WO2023130101A2 - Methods and probes for separating genomic nucleic acid fractions for cancer risk analysis - Google Patents
Methods and probes for separating genomic nucleic acid fractions for cancer risk analysis Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the Global Cancer Observatory International Agency for Research on Cancer, December 2020.
- Cancer involves transformation of normal cells into tumor cells. Such changes are based in part on an interaction between a subject’s genetic factors and carcinogens.
- Survival rates for cancer improve significantly with early cancer detection, and thus, early cancer detection is recognized as an important tool for cancer treatment and management.
- nucleic acid fractions e.g., genomic deoxyribonucleic acid fractions
- Such methods and reagents can facilitate genetic analysis and can lead to accurate detection of cancer even in the early stage.
- a method for preparing nucleic acid (e.g., genomic deoxyribonucleic acid (gDNA)) fractions useful for analyzing genetic loci associated with a risk of a cancer includes obtaining the nucleic acid from a subject; fragmenting the nucleic acid to obtain nucleic acid fractions; mixing the nucleic acid fractions with one or more capture probes in a solution, a respective capture probe coupled with a capture moiety, for forming one or more hybrids between the one or more capture probes and the nucleic acid fractions; and separating the one or more hybrids from the solution by using capture moieties of the one or more capture probes.
- gDNA genomic deoxyribonucleic acid
- the method also includes analyzing the genetic loci in one or more nucleic acid fractions in the one or more hybrids.
- analyzing the genetic loci includes identifying an insertion or deletion at a respective genetic locus of the genetic loci.
- the insertion or deletion at the respective genetic locus of the genetic loci is identified by sequencing.
- the method also includes, subsequent to separating the one or more hybrids from the solution, separating one or more nucleic acid fractions from the one or more hybrids.
- a capture probe of the one or more capture probes comprises a nucleic acid sequence having at least 80% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. [0014] In some embodiments, the capture probe has 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. [0015] In some embodiments, the method also includes mixing the nucleic acid fractions with two or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the nucleic acid fractions with two or more capture probes, a respective capture probe of the two or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the nucleic acid fractions with one hundred or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the nucleic acid fractions with one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the nucleic acid fractions with one thousand or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the nucleic acid fractions with one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the capture moiety is selected from a group consisting of avidin, streptavidin, and biotin.
- the subject is a human subject.
- a reagent kit includes one or more capture probes, a respective capture probe coupled with a capture moiety and having at least 80% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150.
- the reagent kit includes two or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the reagent kit includes two or more capture probes, a respective capture probe of the two or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the reagent kit includes one hundred or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the reagent kit includes one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the reagent kit includes one thousand or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the reagent kit includes one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the capture moiety is selected from a group consisting of avidin, streptavidin, and biotin.
- the capture moiety is selected from a group consisting of avidin, streptavidin, and biotin.
- Figure 1 depicts a method of preparing genomic deoxyribonucleic acid fractions in accordance with some embodiments.
- DETAILED DESCRIPTION [0034] As explained above, the early cancer detection is important for treatment and management of cancer. It is believed that cancer is caused by certain changes to genes and thus, genetic analysis can detect changes in the genes that are associated with cancer. [0035] Although genetic sequencing techiques have improved over years, processing genetic material can further improve the accuracy of the sequencing analysis.
- gDNA genomic doxyribonucleic acid
- enriching gDNA fragments associated with cancer can improve the efficiency and accuracy of early cancer detection.
- invention or “present invention” as used herein is not meant to be limiting to any one specific embodiment of the invention but applies generally to any and all embodiments of the invention as described in the claims and specification.
- the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.
- references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure. It should be understood that the use of “and/or” is defined inclusively such that the term “a, b and/or c” should be read to include the sets of “a,” “b,” “c,” “a and b,” “b and c,” “c and a,” and “a, b and c.” [0038] As used herein, the term “about” means modifying, for example, lengths of nucleotide sequences, degrees of errors, dimensions, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, and like values, and ranges thereof, refers to variation in the numerical quantity that may occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of
- the term “about” also encompasses amounts that differ due to aging of, for example, a composition, formulation, or cell culture with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture. Whether modified by the term “about” the claims appended hereto include equivalents to these quantities.
- the term “about” further may refer to a range of values that are similar to the stated reference value. In certain embodiments, the term “about” refers to a range of values that fall within 50, 25, 10, 9, 8,7, 6, 5,4, 3, 2, 1 percent or less of the stated reference value.
- polymorphism refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals.
- genomic mutation or “genetic variation” and variants thereof include polymorphisms.
- single nucleotide polymorphism refers to a site of one nucleotide that varies between alleles.
- a single nucleotide polymorphism is a single base change or point mutation but variants also include the so-called “indel” mutations (insertions or deletions of 1 to several up to 75 nucleotides), resulting in genetic variation between individuals.
- SNPs which make up about 90% of all human genetic variation, occur every 100 to 300 bases along the 3-billion-base human genome. However, SNPs can occur much more frequently in other organisms like viruses. SNPs can occur in coding or non-coding regions of the genome. A SNP in the coding region may or may not change the amino acid sequence of a protein product. A SNP in a non-coding region can alter promoters or processing sites and may affect gene transcription and/or processing. Knowledge of whether an individual has particular SNPs in a genomic region of interest may provide sufficient information to develop diagnostic, preventive and therapeutic applications for a variety of diseases.
- primer refers to an oligonucleotide that acts as a point of initiation of DNA synthesis in a polymerase chain reaction (PCR).
- a primer is usually about 10 to about 35 nucleotides in length and hybridizes to a region complementary to the target sequence.
- probe refers to an oligonucleotide that hybridizes to a target nucleic acid.
- Target sequence refers to a region of nucleic acid that is to be analyzed and comprises a variant site of interest.
- capture probe and variants thereof refer to an oligonucleotide that hybridizes to a target nucleic acid for separation, isolation, or removal of the target nucleic acid from other nucleic acids.
- the hybridization occurs in such a manner that the probes within a probe set may be modified to form a new, larger molecular entity (e.g., a probe product).
- the probes herein may hybridize to the nucleic acid regions of interest under stringent conditions.
- stringent is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted.
- Stringency typically occurs in a range from about T m ° C to about 20° C to 25° C below Tm.
- a stringent hybridization may be used to isolate and detect identical polynucleotide sequences or to isolate and detect similar or related polynucleotide sequences.
- stringent conditions the nucleotide sequence, in its entirety or portions thereof, will hybridize to its exact complement and closely related sequences.
- Low stringency conditions comprise conditions equivalent to binding or hybridization at 68° C.
- low stringency conditions factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
- conditions which promote hybridization under conditions of high stringency e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc. are well known in the art.
- High stringency conditions when used in reference to nucleic acid hybridization, comprise conditions equivalent to binding or hybridization at 68° C in a solution consisting of 5+SSPE, 1% SDS, 5 ⁇ Denhardt’s reagent and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1+SSPE and 0.1% SDS at 68° C when a probe of about 100 to about 1000 nucleotides in length is employed.
- cancer is the second leading cause of death in the United States. Many studies exist within the literature that attempt to the genetic causes of cancer. These studies have uncovered numerous possible genetic variants or SNPs that are believed to contribute to the etiology of the disease. [0046] However, cancer is not a single disease. Different types of cancer may manifest on different organs, and more importantly, there are typically multiple different types of cancer that may manifest on a same organ. Because different types of cancer may be caused by different genetic mutations, cancer screening can require analysis of a significant portion of patient’s genome.
- FIG. 1 is a flow diagram illustrating a method 100 for preparing nucleic acid (e.g., genomic deoxyribonucleic acid (gDNA)) fractions in accordance with some embodiments. The method is useful for analyzing genetic loci associated with a risk of a cancer.
- nucleic acid e.g., genomic deoxyribonucleic acid (gDNA)
- the method includes (110) obtaining nucleic acid (e.g., genomic deoxyribonucleic acid (gDNA)), from a subject.
- nucleic acid e.g., genomic deoxyribonucleic acid (gDNA)
- the nucleic acid is obtained from one or more genomic samples.
- the method includes isolating the on or more genomic samples to identify and validate genetic mutations.
- the method includes extracting the one or more genomic samples from the subject.
- the genomic samples may be selected from the group consisting of isolated cells, whole blood, serum, plasma, urine, saliva, sweat, fecal matter, and tears.
- a genomic sample is plasma or serum.
- the method includes isolating the plasma or serum from a blood sample of the subject. In some embodiments, the method includes separating genomic materials from whole blood, plasma, or serum. [0052] In some embodiments, the method includes providing a sample of cells from the subject. In some embodiments, the cells are collected by contacting a cellular surface of the subject with a substrate capable of reversibly immobilizing the cells onto a substrate. [0053] The disclosed methods are applicable to a variety of cell types obtained from a variety of samples.
- the cell type for use with the disclosed methods include but is not limited to epithelial cells, endothelial cells, connective tissue cells, skeletal muscle cells, endocrine cells, cardiac cells, urinary cells, melanocytes, keratinocytes, blood cells, white blood cells, buffy coat, hair cells (including, e.g., hair root cells) and/or salival cells.
- the cells are epithelial cells.
- the cells are subcapsular-perivascular (epithelial type 1); pale (epithelial type 2); intermediate (epithelial type 3); dark (epithelial type 4); undifferentiated (epithelial type 5); and large-medullary (epithelial type 6).
- the cells are buccal epithelial cells (e.g., epithelial cells collected using a buccal swab).
- the sample of cells used in the disclosed methods include any combination of the above identified cell types.
- the method includes providing a sample of cells from a subject.
- the cells provided are buccal epithelial cells.
- the cell sample is collected by any of a variety of methods which allow for reversible binding of the subject’s cells to the substrate.
- the substrate is employed in a physical interaction with the sample containing the subject’s cells in order to reversibly bind the cells to the substrate.
- the substrate is employed in a physical interaction with the body of the subject directly in order to reversibly bind the cells to the substrate.
- the sample is a buccal cell sample and the sample of buccal cells is collected by contacting a buccal membrane of the subject (e.g., the inside of their cheek) with a substrate capable of reversibly immobilizing cells that are dislodged from the membrane.
- the swab is rubbed against the inside of the subject’s cheek with a force equivalent to brushing a person’s teeth (e.g., a light amount of force or pressure). Any method which would allow the subject’s cells to be reversibly bound to the substrate is contemplated for use with the disclosed methods.
- the sample is advantageously collected in a non-invasive manner. As such sample collection is accomplished anywhere and by almost anyone.
- the sample is collected at a physician’s office, at a subject’s home, or at a facility where a medical procedure is performed or to be performed.
- the subject the subject’s doctor, nurses or a physician’s assistant or other clinical personnel collects the sample.
- the substrate is made of any of a variety of materials to which cells are reversibly bound.
- Exemplary substrates include those made of rayon, cotton, silica, an elastomer, a shellac, amber, a natural or synthetic rubber, cellulose, BAKELITE, NYLON, a polystyrene, a polyethylene, a polypropylene, a polyacrylonitrile, or other materials or combinations thereof.
- the substrate is a swab having a rayon tip or a cotton tip.
- the substrate containing the sample is freeze-thawed one or more times (e.g., after being frozen, the substrate containing the sample is thawed, used according to the present methods and re-frozen) and or used in the present methods.
- lysis solutions have been described and are known to those of skill in the art. Any of these well-known lysis solutions can be employed with the present methods in order to isolate nucleic acids from a sample.
- Exemplary lysis solutions include those commercially available, such as those sold by INVITROGEN®, QIAGEN®, LIFE TECHNOLOGIES® and other manufacturers, as well as those which can be generated by one of skill in a laboratory setting.
- Lysis buffers have also been well described and a variety of lysis buffers can find use with the disclosed methods, including for example those described in Molecular Cloning (three volume set, Cold Spring Harbor Laboratory Press, 2012) and Current Protocols (Genetics and Genomics; Molecular Biology; 2003-2013), both of which are incorporated herein by reference for all purposes.
- Cell lysis is a commonly practiced method for the recovery of nucleic acids from within cells. In many cases, the cells are contacted with a lysis solution, commonly an alkaline solution comprising a detergent, or a solution of a lysis enzyme. Such lysis solutions typically contain salts, detergents and buffering agents, as well as other agents that one of skill would understand to use.
- the nucleic acids are recovered from the lysis solution.
- cells are resuspended in an aqueous buffer, with a pH in the range of from about pH 4 to about 10, about 5 to about 9, about 6 to about 8 or about 7 to about 9.
- the buffer salt concentration is from about 10 mM to about 200 mM, about 10 mM to about 100 mM or about 20 mM to about 80 mM.
- the buffer further comprises chelating agents such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA).
- the lysis solution further comprises other compounds to assist with nucleic acid release from cells such as polyols, including for example but not limited to sucrose, as well as sugar alcohols such as maltitol, sorbitol, xylitol, erythritol, and/or isomalt.
- polyols are in the range of from about 2% to about 15% w/w, or about 5% to about 15% w/w or about 5% to about 10% w/w.
- the lysis solutions further comprises surfactants, such as for example but not limited to Triton X-100, SDS, CTAB, X-114, CHAPS, DOC, and/or NP-40.
- surfactants are in the range of from about 1% to about 5% w/w, about 1% to about 4% w/w, or about 1% to about 3% w/w.
- the lysis solution further comprises chaotropes, such as for example but not limited to urea, sodium dodecyl sulfate and/or thiourea.
- the chaotrope is used at a concentration in the range of from about 0.5 M to 8 M, about 1 M to about 6 M, about 2 M to about 6 M or about 1 M to 3 M.
- the lysis solution further comprises one or more additional lysis reagents and such lysis reagents are well known in the art.
- such lysis reagents include cell wall lytic enzymes, such as for example but not limited to lysozyme.
- lysis reagents comprise alkaline detergent solutions, such as 0.1 aqueous sodium hydroxide containing 0.5% sodium dodecyl sulphate.
- the lysis solution further comprises aqueous sugar solutions, such as sucrose solution and chelating agents such as EDTA, for example the STET buffer.
- the lysis reagent is prepared by mixing the cell suspension with an equal volume of lysis solution having twice the desired concentration (for example 0.2 sodium hydroxide, 1.0% sodium dodecyl sulphate).
- the mixture comprising lysis solution and lysed cells is contacted with a neutralizing or quenching reagent to adjust the conditions such that the lysis reagent does not adversely affect the desired product.
- the pH is adjusted to a pH of from about 5 to about 9, about 6 to about 8, about 5 to about 7, about 6 to about 7 or about 6.5 to 7.5 to minimize and/or prevent degradation of the cell contents, including for example but not limited to the nucleic acids.
- the neutralizing reagent comprises an acidic buffer, for example an alkali metal acetate/acetic acid buffer.
- lysis conditions such as temperature and composition of the lysis reagent are chosen such that lysis is substantially completed while minimizing degradation of the desired product, including for example but not limited to nucleic acids.
- nucleic acids including for example but not limited to genomic DNA
- the nucleic acids are isolated from lysis buffer prior to performing subsequent analysis.
- the nucleic acids are isolated from the lysis buffer prior to the performance of additional analyses, such as for example but not limited to sequencing.
- additional analyses such as for example but not limited to sequencing.
- Any of a variety of methods useful in the isolation of small quantities of nucleic acids are used by various embodiments of the disclosed methods. These include but are not limited to precipitation, gel filtration, density gradients and solid phase binding.
- Nucleic acid precipitation is a well know method for isolation that is known by those of skill in the art.
- solid phase binding methods are also known in the art including but not limited to solid phase binding methods that make use of solid phases in the form of beads (e.g., silica, magnetic), columns, membranes or any of a variety other physical forms known in the art.
- solid phases used in the disclosed methods reversibly bind nucleic acids.
- solid phases examples include so-called “mixed-bed” solid phases are mixtures of at least two different solid phases, each of which has a capacity to bind to nucleic acids under different solution conditions, and the ability and/or capacity to release the nucleic acid under different conditions; such as those described in US Patent Application No.2002/0001812, incorporated by reference herein in its entirety for all purposes.
- Solid phase affinity for nucleic acids according to the disclosed methods can be through any one of a number of means typically used to bind a solute to a substrate.
- Examples of such means include but are not limited to, ionic interactions (e.g., anion- exchange chromatography) and hydrophobic interactions (e.g., reversed-phase chromatography), pH differentials and changes, salt differentials and changes (e.g., concentration changes, use of chaotropic salts/agents).
