NUM>' and <NUM>' primers, 10x PCR buffer, MgCl<NUM>, dNTPs, and polymerase. Continue with sample to perform target-specific amplification and detection.

Described hereafter is a method for intramolecular ligation followed by amplification of a target by inverse PCR or rolling circle amplification to detect a target nucleic acid.

Prepare total or size selective nucleic acid sample in water or buffer. Add ligation buffer, <NUM>µL ligase (<NUM>-<NUM> U per µL ) and water to reaction volume. Mix well and incubate for <NUM> minutes to over night (<NUM>-<NUM> hours) at <NUM>-<NUM>, followed by ligase inactivation at <NUM> C for <NUM> (or as required for enzyme used). Add PCR buffer, dNTPs, MgCl, inverse PCR primers, and polymerase to the sample. Thermocycle for <NUM> cycles non-template specific amplification.

Claim 1:
A kit for extracting target nucleic acid comprising:
a) an aqueous solution of functional group-coated paramagnetic microparticles;
b) a first binding buffer formulated to comprise a suitable salt, wherein the salt is present at a concentration appropriate for binding a non-target nucleic acid of <NUM> or more base pairs to the paramagnetic microparticles; and
c) a second binding buffer formulated to comprise a suitable salt, wherein the salt is present at a higher concentration than that of the first binding buffer, appropriate for binding a target nucleic acid less than <NUM> base pairs, to the paramagnetic microparticles, wherein the suitable salt in the first binding buffer and in the second binding buffer is at least one compound selected from the group consisting of guanidine salt, sodium iodide, sodium perchlorate, potassium iodide, sodium thiocyanate, urea, sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride, cesium chloride, ammonium acetate, sodium acetate and ammonium perchlorate.