- ionic interactions e.g., anion- exchange chromatography
- hydrophobic interactions e.g., reversed-phase chromatography
- pH differentials and changes e.g., reversed-phase chromatography
- salt differentials and changes e.g., concentration changes, use of chaotropic salts/agents.
- Exemplary pH based solid phases include but are not limited to those used in the INVITROGEN ChargeSwitch Normalized Buccal Kit magnetic beads, to which bind nucleic acids at low pH ( ⁇ 6.5) and releases nucleic acids at high pH (>8.5) and mono-amino-N-aminoethyl (MANAE) which binds nucleic acids at a pH of less than 7.5 and release nucleic acids at a pH of greater than 8.
- MANAE mono-amino-N-aminoethyl
- Exemplary ion exchange based substrates include but are not limited to DEA-SEPHAROSETM, Q- SEPHAROSETM, and DEAE-SEPHADEXTM from PHARMACIA (Piscataway, N.J.), DOWEX® I from The Dow Chemical Company (Midland, Mich.), AMBERLITE® from Rohm & Haas (Philadelphia, Pa.), DUOLITE® from Duolite International, In. (Cleveland, Ohio), DIALON TI and DIALON TII. [0073] Any individual method is contemplated for use alone or in combination with other methods, and such useful combination are well known and appreciated by those of skill in the art.
- the disclosed methods are used to isolate nucleic acids, such as genomic DNA (gDNA) for a variety of nucleic acid analyses, including genomic analyses.
- genomic analyses including genomic analyses.
- such analysis includes detection of variety of genetic mutations, which include but are not limited to deletions, insertions, transitions and transversions.
- the mutation is a single-nucleotide polymorphism (SNP).
- the mutation is an insertion or deletion.
- the subject is (112) a human subject.
- the subject is a non-human subject (e.g., domestic animals, such as pets, livestock, and beasts of burden).
- the method also includes (120) fragmenting the nucleic acid (e.g., gDNA) to obtain nucleic acid (e.g., gDNA) fractions.
- the nucleic acid is fragmented by mechanical shearing.
- the nucleic acid may be subjected to sonication, which can result in nucleic acid fragments typically having approximately 700 bp.
- the sonification is applied at burst cycles.
- the nucleic acid may be subjected to high-frequency acoustic waves, which can fragment the nucleic acid.
- the nucleic acid may be subjected to nebulization by forcing the nucleic acid into a small hole.
- the size of the fragments is typically determined based on the speed of a nucleic acid solution passing through the hole, the viscosity of the nucleic acid solution, the size of the hole, and a temperature.
- the nucleic acid may be fragmented by point-sink shearing, which involves applying hydrodynamic shear forces to the nucleic acid.
- the nucleic acid may be fragmented by one or more enzymes (called restriction enzymes, such as endonucleases or transposases). Each restriction enzyme typically cleaves nucleic acids into fragments at or near specific sites having corresponding nucleic acid sequences. In some cases, a single type of restriction enzyme is used. In some cases, a set of enzymes of different types is used.
- the nucleic acid is enzymatically fragmented.
- An example of enzymatic fragmentation is described further below.
- the method further includes (130) mixing the nucleic acid (e.g., gDNA) fractions with one or more capture probes in a solution for forming one or more hybrids between the one or more capture probes and the nucleic acid (e.g., gDNA) fractions.
- a respective capture probe is coupled with a capture moiety.
- a capture probe of the one or more capture probes includes a nucleic acid sequence having at least 80% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, a capture probe of the one or more capture probes includes a nucleic acid sequence having at least 90% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150.
- a capture probe of the one or more capture probes includes a nucleic acid sequence having at least 95% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, a capture probe of the one or more capture probes includes a nucleic acid sequence having at least 99% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. [0080] In some embodiments, the capture probe includes a nucleic acid sequence having 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. For example, the capture probe may include a nucleic acid sequence that corresponds to SEQ ID NO: 1.
- the method also includes (132) mixing the nucleic acid (e.g., gDNA) fractions with two or more capture probes including (or having) nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0082] In some embodiments, the method also includes mixing the gDNA fractions with two or more capture probes, a respective capture probe of the two or more capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with two or more capture probes, a respective capture probe of the two or more capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with two or more capture probes, a respective capture probe of the two or more capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with two or more capture probes, a respective capture probe of the two or more capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes (134) mixing the gDNA fractions with one hundred or more capture probes including (or having) nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes (136) mixing the gDNA fractions with one thousand or more capture probes including (or having) nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0086] In some embodiments, the method also includes mixing the gDNA fractions with one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with ten thousand or more capture probes including (or having) nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with ten thousand or more capture probes, a respective capture probe of the ten thousand or more capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with ten thousand or more capture probes, a respective capture probe of the ten thousand or more capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with ten thousand or more capture probes, a respective capture probe of the ten thousand or more capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with ten thousand or more capture probes, a respective capture probe of the ten thousand or more capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with hundred thousand or more capture probes, a respective capture probe of the hundred thousand or more capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with hundred thousand or more capture probes, a respective capture probe of the hundred thousand or more capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with hundred thousand or more capture probes, a respective capture probe of the hundred thousand or more capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with hundred thousand or more capture probes, a respective capture probe of the hundred thousand or more capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with 116,150 capture probes, a respective capture probe of the 116,150 capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with 116,150 capture probes, a respective capture probe of the 116,150 capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the method also includes mixing the gDNA fractions with 116,150 capture probes, a respective capture probe of the 116,150 capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with 116,150 capture probes, a respective capture probe of the 116,150 capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
- the capture moiety is selected (138) from a group consisting of avidin, streptavidin, and biotin. In some embodiments, the capture moiety is avidin. In some embodiments, the capture moiety is streptavidin. In some embodiments, the capture moiety is biotin. [0092] The method includes (140) separating the one or more hybrids from the solution by using capture moieties of the one or more capture probes. [0093] In some embodiments, the method also includes, (150) subsequent to separating the one or more hybrids from the solution, separating one or more gDNA fractions from the one or more hybrids.
- the method also includes (160) analyzing the genetic loci in one or more gDNA fractions in the one or more hybrids.
- gDNA genomic DNA
- methods for analyzing such isolated nucleic acids include nucleic acid sequencing methods (including Next Generation Sequencing methods), PCR methods (including real- time PCR analysis, microarray analysis, hybridization analysis) as well as any other nucleic acid sequence analysis methods that are known in the art, which include a variety of other methods where nucleic acid compositions are analyzed and which are known to those of skill in the art.
- genetic mutations described herein may be detected by sequencing.
- High-throughput or Next Generation Sequencing represents an attractive option for detecting mutations within a gene. Distinct from PCR, microarrays, high-resolution melting and mass spectrometry, which all indirectly infer sequence content, NGS directly ascertains the identity of each base and the order in which they fall within a gene.
- the newest platforms on the market have the capacity to cover an exonic region 10,000 times over, meaning the content of each base position in the sequence is measured thousands of different times.
- NGS Next Generation Sequencing
- SBS Sequencing by Synthesis
- MPSS Massively Parallel Signature Sequencing
- Polony sequencing Polony sequencing
- pyrosequencing Reversible dye- terminator sequencing
- SOLiD sequencing Ion semiconductor sequencing
- DNA nanoball sequencing Helioscope single molecule sequencing
- SMRT Single molecule real time sequencing
- RNAP Single molecule real time sequencing
- Nanopore DNA sequencing RNA sequencing
- MPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences.
- Polony sequencing combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1/10 that of Sanger sequencing.
- a parallelized version of pyrosequencing the method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony.
- the sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes.
- SBS is a sequencing technology based on reversible dye-terminators. DNA molecules are first attached to primers on a flowcell and amplified so that local clonal colonies are formed. Four types of reversible terminator bases (RT-bases) are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be extended one nucleotide at a time.
- a camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle.
- SOLiD technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. [00103] Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting bead, each containing only copies of the same DNA molecule, are deposited on a glass slide.
- Ion semiconductor sequencing is based on using standard sequencing chemistry, but with a novel, semiconductor based detection system. This method of sequencing is based on the detection of hydrogen ions that are released during the polymerization of DNA, as opposed to the optical methods used in other sequencing systems.
- a micro well containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred.
- DNA nanoball sequencing is a type of high throughput sequencing technology used to determine the entire genomic sequence of an organism. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Unchained sequencing by ligation is then used to determine the nucleotide sequence. This method of DNA sequencing allows large numbers of DNA nanoballs to be sequenced per run.
- Helicos Biosciences Corporation s single-molecule sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface.
- the next steps involve extension-based sequencing with cyclic washes of the flow cell with fluorescently labeled nucleotides (one nucleotide type at a time, as with the Sanger method).
- the reads are performed by the Helioscope sequencer.
- Single molecule real time (SMRT) sequencing is based on the SBS approach.
- the DNA is synthesized in zero-mode wave-guides (ZMWs) - small well-like containers with the capturing tools located at the bottom of the well.
- the sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labeled nucleotides flowing freely in the solution.
- the wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected.
- RNA polymerase RNA polymerase
- RNAP RNA polymerase
- distal end of sequenced DNA is attached to another bead, with both beads being placed in optical traps.
- RNAP motion during transcription brings the beads in closer and their relative distance changes, which can then be recorded at a single nucleotide resolution.
- the sequence is deduced based on the four readouts with lowered concentrations of each of the four nucleotide types (similarly to Sangers method).
- Nanopore sequencing is based on the readout of electrical signal occurring at nucleotides passing by alpha-hemolysin pores covalently bound with cyclodextrin.
- the DNA passing through the nanopore changes its ion current. This change is dependent on the shape, size and length of the DNA sequence.
- Each type of the nucleotide blocks the ion flow through the pore for a different period of time.
- VisiGen Biotechnologies uses a specially engineered DNA polymerase. This polymerase acts as a sensor - having incorporated a donor fluorescent dye by its active centre. This donor dye acts by FRET (fluorescent resonant energy transfer), inducing fluorescence of differently labeled nucleotides.
- FRET fluorescent resonant energy transfer
- the SBS NGS platform uses a direct sequencing approach to produce a sequencing strategy with very a high precision, rapid pace and low cost.
- One exemplary SBS sequencing is initialized by fragmenting of the template DNA into fragments, amplification, annealing of DNA sequencing primers, and, for example, finally affixing as a high-density array of spots onto a glass chip.
- the array of DNA fragments are sequenced by extending each fragment with modified nucleotides containing cleavable chemical moieties linked to fluorescent dyes capable of discriminating all four possible nucleotides.
- the array is scanned continuously by a high-resolution electronic camera (Measure) to determine the fluorescent intensity of each base (A, C, G or T) that was newly incorporated into the extended DNA fragment. After the incorporation of each modified base the array is exposed to cleavage chemistry to break off the fluorescent dye and end cap allowing additional bases to be added. The process is then repeated until the fragment is completely sequenced or maximal read length has been achieved.
- real-time PCR is used in detecting gene mutations, including for example but not limited to SNPs.
- real-time PCR methods also include the use of molecular beacon technology.
- the molecular beacon technology utilizes hairpin-shaped molecules with an internally-quenched fluorophore whose fluorescence is restored by binding to a DNA target of interest (See, e.g., Kramer, R. et al. Nat. Biotechnol.14:303-308, 1996).
- increased binding of the molecular beacon probe to the accumulating PCR product is used to specifically detect SNPs present in genomic DNA.
- several parts are coordinated, including the DNA fragment that is flanked by the two primers and subsequently amplified, often referred to as the amplicon, the two primers and the detection probe or probes to be used.
- a SNP site in a sample from the subject may be amplified by the amplification methods described herein or any other amplification methods known in the art.
- the nucleic acids in a sample may or may not be amplified prior to contacting the SNP site with a probe described herein, using a universal amplification method (e.g., whole genome amplification and whole genome PCR).
- a universal amplification method e.g., whole genome amplification and whole genome PCR.
- Real-time PCR relies on the visual emission of fluorescent dyes conjugated to short polynucleotides (termed “detection probes”) that associate with genomic alleles in a sequence-specific fashion or on fluorescent molecules that intercalate into double stranded DNA referred to as quantitative or qPCR.
- Real-time PCR probes differing by a single nucleotide can be differentiated in a real-time PCR assay by the conjugation and detection of probes that fluoresce at different wavelengths.
- Real-Time PCR finds use in detection applications (diagnostic applications), quantification applications and genotyping applications.
- Several related methods for performing real-time PCR are disclosed in the art, including assays that rely on TAQMAN® probes (U.S. Pat. Nos.5,210,015 and 5,487,972, and Lee et al., Nucleic Acids Res.21:3761-6, 1993), molecular beacon probes (U.S. Pat. Nos. 5,925,517 and 6,103,476, and Tyagi and Kramer, Nat. Biotechnol.14:303-8, 1996), self- probing amplicons (scorpions) (U.S. Pat. No.6,326,145, and Whitcombe et al., Nat.
- TAQMAN® allelic discrimination assay an oligonucleotide probe labeled with a fluorescent reporter dye at the 5' end of the probe and a quencher dye at the 3' end of the probe is utilized. The proximity of the quencher to the intact probe maintains a low fluorescence for the reporter.
- the 5' nuclease activity of DNA polymerase cleaves the probe, and separates the dye and quencher. This results in an increase in fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye.
- the 5' nuclease activity of DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target and is amplified during PCR. The probe is designed to straddle a target SNP position and hybridize to the nucleic acid molecule only if a particular SNP allele is present.
- Real-time PCR methods include a variety of steps or cycles as part of the methods for amplification.
- cycles include denaturing double-stranded nucleic acids, annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence and synthesizing (i.e., replicating) second-strand DNA from the annealed forward primer and the reverse primer.
- This three-step process is referred to herein as a cycle.
- about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 cycles are employed.
- about 10 to about 60 cycles, about 20 to about 50 or about 30 to about 40 cycles are employed.
- 40 cycles are employed.
- the denaturing double-stranded nucleic acids step occurs at a temperature of about 80oC to 100oC, about 85oC to about 99oC, about 90oC to about 95oC for about 1 second to about 5 seconds, about 2 seconds to about 5 seconds, or about 3 seconds to about 4 seconds. In some embodiments, the denaturing double-stranded nucleic acids step occurs at a temperature of 95oC for about 3 seconds.
- the annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence step occurs at about 40oC to about 80oC, about 50oC to about 70oC, about 55oC to about 65oC for about 15 seconds to about 45 seconds, about 20 seconds to about 40 seconds, about 25 seconds to about 35 seconds. In some embodiments, the annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence step occurs at about 60oC for about 30 seconds.
- the synthesizing (i.e., replicating) second-strand DNA from the annealed forward primer and the reverse primer occurs at about 40oC to about 80oC, about 50oC to about 70oC, about 55oC to about 65oC for about 15 seconds to about 45 seconds, about 20 seconds to about 40 seconds, about 25 seconds to about 35 seconds.
- the annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence step occurs at about 60oC for about 30 seconds.
- the PCR master mix has a final volume of about 5 ⁇ L, about 6 ⁇ L, about 7 ⁇ L, about 8 ⁇ L, about 9 ⁇ L, about 0 ⁇ L, about 11 ⁇ L, about 12 ⁇ L, about 13 ⁇ L, about 14 ⁇ L, about 15 ⁇ L, about 16 ⁇ L, about 17 ⁇ L, about 18 ⁇ L, about 19 ⁇ L or about 20 ⁇ L or more.
- primers are tested and designed in a laboratory setting.
- primers are designed by computer based in silico methods. Primer sequences are based on the sequence of the amplicon or target nucleic acid sequence that is to be amplified. Shorter amplicons typically replicate more efficiently and lead to more efficient amplification as compared to longer amplicons.
- Tm melting temperature
- Primer specificity is defined by its complete sequence in combination with the 3’ end sequence, which is the portion elongated by Taq polymerase.
- the 3’ end should have at least 5 to 7 unique nucleotides not found anywhere else in the target sequence, in order to help reduce false- priming and creation of incorrect amplification products.
- Forward and reverse primers typically bind with similar efficiency to the target.
- tools such as NCBI BLAST (located on the World Wide Web at ncbi.nlm.nih.gov) are employed to performed alignments and assist in primer design.
- primer complexity or linguistic sequence complexity are typically more efficient.
- the linguistic sequence complexity calculation method is used to search for conserved regions between compared sequences for the detection of low-complexity regions including simple sequence repeats, imperfect direct or inverted repeats, polypurine and polypyrimidine triple-stranded cDNA structures, and four-stranded structures (such as G-quadruplexes).
- linguistic complexity (LC) measurements are performed using the alphabet-capacity L-gram method (see, A. Gabrielian, A. Bolshoy, Computer & Chemistry 23:263–274 (1999) and Y.L. Orlov, V.N. Potapov, Complexity: an internet resource for analysis of DNA sequence complexity, Nucleic Acids Res.32: W628–W633(2004)) along the whole sequence length and calculated as the sum of the observed range (xi) from 1 to L size words in the sequence divided by the sum of the expected (E) value for this sequence length.
- G-rich (and C-rich) nucleic acid sequences fold into four-stranded DNA structures that contain stacks of G-quartets (see, the World Wide Web at quadruplex.org).
- these quadruplexes are formed by the intermolecular association of two or four DNA molecules, dimerization of sequences that contain two G-bases, or by the intermolecular folding of a single strand containing four blocks of guanines (see, P.S. Ho, PNAS, 91:9549–9553 (1994); I.A. Il'icheva, V.L. Florent'ev, Russian Journal of Molecular Biology 26:512–531(1992); D. Sen, W.
- GC skew in a sliding window of n where n is a positive integer
- bases is calculated with a step of one base, according to the formula, (G ⁇ C)/(G+C), in which G is the total number of guanines and C is the total number of cytosines for all sequences in the windows (Y. Benita, et al., Nucleic Acids Res.31:e99 (2003)).
- Positive GC-skew values indicated an overabundance of G bases, whereas negative GC-skew values represented an overabundance of C bases.
- other skews are calculated in the sequence.
- Such methods, as well as others, are employed to determine primer complexity in some embodiments.
- real-time PCR is performed using exonuclease primers (TAQMAN® probes).
- the primers utilize the 5' exonuclease activity of thermostable polymerases such as Taq to cleave dual-labeled probes present in the amplification reaction (See, e.g., Wittwer, C. et al. Biotechniques 22:130-138, 1997).
- thermostable polymerases such as Taq to cleave dual-labeled probes present in the amplification reaction
- the primer probes used in this assay are distinct from the PCR primer and are dually-labeled with both a molecule capable of fluorescence and a molecule capable of quenching fluorescence.
- fluorescent probes include the 6-carboxy-fluorescein moiety and the like.
- Exemplary quenchers include Black Hole Quencher 1 moiety and the like.
- detection probes commonly employed by those of skill in the art include but are not limited to hydrolysis probes (also known as TAQMAN® probes, 5’ nuclease probes or dual-labeled probes), hybridization probes, and Scorpion primers (which combine primer and detection probe in one molecule).
- detection probes contain various modifications.
- detection probes include modified nucleic acid residues, such as but not limited to 2'-O-methyl ribonucleotide modifications, phosphorothioate backbone modifications, phosphorodithioate backbone modifications, phosphoramidate backbone modifications, methylphosphonate backbone modifications, 3' terminal phosphate modifications and/or 3' alkyl substitutions.
- the detection probe has increased affinity for a target sequence due to modifications.
- detection probes include detection probes with increased length, as well as detection probes containing chemical modifications.
- modifications include but are not limited to 2'-fluoro (2'-deoxy-2'-fluoro-nucleosides) modifications, LNAs (locked nucleic acids), PNAs (peptide nucleic acids), ZNAs (zip nucleic acids), morpholinos, methylphosphonates, phosphoramidates, polycationic conjugates and 2'-pyrene modifications.
- the detector probes contains one or more modifications including 2' fluoro modifications (aka, 2'-Deoxy-2'-fluoro-nucleosides), LNAs (locked nucleic acids), PNAs (peptide nucleic acids), ZNAs (zip nucleic acids), morpholinos, methylphosphonates, phosphoramidates, and/or polycationic conjugates.
- the detection probes contain detectable moieties, such as those described herein as well as any detectable moieties known to those of skill in the art. Such detectable moieties include for example but are not limited to fluorescent labels and chemiluminescent labels. Examples of such detectable moieties can also include members of FRET pairs.
- the detection probe contains a detectable entity.
- fluorescent labels include but are not limited to AMCA, DEAC (7-Diethylaminocoumarin-3-carboxylic acid); 7-Hydroxy-4-methylcoumarin-3; 7- Hydroxycoumarin-3; MCA (7-Methoxycoumarin-4-acetic acid); 7-Methoxycoumarin-3; AMF (4'-(Aminomethyl)fluorescein); 5-DTAF (5-(4,6-Dichlorotriazinyl)aminofluorescein); 6-DTAF (6-(4,6-Dichlorotriazinyl)aminofluorescein); 6-FAM (6-Carboxyfluorescein; aka FAM; including TAQMAN® FAM TM ); TAQMAN VIC®; 5(6)-FAM cadaverine; 5-FAM cadaverine; 5(6)-FAM ethylenediamme; 5-FAM ethylenediamme; 5-FITC (FITC Isomer I;
- chemiluminescent labels include but are not limited to those labels used with Southern Blot and Western Blot protocols (see, for e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, (3rd ed.) (2001); incorporated by reference herein in its entirety). Examples include but are not limited to -(2'- spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD); acridinium esters and adamantyl-stabilized 1 ,2-dioxetanes, and derivatives thereof. [00141] The labeling of probes is known in the art.
- the labeled probes are used to hybridize within the amplified region during amplification.
- the probes are modified so as to avoid them from acting as primers for amplification.
- the detection probe is labeled with two fluorescent dyes, one capable of quenching the fluorescence of the other dye.
- One dye is attached to the 5' terminus of the probe and the other is attached to an internal site, so that quenching occurs when the probe is in a non-hybridized state.
- real-time PCR probes consist of a pair of dyes (a reporter dye and an acceptor dye) that are involved in fluorescence resonance energy transfer (FRET), whereby the acceptor dye quenches the emission of the reporter dye.
- FRET fluorescence resonance energy transfer
- the fluorescence-labeled probes increase the specificity of amplicon quantification.
- Real-time PCR that are used in some embodiments of the disclosed methods also include the use of one or more hybridization probes (i.e., detection probes), as determined by those skilled in the art, in view of this disclosure.
- hybridization probes include but are not limited to one or more of those provided in the described methods.
- Exemplary probes, such as the HEX channel and/or FAM channel probes, are understood by one skilled in the art.
- detection probes and primers are conveniently selected e.g., using an in silico analysis using primer design software and cross- referencing against the available nucleotide database of genes and genomes deposited at the National Center for Biotechnology Information (NCBI).
- NCBI National Center for Biotechnology Information
- the primers and probes are selected such that they are close together, but not overlapping.
- the primers may have the same (or close TM) (e.g., between about 58 °C and about 60 °C).
- the TM of the probe is approximately 10 °C higher than that selected for the T M of the primers.
- the length of the probes and primers is selected to be between about 17 and 39 base pairs, etc. These and other guidelines are used in some instances by those skilled in the art in selecting appropriate primers and/or probes.
- the SNP described herein may be detected by melting curve analysis using the detection probes above. For example, the melting curves of short oligonucleotide probes hybridized to a region containing the SNP of interest may be analyzed. Two probes are used in these reactions, each one being complimentary to a particular allele at the SNP in question. Perfectly matched probes are more stable and have a higher melting temperature compared to mismatched probes.
- analyzing the genetic loci includes (162) identifying an insertion or deletion at a respective genetic locus of the genetic loci. [00147] In some embodiments, the insertion or deletion at the respective genetic locus of the genetic loci is identified (164) by sequencing. [00148] In some embodiments, analyzing the genetic loci includes determining a number of tandem repeats. In some embodiments, the number of tandem repeats is identified by sequencing. In some embodiments, analyzing the genetic loci includes determining numbers of tandem repeats.
- the methods described herein may include preparing nucleic acid fractions by hybridizing at least one capture probe to a nucleotide molecule from a sample and separating the hybrid of the capture probe and the nucleotide molecule.
- diagnostic testing is employed to determine one or more genetic conditions by detection of any of a variety of mutations.
- diagnostic testing is used to confirm a diagnosis when a particular condition is suspected based on for example physical manifestations, signs and/or symptoms as well as family history information.
- the results of a diagnostic test assist those of skill in the medical arts in determining an appropriate treatment regimen for a given subject and allow for more personalized and more effective treatment regimens.
- a treatment regimen includes any of a variety of pharmaceutical treatments, surgical treatments, lifestyles changes or a combination thereof as determined by one of skill in the art.
- the nucleic acids obtained by the disclosed methods are useful in a variety of diagnostic tests, including tests for detecting mutations such as deletions, insertions, transversions and transitions.
- such diagnostics are useful for identifying unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to be expressed, identifying unaffected individuals who carry one copy of a gene for a disease in which the information could find use in developing a treatment regimen, preimplantation genetic diagnosis, prenatal diagnostic testing, newborn screening, genealogical DNA test (for genetic genealogy purposes), presymptomatic testing for predicting or diagnosing cancer.
- newborns can be screened.
- newborn screening includes any genetic screening employed just after birth in order to identify genetic disorders.
- newborn screening finds use in the identification of genetic disorders so that a treatment regimen is determined early in life.
- Such tests include but are not limited to testing infants for phenylketonuria and congenital hypothyroidism.
- carrier testing is employed to identify people who carry a single copy of a gene mutation.
- the mutation can cause a genetic disorder.
- one copy is sufficient to cause a genetic disorder.
- the presence of two copies is contra-indicated for a particular treatment regimen, such as the presence of a certain genetic mutation (e.g., a mutation in the BRCA gene) and pre-screening prior to prescription of a certain pharmaceutical or biological drug in order to ensure the appropriate treatment regimen is pursued for a given subject.
- predictive and/or presymptomatic types of testing are used to detect gene mutations associated with a variety of disorders. In some cases, these tests are helpful to people who have a family member with a genetic disorder, but who may exhibit no features of the disorder at the time of testing. In some embodiments, predictive testing identifies mutations that increase a person's chances of developing disorders with a genetic basis, including for example but not limited to certain types of cancer.
- presymptomatic testing is useful in determining whether a person will develop a genetic disorder, before any physical signs or symptoms appear.
- the results of predictive and presymptomatic testing provides information about a person’s risk of developing a specific disorder and help with making decisions about an appropriate medical treatment regimen for a subject as well as for a subject’s relatives.
- diagnostic testing also includes pharmacogenomics which includes genetic testing that determines the influence of genetic variation on drug response. Information from such pharmacogenomic analyses finds use in determining and developing an appropriate treatment regimen. Those of skill in the medical arts employ information regarding the presence and/or absence of a genetic variation in designing appropriate treatment regimen.
- diseases whose genetic profiles are determined using the methods of the present disclosure include cancer (e.g., bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, melanoma, oral cancer, oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, uterine cancer).
- cancer e.g., bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, melanoma, oral cancer, oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, uterine cancer.
- diseases whose genetic profiles are determined using the methods of the present disclosure include Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer, Bone Cancer, Brain Tumors, Bronchial Tumors, Burkitt Lymphoma, Cardiac Tumors, Central Nervous System Germ Cell Tumors, Cervical Cancer, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ, Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophage
- the present methods find use in development of personalized medicine treatment regimens by providing the genomic DNA which is used in determining the genetic profile for an individual.
- such genetic profile information is employed by those skilled in the art in order determine and/or develop a treatment regimen.
- the presence and/or absence of various genetic variations and mutations identified in nucleic acids isolated by the described methods are used by those of skill in the art as part of a personalized medicine treatment regimen or plan.
- information obtained using the disclosed methods is compared to databases or other established information in order to determine a diagnosis for a specified disease and or determine a treatment regimen.
- the information regarding the presence or absence of a genetic mutation in a particular subject is compared to a database or other standard source of information in order to make a determination regarding a proposed treatment regimen.
- the presence of a genetic mutation indicates pursuing a particular treatment regimen.
- the absence of a genetic mutation indicates not pursuing a particular treatment regimen.
- information regarding the presence and/or absence of a particular genetic mutation is used to determine the treatment efficacy of treatment with the therapeutic entity, as well as to tailor treatment regimens for treatment with therapeutic entity.
- information regarding the presence and/or absence of a genetic mutation is employed to determine whether to pursue a treatment regimen.
- information regarding the presence and/or absence of a genetic mutation is employed to determine whether to continue a treatment regimen. In some embodiments, the presence and/or absence of a genetic mutation is employed to determine whether to discontinue a treatment regimen. In other embodiments, the presence and/or absence of a genetic mutation is employed to determine whether to modify a treatment regimen. In some embodiments the presence and/or absence of a genetic mutation is used to determine whether to increase or decrease the dosage of a treatment that is being administered as part of a treatment regimen. In other embodiments, the presence and/or absence of a genetic mutation is used to determine whether to change the dosing frequency of a treatment administered as part of a treatment regimen.
- the presence and/or absence of a genetic mutation is used to determine whether to change the number of dosages per day, per week, times per day of a treatment. In some embodiments the presence and/or absence of a genetic mutation is used to determine whether to change the dosage amount of a treatment. In some embodiments, the presence and/or absence of a genetic mutation is determined prior to initiating a treatment regimen and/or after a treatment regimen has begun. In some embodiments, the presence and/or absence of a genetic mutation is determined and compared to predetermined standard information regarding the presence or absence of a genetic mutation.
- a composite of the presence and/or absence of more than one genetic mutation is generated using the disclosed methods and such composite includes any collection of information regarding the presence and/or absence of more than one genetic mutation.
- the presence or absence of genetic mutations in ten or more genes is examined and used for generation of a composite. For example, genetic variants from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or more genes are examined and used for generation of a composite.
- the genetic variants may be selected from ten or more genes.
- Exemplary information in some embodiments includes nucleic acid or protein information, or a combination of information regarding both nucleic acid and/or protein genetic mutations.
- the composite includes information regarding the presence and/or absence of a genetic mutation. In some embodiments, these composites are used for comparison with predetermined standard information in order to pursue, maintain or discontinue a treatment regimen.
- cancer is predicted and/or detected for example through detection of genetic variants of ten or more genes as described herein.
- cancer is predicted and/or detected for example through detection of ten or more genetic variants from different genes, for example, including at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 genes.
- the genetic variants may be detected from nucleic acids separated by using at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 10000 capture probes including sequences selected from SEQ ID Nos: 1 through 116150.
- the detection of the genetic variants is combined with a physical examination in order to diagnose cancer or predict the risk of developing cancer.
- a physical examination can include changes in skin color or enlargement of an organ.
- the detection of the genetic variants is combined with a laboratory tes in order to diagnose cancer or predict the risk of developing cancer.
- a laboratory test can include urine and blood tests, such complete blood count (CBC) test, blood protein test (e.g., immunoglobulin tests), tumor marker tests (e.g., prostate-specific antigen, cancer antigen 125, calcitonin, alpha-fetoprotein, human chrionic gonadotropin, etc.), and circulating tumor cell tests.
- CBC complete blood count
- tumor marker tests e.g., prostate-specific antigen, cancer antigen 125, calcitonin, alpha-fetoprotein, human chrionic gonadotropin, etc.
- the detection of the genetic variants is combined with an imaging test in order to diagnose cancer or predict the risk of developing cancer.
- an imaging test can include ultrasound, x-ray, computerized tomography (CT) scan, bone scan, magnetic resonance imaging (MRI), and positron emission tomography (PET) scan.
- CT computerized tomography
- MRI magnetic resonance imaging
- PET positron emission tomography
- the detection of the genetic variants is combined with a biopsy in order to diagnose cancer or predict the risk of developing cancer.
- the detection of the genetic variants is in combination with one or more indications or signs of cancer development in order to diagnose cancer or predict the risk of developing cancer.
- the sign is an early sign of cancer.
- an early sign of cancer includes but is not limited to fatigue or extreme tiredness, substantial weight loss or gain with no known reason, eating problems, swelling or lumps in the body, thickening or lump in the breast or other part of the body, sustaining pain with no known reason, skin changes, persistent cough or hoarseness, unusual bleeding or bruising with no known reason, change in bowel habits, bladder changes, fever or night sweats, headaches, vision or hearing problems, and sores, bleeding or numbness in or around the mouth.
- the detection of the genetic variants associated with an increased risk of developing cancer described herein can be used to assist with determining a treatment regimen for an individual suspected to have cancer or predicted to develop cancer in the future.
- the detection of the genetic variants as described herein can be used to begin an appropriate treatment early in an individual suspected to be a risk of developing cancer.
- the detection of the genetic variants that predict and increased risk of developing cancer can allow for earlier and/or more frequent monitoring of the individual in order to identify disease onset at an early stage. (i.e., identify early disease onset).
- the detection of the genetic variation described herein can be used to begin early or regular monitoring in an individual suspected to be a risk of developing cancer.
- subjects can be followed on a 6-month to yearly basis for screening cancer.
- the detection of the genetic variants described herein can be used to diagnose cancer in a subject.
- a treatment regimen includes surgical interventions. In some embodiments, after diagnosis, a treatment regimen includes anti-cancer drugs.
- a diagnostic kit for diagnosing, prognosing and/or treating cancer. Any or all of the reagents described above may be packaged into a diagnostic kit. Such kits include any and/or all of the primers, probes, buffers and/or other reagents described herein in any combination. In some embodiments, the kit includes reagents for processing nucleic acids.
- the reagents are for separating at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 10000 nucleic acid fractions by using capture probes with sequences selected from SEQ ID NOs.: 1 through 116150.
- the reagents in the kit are included as lyophilized powders. In some embodiments, the reagents in the kit are included as lyophilized powders with instructions for reconstitution. In some embodiments, the reagents in the kit are included as liquids. In some embodiments, the reagents are included in plastic and/or glass vials or other appropriate containers. In some embodiments the primers and probes are all contained in individual containers in the kit. In some embodiments, the primers are packaged together in one container, and the probes are packaged together in another container. In some embodiments, the primers and probes are packaged together in a single container. [00170] In some embodiments, the kit further includes control gDNA and/or DNA samples.
- control DNA sample is normal (e.g., from a subject who does not have cancer).
- control DNA sample corresponds to a nucleic acid fraction having a sequence selected from SEQ ID NOs.: 1 through 116150 or a complementary sequence thereof.
- the concentration of the control DNA sample is 5 ng/ ⁇ L, 10 ng/ ⁇ L, 20 ng/ ⁇ L, 30 ng/ ⁇ L, 40 ng/ ⁇ L, 50 ng/ ⁇ L, 60 ng/ ⁇ L, 70 ng/ ⁇ L, 80 ng/ ⁇ L, 90 ng/ ⁇ L, 100 ng/ ⁇ L, 110 ng/ ⁇ L, 120 ng/ ⁇ L, 130 ng/ ⁇ L, 140 ng/ ⁇ L, 150 ng/ ⁇ L, 160 ng/ ⁇ L, 170 ng/ ⁇ L, 180 ng/ ⁇ L, 190 ng/ ⁇ L or 200 ng/ ⁇ L.
- the concentration of the control DNA sample is 50 ng/ ⁇ L, 100 ng/ ⁇ L, 150 ng/ ⁇ L or 200 ng/ ⁇ L. In some embodiments, the concentration of the control DNA sample is 100 ng/ ⁇ L. In some embodiments, the control DNA samples have the same concentration. In some embodiments, the control DNA samples have different concentrations.
- the kit can further include buffers, for example, GTXpress TAQMAN® reagent mixture, or any equivalent buffer. In some embodiments, the buffer incldues any buffer described herein. [00173] In some embodiments, the kit further includes reagents for use in purification of DNA.
- the kit further includes instructions for using the kit for the detection of cancer in a subject.
- these instructions include various aspects of the protocols described herein.
- Enzymatic Fragmentation and Nucleic Acid Separation Nucleic acids may be fragmented using one or more enzymes commercially availble (e.g., SureSelect XT HS and XT Low Input Enzymatic Fragmentation Kit from Agilent Technologies, Inc., the Protocol for use of which, such as a version dated September 2018, is incorporated by reference herein in its entirety.).
- fragmented nucleic acides may be separated using the following protocols.
- the separated nucleic acids may be further processed using the protocols described below for further analysis (e.g., sequencing.
- 1. +For XT-HS prepped libraries with DNA concentrations above 187.5 ng/ ⁇ L, prepare 4.0 ⁇ L of a 187.5 ng/ ⁇ L dilution of each library. 2. For XT-HS prepped libraries with DNA concentrations below 187.5 ng/ ⁇ L, use a vacuum concentrator to concentrate the samples at ⁇ 45°C.
- a. Dehydrate using a vacuum concentrator on low heat (less than 45°C). Reconstitute with nuclease-free water to a final concentration of 187.5 ng/ ⁇ L. Pipette up and down along the sides of the tube for optimal recovery. b.
- Capture Library Hybridization Mix appropriate for your Capture Library size according to Table 4 (Capture Libraries ⁇ 3 Mb), or Table 5 (Capture Libraries ⁇ 3 Mb). Mix well by vortexing at high speed for 5 seconds then spin down briefly. Keep the mixture at room temperature briefly, until use in step 9.
- Table 4 Preparation of Capture Library Hybridization Mix for > 3 Mb Capture Libraries (XT kit or XT-HS kit)
- Table 5 Preparation of Capture Library Hybridization Mix for ⁇ 3 Mb Capture Libraries 9. Maintain the gDNA library + Block Mix plate or strip tube at 65°C while you add 20 ⁇ L of the Capture Library Hybridization Mix from step 8 to each sample well. Mix well by pipetting up and down 8 to 10 times.
- the hybridization reaction wells now contain approximately 27 to 29 ⁇ L, depending on the degree of evaporation during the thermal cycler incubation. 10. Seal the wells with strip caps or using the PlateLoc Thermal Microplate Sealer. Make sure that all wells are completely sealed. 11. Incubate the hybridization mixture for 16 to 24 hours at 65°C with a heated lid at 105°C. Prepare streptavidin-coated magnetic beads The hybrid capture protocol uses reagents provided in SureSelect Target Enrichment Box 1 (stored at room temperature) in addition to the streptavidin-coated magnetic beads obtained from another supplier. Binding and wash buffers are identical between XT and XT-HS. 1.
- step d Wait until the solution is clear, then remove and discard the supernatant. . e. Repeat step a through step d two more times for a total of 3 washes. 5. Resuspend the beads in 200 ⁇ L of SureSelect Binding Buffer. Capture the hybridized DNA using streptavidin-coated beads 1. Spin down samples. Estimate and record the volume of hybridization solution that remains after the 16-24 hour incubation. 2. Maintain the hybridization plate or strip tube at 65°C while you use a multichannel pipette to transfer the entire volume (approximately 25 to 29 ⁇ L) of each hybridization mixture to the plate or strip tube wells containing 200 ⁇ L of washed streptavidin beads.
- Post-capture PCR thermal Cycler program Table 6 Post-capture PCR thermal Cycler program
- Table 7 Post-capture PCR cycle number recommendations
- Table 8 Preparation of Post-capture PCR Reaction Mix (XT-HS reagents) 1. Prepare the appropriate volume of PCR reaction mix, as described in Table 8, on ice. Mix well on a vortex mixer. 2. Add 25 ⁇ l of the PCR reaction mix prepared in Table 8 to each sample well containing 25 ⁇ l of bead-bound target-enriched DNA (prepared on page 4 and held on ice). 3. Mix the PCR reactions well by pipetting up and down until the bead suspension is homogeneous. Avoid splashing samples onto well walls; do not spin the samples at this step. 4. Place the plate or strip tube in the SureCycler 8800 thermal cycler.
- Step 2 Purify the amplified captured libraries using AMPure XP beads 1. Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time. 2. Prepare 400 ⁇ l of fresh 70% ethanol per sample, plus excess, for use in step 8. 3.
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Abstract
Probes and methods for separating nucleic acid fractions useful for analyzing genetic loci associated with a risk of a cancer are described.
Description
METHODS AND PROBES FOR SEPARATING GENOMIC NUCLEIC ACID FRACTIONS FOR CANCER RISK ANALYSIS RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63/295,309, filed December 30, 2021, which is incorporated by reference herein in its entirety. FIELD OF THE APPLICATION [0002] This application generally relates to methods for separation, isolation, and enrichment of genomic deoxyribonucleic acid fractions, and in particular to methods for separation, isolation, and enrichment of genomic deoxyribonucleic acid fractions associated with a risk of a cancer. SEQUENCE LISTING SUBMISSION VIA EFS-WEB [0003] A computer readable text file, entitled “SequenceListing.txt,” created on or about December 30, 2021 with a file size of about 38 MB contains the sequence listing for this application and is hereby incorporated by reference in its entirety. BACKGROUND [0004] Cancer is a leading cause of death. In the United States, cancer is the second leading cause of death. (K. D. Kochanek, J. Xu, E. Arias, Mortality in the United States, 2019, National Center for Health Statistics (NCHS) Data Brief, No.395, December 2020). In 2020, there were more than 19 million new cancer cases and more than 9 million deaths associated with cancer globally. (The Global Cancer Observatory, International Agency for Research on Cancer, December 2020). [0005] Cancer involves transformation of normal cells into tumor cells. Such changes are based in part on an interaction between a subject’s genetic factors and carcinogens. [0006] Survival rates for cancer improve significantly with early cancer detection, and thus, early cancer detection is recognized as an important tool for cancer treatment and management.
SUMMARY [0007] The present disclosure provides improved methods and reagents preparing nucleic acid fractions (e.g., genomic deoxyribonucleic acid fractions) for analyzing genetic loci associated with a risk of cancer. Such methods and reagents can facilitate genetic analysis and can lead to accurate detection of cancer even in the early stage. [0008] In accordance with some emboidments, a method for preparing nucleic acid (e.g., genomic deoxyribonucleic acid (gDNA)) fractions useful for analyzing genetic loci associated with a risk of a cancer includes obtaining the nucleic acid from a subject; fragmenting the nucleic acid to obtain nucleic acid fractions; mixing the nucleic acid fractions with one or more capture probes in a solution, a respective capture probe coupled with a capture moiety, for forming one or more hybrids between the one or more capture probes and the nucleic acid fractions; and separating the one or more hybrids from the solution by using capture moieties of the one or more capture probes. [0009] In some embodiments, the method also includes analyzing the genetic loci in one or more nucleic acid fractions in the one or more hybrids. [0010] In some embodiments, analyzing the genetic loci includes identifying an insertion or deletion at a respective genetic locus of the genetic loci. [0011] In some embodiments, the insertion or deletion at the respective genetic locus of the genetic loci is identified by sequencing. [0012] In some embodiments, the method also includes, subsequent to separating the one or more hybrids from the solution, separating one or more nucleic acid fractions from the one or more hybrids. [0013] In some embodiments, a capture probe of the one or more capture probes comprises a nucleic acid sequence having at least 80% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. [0014] In some embodiments, the capture probe has 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. [0015] In some embodiments, the method also includes mixing the nucleic acid fractions with two or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
[0016] In some embodiments, the method also includes mixing the nucleic acid fractions with two or more capture probes, a respective capture probe of the two or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0017] In some embodiments, the method also includes mixing the nucleic acid fractions with one hundred or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0018] In some embodiments, the method also includes mixing the nucleic acid fractions with one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0019] In some embodiments, the method also includes mixing the nucleic acid fractions with one thousand or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0020] In some embodiments, the method also includes mixing the nucleic acid fractions with one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0021] In some embodiments, the capture moiety is selected from a group consisting of avidin, streptavidin, and biotin. [0022] In some embodiments, the subject is a human subject. [0023] In accordance with some emboidments, a reagent kit includes one or more capture probes, a respective capture probe coupled with a capture moiety and having at least 80% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. [0024] In some embodiments, the reagent kit includes two or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
[0025] In some embodiments, the reagent kit includes two or more capture probes, a respective capture probe of the two or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0026] In some embodiments, the reagent kit includes one hundred or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0027] In some embodiments, the reagent kit includes one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0028] In some embodiments, the reagent kit includes one thousand or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0029] In some embodiments, the reagent kit includes one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0030] In some embodiments, the capture moiety is selected from a group consisting of avidin, streptavidin, and biotin. [0031] In accordance with some emboidments, a capture probe coupled with a capture moiety and having at least 80% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. [0032] In some embodiments, the capture moiety is selected from a group consisting of avidin, streptavidin, and biotin. BRIEF DESCRIPTION OF THE DRAWINGS [0033] Figure 1 depicts a method of preparing genomic deoxyribonucleic acid fractions in accordance with some embodiments.
DETAILED DESCRIPTION [0034] As explained above, the early cancer detection is important for treatment and management of cancer. It is believed that cancer is caused by certain changes to genes and thus, genetic analysis can detect changes in the genes that are associated with cancer. [0035] Although genetic sequencing techiques have improved over years, processing genetic material can further improve the accuracy of the sequencing analysis. For example, enriching genomic doxyribonucleic acid (gDNA) fragments can allow sequencing of only relevant gDNA fragments, thereby further improving the efficiency and accuracy of genetic analysis. In particular, enriching gDNA fragments associated with cancer can improve the efficiency and accuracy of early cancer detection. [0036] The term “invention” or “present invention” as used herein is not meant to be limiting to any one specific embodiment of the invention but applies generally to any and all embodiments of the invention as described in the claims and specification. [0037] As used herein, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure. It should be understood that the use of “and/or” is defined inclusively such that the term “a, b and/or c” should be read to include the sets of “a,” “b,” “c,” “a and b,” “b and c,” “c and a,” and “a, b and c.” [0038] As used herein, the term “about” means modifying, for example, lengths of nucleotide sequences, degrees of errors, dimensions, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, and like values, and ranges thereof, refers to variation in the numerical quantity that may occur, for example, through typical measuring and handling procedures used for making compounds, compositions, concentrates or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term “about” also encompasses amounts that differ due to aging of, for example, a composition, formulation, or cell culture with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture. Whether modified by the term “about” the claims appended hereto
include equivalents to these quantities. The term “about” further may refer to a range of values that are similar to the stated reference value. In certain embodiments, the term “about” refers to a range of values that fall within 50, 25, 10, 9, 8,7, 6, 5,4, 3, 2, 1 percent or less of the stated reference value. [0039] As used herein, the term “polymorphism” and variants thereof refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. The terms “genetic mutation” or “genetic variation” and variants thereof include polymorphisms. [0040] As used herein the term “single nucleotide polymorphism” (“SNP”) and variants thereof refers to a site of one nucleotide that varies between alleles. A single nucleotide polymorphism (SNP) is a single base change or point mutation but variants also include the so-called “indel” mutations (insertions or deletions of 1 to several up to 75 nucleotides), resulting in genetic variation between individuals. SNPs, which make up about 90% of all human genetic variation, occur every 100 to 300 bases along the 3-billion-base human genome. However, SNPs can occur much more frequently in other organisms like viruses. SNPs can occur in coding or non-coding regions of the genome. A SNP in the coding region may or may not change the amino acid sequence of a protein product. A SNP in a non-coding region can alter promoters or processing sites and may affect gene transcription and/or processing. Knowledge of whether an individual has particular SNPs in a genomic region of interest may provide sufficient information to develop diagnostic, preventive and therapeutic applications for a variety of diseases. [0041] The term “primer” and variants thereof refer to an oligonucleotide that acts as a point of initiation of DNA synthesis in a polymerase chain reaction (PCR). A primer is usually about 10 to about 35 nucleotides in length and hybridizes to a region complementary to the target sequence. [0042] The term “probe” and variants thereof refer to an oligonucleotide that hybridizes to a target nucleic acid. Target sequence refers to a region of nucleic acid that is to be analyzed and comprises a variant site of interest. The term “capture probe” and variants thereof refer to an oligonucleotide that hybridizes to a target nucleic acid for separation, isolation, or removal of the target nucleic acid from other nucleic acids.
[0043] The hybridization occurs in such a manner that the probes within a probe set may be modified to form a new, larger molecular entity (e.g., a probe product). The probes herein may hybridize to the nucleic acid regions of interest under stringent conditions. As used herein the term “stringency” is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. “Stringency” typically occurs in a range from about Tm° C to about 20° C to 25° C below Tm. A stringent hybridization may be used to isolate and detect identical polynucleotide sequences or to isolate and detect similar or related polynucleotide sequences. Under “stringent conditions” the nucleotide sequence, in its entirety or portions thereof, will hybridize to its exact complement and closely related sequences. Low stringency conditions comprise conditions equivalent to binding or hybridization at 68° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/l NaH2PO4.H2O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5× Denhardt’s reagent (50× Denhardt’s contains per 500 ml: 5 g Ficoll (Type 400), 5 g BSA) and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 2.0+SSPE, 0.1% SDS at room temperature when a probe of about 100 to about 1000 nucleotides in length is employed. It is well known in the art that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, conditions which promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.) are well known in the art. High stringency conditions, when used in reference to nucleic acid hybridization, comprise conditions equivalent to binding or hybridization at 68° C in a solution consisting of 5+SSPE, 1% SDS, 5× Denhardt’s reagent and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1+SSPE and 0.1% SDS at 68° C when a probe of about 100 to about 1000 nucleotides in length is employed. [0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein
can be used in the practice or testing of the present invention, various embodiments of methods and materials are specifically described herein. [0045] As explained above, cancer is the second leading cause of death in the United States. Many studies exist within the literature that attempt to the genetic causes of cancer. These studies have uncovered numerous possible genetic variants or SNPs that are believed to contribute to the etiology of the disease. [0046] However, cancer is not a single disease. Different types of cancer may manifest on different organs, and more importantly, there are typically multiple different types of cancer that may manifest on a same organ. Because different types of cancer may be caused by different genetic mutations, cancer screening can require analysis of a significant portion of patient’s genome. [0047] Although genetic sequencing techniques have improved over years, clinical decisions demand genetic analysis with high accuracy. Processing genetic material can further improve the accuracy of the genetic analysis. For example, enriching genomic doxyribonucleic acid (gDNA) fragments can allow sequencing of only relevant gDNA fragments, thereby further improving the efficiency and accuracy of genetic analysis. In particular, enriching gDNA fragments associated with cancer can improve the efficiency and accuracy of early cancer detection. [0048] Figure 1 is a flow diagram illustrating a method 100 for preparing nucleic acid (e.g., genomic deoxyribonucleic acid (gDNA)) fractions in accordance with some embodiments. The method is useful for analyzing genetic loci associated with a risk of a cancer. [0049] The method includes (110) obtaining nucleic acid (e.g., genomic deoxyribonucleic acid (gDNA)), from a subject. In some embodmients, the nucleic acid is obtained from one or more genomic samples. [0050] In some embodiments, the method includes isolating the on or more genomic samples to identify and validate genetic mutations. In some embodiments, the method includes extracting the one or more genomic samples from the subject. In some embodiments, the genomic samples may be selected from the group consisting of isolated cells, whole blood, serum, plasma, urine, saliva, sweat, fecal matter, and tears.
[0051] In some embodiments, a genomic sample is plasma or serum. In some embodiments, the method includes isolating the plasma or serum from a blood sample of the subject. In some embodiments, the method includes separating genomic materials from whole blood, plasma, or serum. [0052] In some embodiments, the method includes providing a sample of cells from the subject. In some embodiments, the cells are collected by contacting a cellular surface of the subject with a substrate capable of reversibly immobilizing the cells onto a substrate. [0053] The disclosed methods are applicable to a variety of cell types obtained from a variety of samples. In some embodiments, the cell type for use with the disclosed methods include but is not limited to epithelial cells, endothelial cells, connective tissue cells, skeletal muscle cells, endocrine cells, cardiac cells, urinary cells, melanocytes, keratinocytes, blood cells, white blood cells, buffy coat, hair cells (including, e.g., hair root cells) and/or salival cells. In some embodiments, the cells are epithelial cells. In some embodiments, the cells are subcapsular-perivascular (epithelial type 1); pale (epithelial type 2); intermediate (epithelial type 3); dark (epithelial type 4); undifferentiated (epithelial type 5); and large-medullary (epithelial type 6). In some embodiments, the cells are buccal epithelial cells (e.g., epithelial cells collected using a buccal swab). In some embodiments, the sample of cells used in the disclosed methods include any combination of the above identified cell types. [0054] In some embodiments, the method includes providing a sample of cells from a subject. In some embodiments, the cells provided are buccal epithelial cells. [0055] In some embodiments, the cell sample is collected by any of a variety of methods which allow for reversible binding of the subject’s cells to the substrate. In some embodiments, the substrate is employed in a physical interaction with the sample containing the subject’s cells in order to reversibly bind the cells to the substrate. In some embodiments, the substrate is employed in a physical interaction with the body of the subject directly in order to reversibly bind the cells to the substrate. In some embodiments, the sample is a buccal cell sample and the sample of buccal cells is collected by contacting a buccal membrane of the subject (e.g., the inside of their cheek) with a substrate capable of reversibly immobilizing cells that are dislodged from the membrane. In such embodiments, the swab is rubbed against the inside of the subject’s cheek with a force equivalent to brushing a person’s teeth (e.g., a light amount of force or pressure). Any method which would allow the subject’s
cells to be reversibly bound to the substrate is contemplated for use with the disclosed methods. [0056] In some embodiments, the sample is advantageously collected in a non-invasive manner. As such sample collection is accomplished anywhere and by almost anyone. For example, in some embodiments, the sample is collected at a physician’s office, at a subject’s home, or at a facility where a medical procedure is performed or to be performed. In some embodiments the subject, the subject’s doctor, nurses or a physician’s assistant or other clinical personnel collects the sample. [0057] In some embodiments the substrate is made of any of a variety of materials to which cells are reversibly bound. Exemplary substrates include those made of rayon, cotton, silica, an elastomer, a shellac, amber, a natural or synthetic rubber, cellulose, BAKELITE, NYLON, a polystyrene, a polyethylene, a polypropylene, a polyacrylonitrile, or other materials or combinations thereof. In some embodiments, the substrate is a swab having a rayon tip or a cotton tip. [0058] In some embodiments, the substrate containing the sample is freeze-thawed one or more times (e.g., after being frozen, the substrate containing the sample is thawed, used according to the present methods and re-frozen) and or used in the present methods. [0059] In another aspect, a variety of lysis solutions have been described and are known to those of skill in the art. Any of these well-known lysis solutions can be employed with the present methods in order to isolate nucleic acids from a sample. Exemplary lysis solutions include those commercially available, such as those sold by INVITROGEN®, QIAGEN®, LIFE TECHNOLOGIES® and other manufacturers, as well as those which can be generated by one of skill in a laboratory setting. Lysis buffers have also been well described and a variety of lysis buffers can find use with the disclosed methods, including for example those described in Molecular Cloning (three volume set, Cold Spring Harbor Laboratory Press, 2012) and Current Protocols (Genetics and Genomics; Molecular Biology; 2003-2013), both of which are incorporated herein by reference for all purposes. [0060] Cell lysis is a commonly practiced method for the recovery of nucleic acids from within cells. In many cases, the cells are contacted with a lysis solution, commonly an alkaline solution comprising a detergent, or a solution of a lysis enzyme. Such lysis solutions typically contain salts, detergents and buffering agents, as well as other agents that one of
skill would understand to use. After full and/or partial lysis, the nucleic acids are recovered from the lysis solution. [0061] In some embodiments, cells are resuspended in an aqueous buffer, with a pH in the range of from about pH 4 to about 10, about 5 to about 9, about 6 to about 8 or about 7 to about 9. [0062] In some embodiments, the buffer salt concentration is from about 10 mM to about 200 mM, about 10 mM to about 100 mM or about 20 mM to about 80 mM. [0063] In some embodiments, the buffer further comprises chelating agents such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA). [0064] In some embodiments, the lysis solution further comprises other compounds to assist with nucleic acid release from cells such as polyols, including for example but not limited to sucrose, as well as sugar alcohols such as maltitol, sorbitol, xylitol, erythritol, and/or isomalt. In some embodiments, polyols are in the range of from about 2% to about 15% w/w, or about 5% to about 15% w/w or about 5% to about 10% w/w. [0065] In some embodiments, the lysis solutions further comprises surfactants, such as for example but not limited to Triton X-100, SDS, CTAB, X-114, CHAPS, DOC, and/or NP-40. In some embodiments such surfactants are in the range of from about 1% to about 5% w/w, about 1% to about 4% w/w, or about 1% to about 3% w/w. [0066] In embodiments, the lysis solution further comprises chaotropes, such as for example but not limited to urea, sodium dodecyl sulfate and/or thiourea. In some embodiments, the chaotrope is used at a concentration in the range of from about 0.5 M to 8 M, about 1 M to about 6 M, about 2 M to about 6 M or about 1 M to 3 M. [0067] In some embodiments, the lysis solution further comprises one or more additional lysis reagents and such lysis reagents are well known in the art. In some embodiments, such lysis reagents include cell wall lytic enzymes, such as for example but not limited to lysozyme. In some embodiments, lysis reagents comprise alkaline detergent solutions, such as 0.1 aqueous sodium hydroxide containing 0.5% sodium dodecyl sulphate. [0068] In some embodiments, the lysis solution further comprises aqueous sugar solutions, such as sucrose solution and chelating agents such as EDTA, for example the STET buffer. In certain embodiments, the lysis reagent is prepared by mixing the cell suspension with an
equal volume of lysis solution having twice the desired concentration (for example 0.2 sodium hydroxide, 1.0% sodium dodecyl sulphate). [0069] In some embodiments, after the desired extent of lysis has been achieved, the mixture comprising lysis solution and lysed cells is contacted with a neutralizing or quenching reagent to adjust the conditions such that the lysis reagent does not adversely affect the desired product. In some embodiments, the pH is adjusted to a pH of from about 5 to about 9, about 6 to about 8, about 5 to about 7, about 6 to about 7 or about 6.5 to 7.5 to minimize and/or prevent degradation of the cell contents, including for example but not limited to the nucleic acids. In some embodiments, when the lysis reagent comprises an alkaline solution, the neutralizing reagent comprises an acidic buffer, for example an alkali metal acetate/acetic acid buffer. In some embodiments, lysis conditions, such as temperature and composition of the lysis reagent are chosen such that lysis is substantially completed while minimizing degradation of the desired product, including for example but not limited to nucleic acids. [0070] Any combination of the above can be employed by one of skill in the art, as well as combined with other known and routine methods, and such combinations are contemplated by the present invention. [0071] In another aspect, the nucleic acids, including for example but not limited to genomic DNA, are isolated from lysis buffer prior to performing subsequent analysis. In some embodiments, the nucleic acids are isolated from the lysis buffer prior to the performance of additional analyses, such as for example but not limited to sequencing. Any of a variety of methods useful in the isolation of small quantities of nucleic acids are used by various embodiments of the disclosed methods. These include but are not limited to precipitation, gel filtration, density gradients and solid phase binding. Such methods have also been described in for example, Molecular Cloning (three volume set, Cold Spring Harbor Laboratory Press, 2012) and Current Protocols (Genetics and Genomics; Molecular Biology; 2003-2013), incorporated herein by reference for all purposes. [0072] Nucleic acid precipitation is a well know method for isolation that is known by those of skill in the art. A variety of solid phase binding methods are also known in the art including but not limited to solid phase binding methods that make use of solid phases in the form of beads (e.g., silica, magnetic), columns, membranes or any of a variety other physical forms known in the art. In some embodiments, solid phases used in the disclosed methods reversibly bind nucleic acids. Examples of such solid phases include so-called “mixed-bed”
solid phases are mixtures of at least two different solid phases, each of which has a capacity to bind to nucleic acids under different solution conditions, and the ability and/or capacity to release the nucleic acid under different conditions; such as those described in US Patent Application No.2002/0001812, incorporated by reference herein in its entirety for all purposes. Solid phase affinity for nucleic acids according to the disclosed methods can be through any one of a number of means typically used to bind a solute to a substrate. Examples of such means include but are not limited to, ionic interactions (e.g., anion- exchange chromatography) and hydrophobic interactions (e.g., reversed-phase chromatography), pH differentials and changes, salt differentials and changes (e.g., concentration changes, use of chaotropic salts/agents). Exemplary pH based solid phases include but are not limited to those used in the INVITROGEN ChargeSwitch Normalized Buccal Kit magnetic beads, to which bind nucleic acids at low pH (<6.5) and releases nucleic acids at high pH (>8.5) and mono-amino-N-aminoethyl (MANAE) which binds nucleic acids at a pH of less than 7.5 and release nucleic acids at a pH of greater than 8. Exemplary ion exchange based substrates include but are not limited to DEA-SEPHAROSE™, Q- SEPHAROSE™, and DEAE-SEPHADEX™ from PHARMACIA (Piscataway, N.J.), DOWEX® I from The Dow Chemical Company (Midland, Mich.), AMBERLITE® from Rohm & Haas (Philadelphia, Pa.), DUOLITE® from Duolite International, In. (Cleveland, Ohio), DIALON TI and DIALON TII. [0073] Any individual method is contemplated for use alone or in combination with other methods, and such useful combination are well known and appreciated by those of skill in the art. [0074] In some embodiments, the disclosed methods are used to isolate nucleic acids, such as genomic DNA (gDNA) for a variety of nucleic acid analyses, including genomic analyses. In some embodiments, such analysis includes detection of variety of genetic mutations, which include but are not limited to deletions, insertions, transitions and transversions. In some embodiments, the mutation is a single-nucleotide polymorphism (SNP). In some embodiments, the mutation is an insertion or deletion. [0075] In some embodiments, the subject is (112) a human subject. In some embodiments, the subject is a non-human subject (e.g., domestic animals, such as pets, livestock, and beasts of burden).
[0076] The method also includes (120) fragmenting the nucleic acid (e.g., gDNA) to obtain nucleic acid (e.g., gDNA) fractions. In some embodiments, the nucleic acid is fragmented by mechanical shearing. For example, the nucleic acid may be subjected to sonication, which can result in nucleic acid fragments typically having approximately 700 bp. In some cases, the sonification is applied at burst cycles. Similarly, the nucleic acid may be subjected to high-frequency acoustic waves, which can fragment the nucleic acid. In another example, the nucleic acid may be subjected to nebulization by forcing the nucleic acid into a small hole. The size of the fragments is typically determined based on the speed of a nucleic acid solution passing through the hole, the viscosity of the nucleic acid solution, the size of the hole, and a temperature. In yet another example, the nucleic acid may be fragmented by point-sink shearing, which involves applying hydrodynamic shear forces to the nucleic acid. Alternatively, the nucleic acid may be fragmented by one or more enzymes (called restriction enzymes, such as endonucleases or transposases). Each restriction enzyme typically cleaves nucleic acids into fragments at or near specific sites having corresponding nucleic acid sequences. In some cases, a single type of restriction enzyme is used. In some cases, a set of enzymes of different types is used. Examples of commercially available reagents for enzymatic fragmentation include HyperPlus (KAPA Biosystems), SureSelect QXT (Agilent Technologies), Fragmentase (New England Biolabs), and Nextera Tagmentation (Illumina). [0077] In some embodiments, the nucleic acid is enzymatically fragmented. An example of enzymatic fragmentation is described further below. [0078] The method further includes (130) mixing the nucleic acid (e.g., gDNA) fractions with one or more capture probes in a solution for forming one or more hybrids between the one or more capture probes and the nucleic acid (e.g., gDNA) fractions. A respective capture probe is coupled with a capture moiety. The one or more capture probes, once mixed with the nucleic acid fractions, may bind with matching nucleic acid fractions under the hybridization conditions described herein. [0079] In some embodiments, a capture probe of the one or more capture probes includes a nucleic acid sequence having at least 80% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, a capture probe of the one or more capture probes includes a nucleic acid sequence having at least 90% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, a capture probe of the one or more capture probes includes a nucleic acid
sequence having at least 95% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, a capture probe of the one or more capture probes includes a nucleic acid sequence having at least 99% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. [0080] In some embodiments, the capture probe includes a nucleic acid sequence having 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150. For example, the capture probe may include a nucleic acid sequence that corresponds to SEQ ID NO: 1. [0081] In some embodiments, the method also includes (132) mixing the nucleic acid (e.g., gDNA) fractions with two or more capture probes including (or having) nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0082] In some embodiments, the method also includes mixing the gDNA fractions with two or more capture probes, a respective capture probe of the two or more capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with two or more capture probes, a respective capture probe of the two or more capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with two or more capture probes, a respective capture probe of the two or more capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with two or more capture probes, a respective capture probe of the two or more capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0083] In some embodiments, the method also includes (134) mixing the gDNA fractions with one hundred or more capture probes including (or having) nucleic acid sequences
selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0084] In some embodiments, the method also includes mixing the gDNA fractions with one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0085] In some embodiments, the method also includes (136) mixing the gDNA fractions with one thousand or more capture probes including (or having) nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0086] In some embodiments, the method also includes mixing the gDNA fractions with one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected
from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0087] In some embodiments, the method also includes mixing the gDNA fractions with ten thousand or more capture probes including (or having) nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0088] In some embodiments, the method also includes mixing the gDNA fractions with ten thousand or more capture probes, a respective capture probe of the ten thousand or more capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with ten thousand or more capture probes, a respective capture probe of the ten thousand or more capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with ten thousand or more capture probes, a respective capture probe of the ten thousand or more capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with ten thousand or more capture probes, a respective capture probe of the ten thousand or more capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
[0089] In some embodiments, the method also includes mixing the gDNA fractions with hundred thousand or more capture probes, a respective capture probe of the hundred thousand or more capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with hundred thousand or more capture probes, a respective capture probe of the hundred thousand or more capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with hundred thousand or more capture probes, a respective capture probe of the hundred thousand or more capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with hundred thousand or more capture probes, a respective capture probe of the hundred thousand or more capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0090] In some embodiments, the method also includes mixing the gDNA fractions with 116,150 capture probes, a respective capture probe of the 116,150 capture probes including (or having) a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with 116,150 capture probes, a respective capture probe of the 116,150 capture probes including (or having) a nucleic acid sequence having at least 90% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with 116,150 capture probes, a respective capture probe of the 116,150 capture probes including (or having) a nucleic acid sequence having at least 95% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. In some embodiments, the method also includes mixing the gDNA fractions with 116,150 capture probes, a respective capture
probe of the 116,150 capture probes including (or having) a nucleic acid sequence having at least 99% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150. [0091] In some embodiments, the capture moiety is selected (138) from a group consisting of avidin, streptavidin, and biotin. In some embodiments, the capture moiety is avidin. In some embodiments, the capture moiety is streptavidin. In some embodiments, the capture moiety is biotin. [0092] The method includes (140) separating the one or more hybrids from the solution by using capture moieties of the one or more capture probes. [0093] In some embodiments, the method also includes, (150) subsequent to separating the one or more hybrids from the solution, separating one or more gDNA fractions from the one or more hybrids. [0094] In some embodiments, the method also includes (160) analyzing the genetic loci in one or more gDNA fractions in the one or more hybrids. [0095] A variety of methods for analyzing such isolated nucleic acids, for example but not limited to genomic DNA (gDNA) are known in the art and include nucleic acid sequencing methods (including Next Generation Sequencing methods), PCR methods (including real- time PCR analysis, microarray analysis, hybridization analysis) as well as any other nucleic acid sequence analysis methods that are known in the art, which include a variety of other methods where nucleic acid compositions are analyzed and which are known to those of skill in the art. See, for example, Molecular Cloning (three volume set, Cold Spring Harbor Laboratory Press, 2012) and Current Protocols (Genetics and Genomics; Molecular Biology; 2003-2013). [0096] In one aspect, genetic mutations described herein may be detected by sequencing. For example, High-throughput or Next Generation Sequencing (NGS) represents an attractive option for detecting mutations within a gene. Distinct from PCR, microarrays, high-resolution melting and mass spectrometry, which all indirectly infer sequence content, NGS directly ascertains the identity of each base and the order in which they fall within a gene. The newest platforms on the market have the capacity to cover an exonic region 10,000 times over, meaning the content of each base position in the sequence is measured thousands of different times. This high level of coverage ensures that the consensus sequence is extremely accurate
and enables the detection of rare variants within a heterogeneous sample. For example, in a sample extracted from formalin-fixed, paraffin-embedded (FFPE) tissue, often a mutation of interest is only present at a frequency of 1%. When this sample is sequenced at 10,000X coverage, then even the rare allele, comprising only 1% of the sample, is uniquely measured 100 times over. Thus, NGS provides reliably accurate results with very high sensitivity, making it ideal for clinical diagnostic testing of FFPEs and other mixed samples. [0097] Examples of sequencing techniques, often referred to as Next Generation Sequencing (NGS) techniques include, but are not limited to Sequencing by Synthesis (SBS), Massively Parallel Signature Sequencing (MPSS), Polony sequencing, pyrosequencing, Reversible dye- terminator sequencing, SOLiD sequencing, Ion semiconductor sequencing, DNA nanoball sequencing, Helioscope single molecule sequencing, Single molecule real time (SMRT) sequencing, Single molecule real time (RNAP) sequencing, and Nanopore DNA sequencing. [0098] MPSS was a bead-based method that used a complex approach of adapter ligation followed by adapter decoding, reading the sequence in increments of four nucleotides; this method made it susceptible to sequence-specific bias or loss of specific sequences. [0099] Polony sequencing, combined an in vitro paired-tag library with emulsion PCR, an automated microscope, and ligation-based sequencing chemistry to sequence an E. coli genome at an accuracy of > 99.9999% and a cost approximately 1/10 that of Sanger sequencing. [00100] A parallelized version of pyrosequencing, the method amplifies DNA inside water droplets in an oil solution (emulsion PCR), with each droplet containing a single DNA template attached to a single primer-coated bead that then forms a clonal colony. The sequencing machine contains many picolitre-volume wells each containing a single bead and sequencing enzymes. Pyrosequencing uses luciferase to generate light for detection of the individual nucleotides added to the nascent DNA, and the combined data are used to generate sequence read-outs. This technology provides intermediate read length and price per base compared to Sanger sequencing on one end and Solexa and SOLiD on the other. [00101] SBS is a sequencing technology based on reversible dye-terminators. DNA molecules are first attached to primers on a flowcell and amplified so that local clonal colonies are formed. Four types of reversible terminator bases (RT-bases) are added, and non-incorporated nucleotides are washed away. Unlike pyrosequencing, the DNA can only be
extended one nucleotide at a time. A camera takes images of the fluorescently labeled nucleotides, then the dye along with the terminal 3' blocker is chemically removed from the DNA, allowing the next cycle. [00102] SOLiD technology employs sequencing by ligation. Here, a pool of all possible oligonucleotides of a fixed length are labeled according to the sequenced position. [00103] Oligonucleotides are annealed and ligated; the preferential ligation by DNA ligase for matching sequences results in a signal informative of the nucleotide at that position. Before sequencing, the DNA is amplified by emulsion PCR. The resulting bead, each containing only copies of the same DNA molecule, are deposited on a glass slide. The result is sequences of quantities and lengths comparable to Illumina sequencing. [00104] Ion semiconductor sequencing is based on using standard sequencing chemistry, but with a novel, semiconductor based detection system. This method of sequencing is based on the detection of hydrogen ions that are released during the polymerization of DNA, as opposed to the optical methods used in other sequencing systems. A micro well containing a template DNA strand to be sequenced is flooded with a single type of nucleotide. If the introduced nucleotide is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence multiple nucleotides will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal. [00105] DNA nanoball sequencing is a type of high throughput sequencing technology used to determine the entire genomic sequence of an organism. The method uses rolling circle replication to amplify small fragments of genomic DNA into DNA nanoballs. Unchained sequencing by ligation is then used to determine the nucleotide sequence. This method of DNA sequencing allows large numbers of DNA nanoballs to be sequenced per run. [00106] Helicos Biosciences Corporation’s single-molecule sequencing uses DNA fragments with added polyA tail adapters, which are attached to the flow cell surface. The next steps involve extension-based sequencing with cyclic washes of the flow cell with
fluorescently labeled nucleotides (one nucleotide type at a time, as with the Sanger method). The reads are performed by the Helioscope sequencer. [00107] Single molecule real time (SMRT) sequencing is based on the SBS approach. The DNA is synthesized in zero-mode wave-guides (ZMWs) - small well-like containers with the capturing tools located at the bottom of the well. The sequencing is performed with use of unmodified polymerase (attached to the ZMW bottom) and fluorescently labeled nucleotides flowing freely in the solution. The wells are constructed in a way that only the fluorescence occurring by the bottom of the well is detected. The fluorescent label is detached from the nucleotide at its incorporation into the DNA strand, leaving an unmodified DNA strand. [00108] Single molecule real time sequencing based on RNA polymerase (RNAP), which is attached to a polystyrene bead, with distal end of sequenced DNA is attached to another bead, with both beads being placed in optical traps. RNAP motion during transcription brings the beads in closer and their relative distance changes, which can then be recorded at a single nucleotide resolution. The sequence is deduced based on the four readouts with lowered concentrations of each of the four nucleotide types (similarly to Sangers method). [00109] Nanopore sequencing is based on the readout of electrical signal occurring at nucleotides passing by alpha-hemolysin pores covalently bound with cyclodextrin. The DNA passing through the nanopore changes its ion current. This change is dependent on the shape, size and length of the DNA sequence. Each type of the nucleotide blocks the ion flow through the pore for a different period of time. [00110] VisiGen Biotechnologies uses a specially engineered DNA polymerase. This polymerase acts as a sensor - having incorporated a donor fluorescent dye by its active centre. This donor dye acts by FRET (fluorescent resonant energy transfer), inducing fluorescence of differently labeled nucleotides. This approach allows reads performed at the speed at which polymerase incorporates nucleotides into the sequence (several hundred per second). The nucleotide fluorochrome is released after the incorporation into the DNA strand. [00111] Mass spectrometry may be used to determine mass differences between DNA fragments produced in chain-termination reactions. In some cases, certain sequence information may be obtained by mass spectrometry.
[00112] SBS technology is capable of overcoming the limitations of existing pyrosequencing based NGS platforms. [00113] Such technologies rely on complex enzymatic cascades for read out, are unreliable for the accurate determination of the number of nucleotides in homopolymeric regions and require excessive amounts of time to run individual nucleotides across growing DNA strands. The SBS NGS platform uses a direct sequencing approach to produce a sequencing strategy with very a high precision, rapid pace and low cost. [00114] One exemplary SBS sequencing is initialized by fragmenting of the template DNA into fragments, amplification, annealing of DNA sequencing primers, and, for example, finally affixing as a high-density array of spots onto a glass chip. The array of DNA fragments are sequenced by extending each fragment with modified nucleotides containing cleavable chemical moieties linked to fluorescent dyes capable of discriminating all four possible nucleotides. The array is scanned continuously by a high-resolution electronic camera (Measure) to determine the fluorescent intensity of each base (A, C, G or T) that was newly incorporated into the extended DNA fragment. After the incorporation of each modified base the array is exposed to cleavage chemistry to break off the fluorescent dye and end cap allowing additional bases to be added. The process is then repeated until the fragment is completely sequenced or maximal read length has been achieved. [00115] In another aspect, real-time PCR is used in detecting gene mutations, including for example but not limited to SNPs. In some embodiments, detection of SNPs in specific gene candidates is performed using real-time PCR, based on the use of intramolecular quenching of a fluorescent molecule by use of a tethered quenching moiety. Thus, according to exemplary embodiments, real-time PCR methods also include the use of molecular beacon technology. The molecular beacon technology utilizes hairpin-shaped molecules with an internally-quenched fluorophore whose fluorescence is restored by binding to a DNA target of interest (See, e.g., Kramer, R. et al. Nat. Biotechnol.14:303-308, 1996). In some embodiments, increased binding of the molecular beacon probe to the accumulating PCR product is used to specifically detect SNPs present in genomic DNA. [00116] For the design of Real-Time PCR assays, several parts are coordinated, including the DNA fragment that is flanked by the two primers and subsequently amplified, often referred to as the amplicon, the two primers and the detection probe or probes to be used.
[00117] In some embodiments, a SNP site in a sample from the subject may be amplified by the amplification methods described herein or any other amplification methods known in the art. The nucleic acids in a sample may or may not be amplified prior to contacting the SNP site with a probe described herein, using a universal amplification method (e.g., whole genome amplification and whole genome PCR). [00118] Real-time PCR relies on the visual emission of fluorescent dyes conjugated to short polynucleotides (termed “detection probes”) that associate with genomic alleles in a sequence-specific fashion or on fluorescent molecules that intercalate into double stranded DNA referred to as quantitative or qPCR. Real-time PCR probes differing by a single nucleotide can be differentiated in a real-time PCR assay by the conjugation and detection of probes that fluoresce at different wavelengths. Real-Time PCR finds use in detection applications (diagnostic applications), quantification applications and genotyping applications. [00119] Several related methods for performing real-time PCR are disclosed in the art, including assays that rely on TAQMAN® probes (U.S. Pat. Nos.5,210,015 and 5,487,972, and Lee et al., Nucleic Acids Res.21:3761-6, 1993), molecular beacon probes (U.S. Pat. Nos. 5,925,517 and 6,103,476, and Tyagi and Kramer, Nat. Biotechnol.14:303-8, 1996), self- probing amplicons (scorpions) (U.S. Pat. No.6,326,145, and Whitcombe et al., Nat. Biotechnol.17:804-7, 1999), Amplisensor (Chen et al., Appl. Environ. Microbiol.64:4210-6, 1998), Amplifluor (U.S. Pat. No.6,117,635, and Nazarenko et al., Nucleic Acids Res. 25:2516-21, 1997, displacement hybridization probes (Li et al., Nucleic Acids Res.30:E5, 2002), DzyNA-PCR (Todd et al., Clin. Chem.46:625-30, 2000), fluorescent restriction enzyme detection (Cairns et al., Biochem. Biophys. Res. Commun.318:684-90, 2004) and adjacent hybridization probes (U.S. Pat. No.6,174,670 and Wittwer et al., Biotechniques 22:130-1, 134-8, 1997). [00120] One of the many suitable genotyping procedures is the TAQMAN® allelic discrimination assay. In some instances of this assay, an oligonucleotide probe labeled with a fluorescent reporter dye at the 5' end of the probe and a quencher dye at the 3' end of the probe is utilized. The proximity of the quencher to the intact probe maintains a low fluorescence for the reporter. During the PCR reaction, the 5' nuclease activity of DNA polymerase cleaves the probe, and separates the dye and quencher. This results in an increase in fluorescence of the reporter. Accumulation of PCR product is detected directly by
monitoring the increase in fluorescence of the reporter dye. The 5' nuclease activity of DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target and is amplified during PCR. The probe is designed to straddle a target SNP position and hybridize to the nucleic acid molecule only if a particular SNP allele is present. [00121] Real-time PCR methods include a variety of steps or cycles as part of the methods for amplification. These cycles include denaturing double-stranded nucleic acids, annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence and synthesizing (i.e., replicating) second-strand DNA from the annealed forward primer and the reverse primer. This three-step process is referred to herein as a cycle. [00122] In some embodiments, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 cycles are employed. In some embodiments, about 10 to about 60 cycles, about 20 to about 50 or about 30 to about 40 cycles are employed. In some embodiments, 40 cycles are employed. [00123] In some embodiments, the denaturing double-stranded nucleic acids step occurs at a temperature of about 80ºC to 100ºC, about 85ºC to about 99ºC, about 90ºC to about 95ºC for about 1 second to about 5 seconds, about 2 seconds to about 5 seconds, or about 3 seconds to about 4 seconds. In some embodiments, the denaturing double-stranded nucleic acids step occurs at a temperature of 95ºC for about 3 seconds. [00124] In some embodiments, the annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence step occurs at about 40ºC to about 80ºC, about 50ºC to about 70ºC, about 55ºC to about 65ºC for about 15 seconds to about 45 seconds, about 20 seconds to about 40 seconds, about 25 seconds to about 35 seconds. In some embodiments, the annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence step occurs at about 60ºC for about 30 seconds. [00125] In some embodiments, the synthesizing (i.e., replicating) second-strand DNA from the annealed forward primer and the reverse primer occurs at about 40ºC to about 80ºC, about 50ºC to about 70ºC, about 55ºC to about 65ºC for about 15 seconds to about 45 seconds, about 20 seconds to about 40 seconds, about 25 seconds to about 35 seconds. In some embodiments, the annealing a forward primer, a reverse primer and a detection probe to the target genomic DNA sequence step occurs at about 60ºC for about 30 seconds.
[00126] In some embodiments, it was found that about 1 µL, about 2 µL, about 3 µL, about 4 µL or about 5 µL of a genomic DNA sample prepared according to the present methods described herein, are combined with only about 0.05 µL, about 0.10 µL about 0.15 µL, about 0.20 µL, about 0.25 µL or about 0.25 µL of a 30X, 35X, 40X, 45X, 50X or 100X real-time PCR assay mix and distilled water to form the PCR master mix. In some embodiments, the PCR master mix has a final volume of about 5 µL, about 6 µL, about 7 µL, about 8 µL, about 9 µL, about 0 µL, about 11 µL, about 12 µL, about 13 µL, about 14 µL, about 15 µL, about 16 µL, about 17 µL, about 18 µL, about 19 µL or about 20 µL or more. In some embodiments, it was found that 2 µL of a genomic DNA sample prepared as described above, are combined with only about 0.15 µL of a 40X real-time PCR assay mix and 2.85 µL of distilled water in order to form the PCR master mix. [00127] While exemplary reactions are described herein, one of skill would understand how to modify the temperatures and times based on the probe design. Moreover, the present methods contemplate any combination of the above times and temperatures. [00128] In some embodiments, primers are tested and designed in a laboratory setting. In some embodiments, primers are designed by computer based in silico methods. Primer sequences are based on the sequence of the amplicon or target nucleic acid sequence that is to be amplified. Shorter amplicons typically replicate more efficiently and lead to more efficient amplification as compared to longer amplicons. [00129] In designing primers, one of skill in the art would understand the need to take into account melting temperature (Tm; the temperature at which half of the primer-target duplex is dissociated and becomes single stranded and is an indication of duplex stability; increased Tm indicates increased stability) based on GC and AT content of the primers being designed as well as secondary structure considerations (increased GC content can lead to increased secondary structure). Tm’s can be calculated using a variety of methods known in the art and those of skill would readily understand such various methods for calculating Tm; such methods include for example but are not limited to those available in online tools such as the Tm calculators available on the World Wide Web at promega.com/techserv/tools/biomath/calc11.htm. Primer specificity is defined by its complete sequence in combination with the 3’ end sequence, which is the portion elongated by Taq polymerase. In some embodiments, the 3’ end should have at least 5 to 7 unique nucleotides not found anywhere else in the target sequence, in order to help reduce false-
priming and creation of incorrect amplification products. Forward and reverse primers typically bind with similar efficiency to the target. In some instances, tools such as NCBI BLAST (located on the World Wide Web at ncbi.nlm.nih.gov) are employed to performed alignments and assist in primer design. [00130] Those of skill in the art would be well aware of the basics regarding primer design for a target nucleic acid sequence and a variety of reference manuals and texts have extensive teachings on such methods, including for example, Molecular Cloning (three volume set, Cold Spring Harbor Laboratory Press, 2012) and Current Protocols (Genetics and Genomics; Molecular Biology; 2003-2013) and Real-Time PCR in Microbiology: From Diagnostics to Characterization (Ian M. MacKay, Calster Academic Press; 2007); PrimerAnalyser Java tool available on the World Wide Web at primerdigital.com/tools/PrimerAnalyser.html and Kalendar R, et al. (Genomics, 98(2): 137- 144 (2011)), all of which are incorporated herein in their entireties for all purposes. [00131] An additional aspect of primer design is primer complexity or linguistic sequence complexity (see, Kalendar R, et al. (Genomics, 98(2): 137-144 (2011)). Primers with greater linguistic sequence complexity (e.g., nucleotide arrangement and composition) are typically more efficient. In some embodiments, the linguistic sequence complexity calculation method is used to search for conserved regions between compared sequences for the detection of low-complexity regions including simple sequence repeats, imperfect direct or inverted repeats, polypurine and polypyrimidine triple-stranded cDNA structures, and four-stranded structures (such as G-quadruplexes). In some embodiments, linguistic complexity (LC) measurements are performed using the alphabet-capacity L-gram method (see, A. Gabrielian, A. Bolshoy, Computer & Chemistry 23:263–274 (1999) and Y.L. Orlov, V.N. Potapov, Complexity: an internet resource for analysis of DNA sequence complexity, Nucleic Acids Res.32: W628–W633(2004)) along the whole sequence length and calculated as the sum of the observed range (xi) from 1 to L size words in the sequence divided by the sum of the expected (E) value for this sequence length. Some G-rich (and C-rich) nucleic acid sequences fold into four-stranded DNA structures that contain stacks of G-quartets (see, the World Wide Web at quadruplex.org). In some instances, these quadruplexes are formed by the intermolecular association of two or four DNA molecules, dimerization of sequences that contain two G-bases, or by the intermolecular folding of a single strand containing four blocks of guanines (see, P.S. Ho, PNAS, 91:9549–9553 (1994); I.A. Il'icheva, V.L. Florent'ev, Russian Journal of Molecular Biology 26:512–531(1992); D. Sen, W. Gilbert, Methods
Enzymol.211:191–199 (1992); P.A. Rachwal, K.R. Fox, Methods 43:291–301 (2007); S. Burge, G.N. Parkinson, P. Hazel, A.K. Todd, K. Neidle, Nucleic Acids Res.34:5402–5415 (2006); A. Guédin, J. Gros, P. Alberti, J. Mergny, Nucleic Acids Res.38:7858–7868 (2010); O. Stegle, L. Payet, J.L. Mergny, D.J. MacKay, J.H. Leon, Bioinformatics 25:i374–i382 (2009); in some instances, these are eliminated from primer design because of their low linguistic complexity, LC=32% for (TTAGGG)4. [00132] These methods include various bioinformatics tools for pattern analysis in sequences having GC skew, (G−C)/(G+C), AT skew, (A−T)/(A+T), CG−AT skew, (S−W)/(S+W), or purine–pyrimidine (R−Y)/(R+Y) skew regarding CG content and melting temperature and provide tools for determining linguistic sequence complexity profiles. For example the GC skew in a sliding window of n, where n is a positive integer, bases is calculated with a step of one base, according to the formula, (G−C)/(G+C), in which G is the total number of guanines and C is the total number of cytosines for all sequences in the windows (Y. Benita, et al., Nucleic Acids Res.31:e99 (2003)). Positive GC-skew values indicated an overabundance of G bases, whereas negative GC-skew values represented an overabundance of C bases. Similarly, other skews are calculated in the sequence. Such methods, as well as others, are employed to determine primer complexity in some embodiments. [00133] According to non-limiting example embodiments, real-time PCR is performed using exonuclease primers (TAQMAN® probes). In such embodiments, the primers utilize the 5' exonuclease activity of thermostable polymerases such as Taq to cleave dual-labeled probes present in the amplification reaction (See, e.g., Wittwer, C. et al. Biotechniques 22:130-138, 1997). While complementary to the PCR product, the primer probes used in this assay are distinct from the PCR primer and are dually-labeled with both a molecule capable of fluorescence and a molecule capable of quenching fluorescence. When the probes are intact, intramolecular quenching of the fluorescent signal within the DNA probe leads to little signal. When the fluorescent molecule is liberated by the exonuclease activity of Taq during amplification, the quenching is greatly reduced leading to increased fluorescent signal. Non- limiting examples of fluorescent probes include the 6-carboxy-fluorescein moiety and the like. Exemplary quenchers include Black Hole Quencher 1 moiety and the like. [00134] A variety of PCR primers can find use with the disclosed methods.
[00135] A variety of detection probes can find use with the disclosed methods and are employed for genotyping and or for quantification. Detection probes commonly employed by those of skill in the art include but are not limited to hydrolysis probes (also known as TAQMAN® probes, 5’ nuclease probes or dual-labeled probes), hybridization probes, and Scorpion primers (which combine primer and detection probe in one molecule). [00136] In some embodiments, detection probes contain various modifications. In some embodiments, detection probes include modified nucleic acid residues, such as but not limited to 2'-O-methyl ribonucleotide modifications, phosphorothioate backbone modifications, phosphorodithioate backbone modifications, phosphoramidate backbone modifications, methylphosphonate backbone modifications, 3' terminal phosphate modifications and/or 3' alkyl substitutions. [00137] In some embodiments, the detection probe has increased affinity for a target sequence due to modifications. Such detection probes include detection probes with increased length, as well as detection probes containing chemical modifications. Such modifications include but are not limited to 2'-fluoro (2'-deoxy-2'-fluoro-nucleosides) modifications, LNAs (locked nucleic acids), PNAs (peptide nucleic acids), ZNAs (zip nucleic acids), morpholinos, methylphosphonates, phosphoramidates, polycationic conjugates and 2'-pyrene modifications. In some embodiments, the detector probes contains one or more modifications including 2' fluoro modifications (aka, 2'-Deoxy-2'-fluoro-nucleosides), LNAs (locked nucleic acids), PNAs (peptide nucleic acids), ZNAs (zip nucleic acids), morpholinos, methylphosphonates, phosphoramidates, and/or polycationic conjugates. [00138] In some embodiments, the detection probes contain detectable moieties, such as those described herein as well as any detectable moieties known to those of skill in the art. Such detectable moieties include for example but are not limited to fluorescent labels and chemiluminescent labels. Examples of such detectable moieties can also include members of FRET pairs. In some embodiments, the detection probe contains a detectable entity. [00139] Examples of fluorescent labels include but are not limited to AMCA, DEAC (7-Diethylaminocoumarin-3-carboxylic acid); 7-Hydroxy-4-methylcoumarin-3; 7- Hydroxycoumarin-3; MCA (7-Methoxycoumarin-4-acetic acid); 7-Methoxycoumarin-3; AMF (4'-(Aminomethyl)fluorescein); 5-DTAF (5-(4,6-Dichlorotriazinyl)aminofluorescein); 6-DTAF (6-(4,6-Dichlorotriazinyl)aminofluorescein); 6-FAM (6-Carboxyfluorescein; aka FAM; including TAQMAN® FAMTM); TAQMAN VIC®; 5(6)-FAM cadaverine; 5-FAM
cadaverine; 5(6)-FAM ethylenediamme; 5-FAM ethylenediamme; 5-FITC (FITC Isomer I; fluorescein-5-isothiocyanate); 5-FITC cadaverin; Fluorescein-5-maleimide; 5-IAF (5- Iodoacetamidofluorescein); 6-JOE (6-Carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein); 5- CR110 (5-Carboxyrhodamine 110); 6-CR110 (6-Carboxyrhodamine 110); 5-CR6G (5- Carboxyrhodamine 6G); 6-CR6G (6-Carboxyrhodamine 6G); 5(6)-Carboxyrhodamine 6G cadaverine; 5(6)-Carboxyrhodamine 6G ethylenediamme; 5-ROX (5-Carboxy-X-rhodamine); 6-ROX (6-Carboxy-X-rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6- TAMRA (6-Carboxytetramethylrhodamine); 5-TAMRA cadaverine; 6-TAMRA cadaverine; 5-TAMRA ethylenediamme; 6-TAMRA ethylenediamme; 5-TMR C6 maleimide; 6-TMR C6 maleimide; TR C2 maleimide; TR cadaverine; 5-TRITC; G isomer (Tetramethylrhodamine- 5-isothiocyanate); 6-TRITC; R isomer (Tetramethylrhodamine-6-isothiocyanate); Dansyl cadaverine (5-Dimethylaminonaphthalene-l-(N-(5-aminopentyl))sulfonamide); EDANS C2 maleimide; fluorescamine; NBD; and pyrromethene and derivatives thereof. [00140] Examples of chemiluminescent labels include but are not limited to those labels used with Southern Blot and Western Blot protocols (see, for e.g., Sambrook and Russell, Molecular Cloning: A Laboratory Manual, (3rd ed.) (2001); incorporated by reference herein in its entirety). Examples include but are not limited to -(2'- spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD); acridinium esters and adamantyl-stabilized 1 ,2-dioxetanes, and derivatives thereof. [00141] The labeling of probes is known in the art. The labeled probes are used to hybridize within the amplified region during amplification. The probes are modified so as to avoid them from acting as primers for amplification. The detection probe is labeled with two fluorescent dyes, one capable of quenching the fluorescence of the other dye. One dye is attached to the 5' terminus of the probe and the other is attached to an internal site, so that quenching occurs when the probe is in a non-hybridized state. [00142] Typically, real-time PCR probes consist of a pair of dyes (a reporter dye and an acceptor dye) that are involved in fluorescence resonance energy transfer (FRET), whereby the acceptor dye quenches the emission of the reporter dye. In general, the fluorescence-labeled probes increase the specificity of amplicon quantification. [00143] Real-time PCR that are used in some embodiments of the disclosed methods also include the use of one or more hybridization probes (i.e., detection probes), as determined by those skilled in the art, in view of this disclosure. By way of non-limiting
example, such hybridization probes include but are not limited to one or more of those provided in the described methods. Exemplary probes, such as the HEX channel and/or FAM channel probes, are understood by one skilled in the art. [00144] According to example embodiments, detection probes and primers are conveniently selected e.g., using an in silico analysis using primer design software and cross- referencing against the available nucleotide database of genes and genomes deposited at the National Center for Biotechnology Information (NCBI). Some additional guidelines may be used for selection of primers and/or probes in some embodiments. For example, in some embodiments, the primers and probes are selected such that they are close together, but not overlapping. In some embodiments, the primers may have the same (or close TM) (e.g., between about 58 °C and about 60 °C). In some embodiments, the TM of the probe is approximately 10 °C higher than that selected for the TM of the primers. In some embodiments, the length of the probes and primers is selected to be between about 17 and 39 base pairs, etc. These and other guidelines are used in some instances by those skilled in the art in selecting appropriate primers and/or probes. [00145] In some embodiments, the SNP described herein may be detected by melting curve analysis using the detection probes above. For example, the melting curves of short oligonucleotide probes hybridized to a region containing the SNP of interest may be analyzed. Two probes are used in these reactions, each one being complimentary to a particular allele at the SNP in question. Perfectly matched probes are more stable and have a higher melting temperature compared to mismatched probes. Hence, SNP genotypes are inferred according to the characteristic melting curves produced by annealing and melting either matched or mismatched oligonucleotide probes. [00146] In some embodiments, analyzing the genetic loci includes (162) identifying an insertion or deletion at a respective genetic locus of the genetic loci. [00147] In some embodiments, the insertion or deletion at the respective genetic locus of the genetic loci is identified (164) by sequencing. [00148] In some embodiments, analyzing the genetic loci includes determining a number of tandem repeats. In some embodiments, the number of tandem repeats is identified by sequencing. In some embodiments, analyzing the genetic loci includes determining numbers of tandem repeats.
[00149] In one aspect, the methods described herein may include preparing nucleic acid fractions by hybridizing at least one capture probe to a nucleotide molecule from a sample and separating the hybrid of the capture probe and the nucleotide molecule. [00150] In another aspect, diagnostic testing is employed to determine one or more genetic conditions by detection of any of a variety of mutations. In some embodiments, diagnostic testing is used to confirm a diagnosis when a particular condition is suspected based on for example physical manifestations, signs and/or symptoms as well as family history information. In some embodiments, the results of a diagnostic test assist those of skill in the medical arts in determining an appropriate treatment regimen for a given subject and allow for more personalized and more effective treatment regimens. In some embodiments, a treatment regimen includes any of a variety of pharmaceutical treatments, surgical treatments, lifestyles changes or a combination thereof as determined by one of skill in the art. [00151] The nucleic acids obtained by the disclosed methods are useful in a variety of diagnostic tests, including tests for detecting mutations such as deletions, insertions, transversions and transitions. In some embodiments, such diagnostics are useful for identifying unaffected individuals who carry one copy of a gene for a disease that requires two copies for the disease to be expressed, identifying unaffected individuals who carry one copy of a gene for a disease in which the information could find use in developing a treatment regimen, preimplantation genetic diagnosis, prenatal diagnostic testing, newborn screening, genealogical DNA test (for genetic genealogy purposes), presymptomatic testing for predicting or diagnosing cancer. [00152] In some embodiments, newborns can be screened. In some embodiments, newborn screening includes any genetic screening employed just after birth in order to identify genetic disorders. In some embodiments, newborn screening finds use in the identification of genetic disorders so that a treatment regimen is determined early in life. Such tests include but are not limited to testing infants for phenylketonuria and congenital hypothyroidism. [00153] In some embodiments, carrier testing is employed to identify people who carry a single copy of a gene mutation. In some cases, when present in two copies, the mutation can cause a genetic disorder. In some cases, one copy is sufficient to cause a genetic disorder. In some cases, the presence of two copies is contra-indicated for a particular treatment regimen, such as the presence of a certain genetic mutation (e.g., a mutation in the
BRCA gene) and pre-screening prior to prescription of a certain pharmaceutical or biological drug in order to ensure the appropriate treatment regimen is pursued for a given subject. In some embodiments, such information is also useful for individual contemplating procreation and assists individuals with making informed decisions as well as assisting those skilled in the medical arts in providing important advice to individual subjects as well as subjects’ relatives. [00154] In some embodiments, predictive and/or presymptomatic types of testing are used to detect gene mutations associated with a variety of disorders. In some cases, these tests are helpful to people who have a family member with a genetic disorder, but who may exhibit no features of the disorder at the time of testing. In some embodiments, predictive testing identifies mutations that increase a person's chances of developing disorders with a genetic basis, including for example but not limited to certain types of cancer. In some embodiments, presymptomatic testing is useful in determining whether a person will develop a genetic disorder, before any physical signs or symptoms appear. The results of predictive and presymptomatic testing provides information about a person’s risk of developing a specific disorder and help with making decisions about an appropriate medical treatment regimen for a subject as well as for a subject’s relatives. [00155] In some embodiments, diagnostic testing also includes pharmacogenomics which includes genetic testing that determines the influence of genetic variation on drug response. Information from such pharmacogenomic analyses finds use in determining and developing an appropriate treatment regimen. Those of skill in the medical arts employ information regarding the presence and/or absence of a genetic variation in designing appropriate treatment regimen. [00156] In some embodiments, diseases whose genetic profiles are determined using the methods of the present disclosure include cancer (e.g., bladder cancer, breast cancer, colorectal cancer, kidney cancer, lung cancer, lymphoma, melanoma, oral cancer, oropharyngeal cancer, pancreatic cancer, prostate cancer, thyroid cancer, uterine cancer). In some embodiments, diseases whose genetic profiles are determined using the methods of the present disclosure include Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Bile Duct Cancer, Bone Cancer, Brain Tumors, Bronchial Tumors, Burkitt Lymphoma, Cardiac Tumors, Central Nervous System
Germ Cell Tumors, Cervical Cancer, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ, Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Eye Cancer, Fallopian Tube Cancer, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid, Gastrointestinal Stromal Tumors (GIST), Germ Cell Tumor, Gestational Trophoblastic Disease, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin Lymphoma, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors, Kaposi Sarcoma, Kidney Cancer, Laryngeal Cancer, Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Medulloblastoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer, Midline Tract Carcinoma, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic/Myeloproliferative Neoplasms, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, Ovarian Germ Cell Tumors, Pancreatic Neuroendocrine Tumors, Papillomatosis, Paraganglioma, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Rectal Cancer, Renal Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma, Sézary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Tracheobronchial Tumors, Transitional Cell Cancer, Ureter and Renal Pelvis, Urethral Cancer, Uterine Sarcoma, Vaginal Cancer, Vascular Tumors, and Vulvar Cancer. [00157] In some embodiments, the present methods find use in development of personalized medicine treatment regimens by providing the genomic DNA which is used in determining the genetic profile for an individual. In some embodiments, such genetic profile information is employed by those skilled in the art in order determine and/or develop a treatment regimen. In some embodiments, the presence and/or absence of various genetic variations and mutations identified in nucleic acids isolated by the described methods are
used by those of skill in the art as part of a personalized medicine treatment regimen or plan. For example, in some embodiments, information obtained using the disclosed methods is compared to databases or other established information in order to determine a diagnosis for a specified disease and or determine a treatment regimen. In some cases, the information regarding the presence or absence of a genetic mutation in a particular subject is compared to a database or other standard source of information in order to make a determination regarding a proposed treatment regimen. In some cases, the presence of a genetic mutation indicates pursuing a particular treatment regimen. In some cases, the absence of a genetic mutation indicates not pursuing a particular treatment regimen. [00158] In some embodiments, information regarding the presence and/or absence of a particular genetic mutation is used to determine the treatment efficacy of treatment with the therapeutic entity, as well as to tailor treatment regimens for treatment with therapeutic entity. In some embodiments, information regarding the presence and/or absence of a genetic mutation is employed to determine whether to pursue a treatment regimen. In some embodiments, information regarding the presence and/or absence of a genetic mutation is employed to determine whether to continue a treatment regimen. In some embodiments, the presence and/or absence of a genetic mutation is employed to determine whether to discontinue a treatment regimen. In other embodiments, the presence and/or absence of a genetic mutation is employed to determine whether to modify a treatment regimen. In some embodiments the presence and/or absence of a genetic mutation is used to determine whether to increase or decrease the dosage of a treatment that is being administered as part of a treatment regimen. In other embodiments, the presence and/or absence of a genetic mutation is used to determine whether to change the dosing frequency of a treatment administered as part of a treatment regimen. In some embodiments, the presence and/or absence of a genetic mutation is used to determine whether to change the number of dosages per day, per week, times per day of a treatment. In some embodiments the presence and/or absence of a genetic mutation is used to determine whether to change the dosage amount of a treatment. In some embodiments, the presence and/or absence of a genetic mutation is determined prior to initiating a treatment regimen and/or after a treatment regimen has begun. In some embodiments, the presence and/or absence of a genetic mutation is determined and compared to predetermined standard information regarding the presence or absence of a genetic mutation.
[00159] In some embodiments, a composite of the presence and/or absence of more than one genetic mutation is generated using the disclosed methods and such composite includes any collection of information regarding the presence and/or absence of more than one genetic mutation. In some embodiments, the presence or absence of genetic mutations in ten or more genes is examined and used for generation of a composite. For example, genetic variants from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or more genes are examined and used for generation of a composite. In additional embodiments, the genetic variants may be selected from ten or more genes. In some embodiments, the presence or absence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 10000 genetic mutations, for example, is examined and used for generation of a composite. Exemplary information in some embodiments includes nucleic acid or protein information, or a combination of information regarding both nucleic acid and/or protein genetic mutations. Generally, the composite includes information regarding the presence and/or absence of a genetic mutation. In some embodiments, these composites are used for comparison with predetermined standard information in order to pursue, maintain or discontinue a treatment regimen. [00160] In some embodiments, cancer is predicted and/or detected for example through detection of genetic variants of ten or more genes as described herein. In some embodiments, cancer is predicted and/or detected for example through detection of ten or more genetic variants from different genes, for example, including at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 genes. In some embodiments, the presence or absence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 10000 genetic mutations, for example, are detected. [00161] In some embodiments, the genetic variants are detected from nucleic acids separated by using the capture probed described herein. The genetic variants may be detected from nucleic acids separated by using at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 70, 80, 90, 100, 150,
200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 10000 capture probes including sequences selected from SEQ ID Nos: 1 through 116150. [00162] In some embodiments, the detection of the genetic variants is combined with a physical examination in order to diagnose cancer or predict the risk of developing cancer. Such a physical examination can include changes in skin color or enlargement of an organ. In some embodiments, the detection of the genetic variants is combined with a laboratory tes in order to diagnose cancer or predict the risk of developing cancer. Such a laboratory test can include urine and blood tests, such complete blood count (CBC) test, blood protein test (e.g., immunoglobulin tests), tumor marker tests (e.g., prostate-specific antigen, cancer antigen 125, calcitonin, alpha-fetoprotein, human chrionic gonadotropin, etc.), and circulating tumor cell tests. In some embodiments, the detection of the genetic variants is combined with an imaging test in order to diagnose cancer or predict the risk of developing cancer. Such an imaging test can include ultrasound, x-ray, computerized tomography (CT) scan, bone scan, magnetic resonance imaging (MRI), and positron emission tomography (PET) scan. In some embodiments, the detection of the genetic variants is combined with a biopsy in order to diagnose cancer or predict the risk of developing cancer. [00163] In some embodiments, the detection of the genetic variants is in combination with one or more indications or signs of cancer development in order to diagnose cancer or predict the risk of developing cancer. In some embodiments, the sign is an early sign of cancer. In some embodiments, an early sign of cancer includes but is not limited to fatigue or extreme tiredness, substantial weight loss or gain with no known reason, eating problems, swelling or lumps in the body, thickening or lump in the breast or other part of the body, sustaining pain with no known reason, skin changes, persistent cough or hoarseness, unusual bleeding or bruising with no known reason, change in bowel habits, bladder changes, fever or night sweats, headaches, vision or hearing problems, and sores, bleeding or numbness in or around the mouth. [00164] In some embodiments, the detection of the genetic variants associated with an increased risk of developing cancer described herein can be used to assist with determining a treatment regimen for an individual suspected to have cancer or predicted to develop cancer in the future.
[00165] In some embodiments, the detection of the genetic variants as described herein can be used to begin an appropriate treatment early in an individual suspected to be a risk of developing cancer. In some embodiments, the detection of the genetic variants that predict and increased risk of developing cancer can allow for earlier and/or more frequent monitoring of the individual in order to identify disease onset at an early stage. (i.e., identify early disease onset). [00166] In another aspect, the detection of the genetic variation described herein can be used to begin early or regular monitoring in an individual suspected to be a risk of developing cancer. In some embodiments, subjects can be followed on a 6-month to yearly basis for screening cancer. [00167] In another aspect, the detection of the genetic variants described herein can be used to diagnose cancer in a subject. In some embodiments, after diagnosis, a treatment regimen includes surgical interventions. In some embodiments, after diagnosis, a treatment regimen includes anti-cancer drugs. [00168] In another aspect, the disclosure provides a diagnostic kit for diagnosing, prognosing and/or treating cancer. Any or all of the reagents described above may be packaged into a diagnostic kit. Such kits include any and/or all of the primers, probes, buffers and/or other reagents described herein in any combination. In some embodiments, the kit includes reagents for processing nucleic acids. In some embodiments, the reagents are for separating at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, or 10000 nucleic acid fractions by using capture probes with sequences selected from SEQ ID NOs.: 1 through 116150. [00169] In some embodiments, the reagents in the kit are included as lyophilized powders. In some embodiments, the reagents in the kit are included as lyophilized powders with instructions for reconstitution. In some embodiments, the reagents in the kit are included as liquids. In some embodiments, the reagents are included in plastic and/or glass vials or other appropriate containers. In some embodiments the primers and probes are all contained in individual containers in the kit. In some embodiments, the primers are packaged together in one container, and the probes are packaged together in another container. In some embodiments, the primers and probes are packaged together in a single container.
[00170] In some embodiments, the kit further includes control gDNA and/or DNA samples. In some embodiments, the control DNA sample is normal (e.g., from a subject who does not have cancer). In some embodiments, the control DNA sample corresponds to a nucleic acid fraction having a sequence selected from SEQ ID NOs.: 1 through 116150 or a complementary sequence thereof. [00171] In some embodiments, the concentration of the control DNA sample is 5 ng/µL, 10 ng/µL, 20 ng/µL, 30 ng/µL, 40 ng/µL, 50 ng/µL, 60 ng/µL, 70 ng/µL, 80 ng/µL, 90 ng/µL, 100 ng/µL, 110 ng/µL, 120 ng/µL, 130 ng/µL, 140 ng/µL, 150 ng/µL, 160 ng/µL, 170 ng/µL, 180 ng/µL, 190 ng/µL or 200 ng/µL. In some embodiments, the concentration of the control DNA sample is 50 ng/µL, 100 ng/µL, 150 ng/µL or 200 ng/µL. In some embodiments, the concentration of the control DNA sample is 100 ng/µL. In some embodiments, the control DNA samples have the same concentration. In some embodiments, the control DNA samples have different concentrations. [00172] In some embodiments, the kit can further include buffers, for example, GTXpress TAQMAN® reagent mixture, or any equivalent buffer. In some embodiments, the buffer incldues any buffer described herein. [00173] In some embodiments, the kit further includes reagents for use in purification of DNA. [00174] In some embodiments, the kit further includes instructions for using the kit for the detection of cancer in a subject. In some embodiments, these instructions include various aspects of the protocols described herein. Examples [00175] Enzymatic Fragmentation and Nucleic Acid Separation [00176] Nucleic acids may be fragmented using one or more enzymes commercially availble (e.g., SureSelect XT HS and XT Low Input Enzymatic Fragmentation Kit from Agilent Technologies, Inc., the Protocol for use of which, such as a version dated September 2018, is incorporated by reference herein in its entirety.). [00177] In some cases, fragmented nucleic acides may be separated using the following protocols. In some cases, the separated nucleic acids may be further processed using the protocols described below for further analysis (e.g., sequencing.
1. +For XT-HS prepped libraries with DNA concentrations above 187.5 ng/μL, prepare 4.0 μL of a 187.5 ng/μL dilution of each library. 2. For XT-HS prepped libraries with DNA concentrations below 187.5 ng/μL, use a vacuum concentrator to concentrate the samples at ≤45°C. a. Dehydrate using a vacuum concentrator on low heat (less than 45°C). Reconstitute with nuclease-free water to a final concentration of 187.5 ng/μL. Pipette up and down along the sides of the tube for optimal recovery. b. Mix well on a vortex mixer and spin in a centrifuge for 1 minute. 3. Transfer each 4.0 μL gDNA library sample (750 ng) to a separate well of a 96-well plate or strip tube. Seal the wells and keep on ice 4. Prepare the Hybridization Buffer by mixing the components in Table1 at room temperature. Warm the Hybridization Buffer at 65°C for 5minutes. Keep the prepared Hybridization Buffer at room temperature until it is used in step 9. Table 1 Preparation of Hybridization Buffer (XT reagents)
5. To each gDNA library sample well prepared in step 3, add 5 μL of the SureSelect XT HS Block Mix in the kit. Pipette up and down to mix. 6. Cap the wells, then transfer the sealed plate or strip tube to the thermal cycler and run the following program shown in Table 2. Use a heated lid, set at 105°C, to hold the temperature at 65°C. Make sure that the DNA + Block Mix samples are held at 65°C for at least 5 minutes before adding the remaining hybridization reaction components in step 10 below. Table 2 Thermal cycler program for DNA + Blocker Mix prior to hybridization
7. Prepare the appropriate dilution of SureSelect RNase Block, based on the size of your Capture Library, according to Table 3. Prepare the amount required for the number of hybridization reactions in the run, plus excess. Table 3 Preparation of RNase Block dilution (XT kit or XT-HS kit)
8. Prepare the Capture Library Hybridization Mix appropriate for your Capture Library size according to Table 4 (Capture Libraries ≥ 3 Mb), or Table 5 (Capture Libraries<3 Mb). Mix well by vortexing at high speed for 5 seconds then spin down briefly. Keep the mixture at room temperature briefly, until use in step 9. Table 4 Preparation of Capture Library Hybridization Mix for > 3 Mb Capture Libraries (XT kit or XT-HS kit)
Table 5 Preparation of Capture Library Hybridization Mix for < 3 Mb Capture Libraries
9. Maintain the gDNA library + Block Mix plate or strip tube at 65°C while you add 20 μL of the Capture Library Hybridization Mix from step 8 to each sample well. Mix well by pipetting up and down 8 to 10 times. The hybridization reaction wells now contain approximately 27 to 29 μL, depending on the degree of evaporation during the thermal cycler incubation.
10. Seal the wells with strip caps or using the PlateLoc Thermal Microplate Sealer. Make sure that all wells are completely sealed. 11. Incubate the hybridization mixture for 16 to 24 hours at 65°C with a heated lid at 105°C. Prepare streptavidin-coated magnetic beads The hybrid capture protocol uses reagents provided in SureSelect Target Enrichment Box 1 (stored at room temperature) in addition to the streptavidin-coated magnetic beads obtained from another supplier. Binding and wash buffers are identical between XT and XT-HS. 1. Prewarm SureSelect Wash Buffer 2 at 65°C in a circulating water bath or heat block for use in “Step 3. Capture the hybridized DNA using streptavidin-coated beads” from ThermoFisher. 2. Vigorously resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads on a vortex mixer. The magnetic beads settle during storage. 3. For each hybridization sample, add 50 μL of the resuspended beads to wells of a fresh PCR plate or strip tube. 4. Wash the beads: a. Add 200 μL of SureSelect Binding Buffer. b. Mix by pipetting up and down until beads are fully resuspended. c. Put the plate or strip tube into a magnetic separator device. d. Wait until the solution is clear, then remove and discard the supernatant. . e. Repeat step a through step d two more times for a total of 3 washes. 5. Resuspend the beads in 200 μL of SureSelect Binding Buffer. Capture the hybridized DNA using streptavidin-coated beads 1. Spin down samples. Estimate and record the volume of hybridization solution that remains after the 16-24 hour incubation. 2. Maintain the hybridization plate or strip tube at 65°C while you use a multichannel pipette to transfer the entire volume (approximately 25 to 29 μL) of each hybridization mixture to the plate or strip tube wells containing 200 μL of washed streptavidin beads. Mix well by slowly pipetting up and down until beads are fully Resuspended. 3. Cap the wells, then incubate the capture plate or strip tube on a 96-well plate mixer, mixing vigorously (1400–1800 rpm) for 30 minutes at room temperature. Make sure the samples are properly mixing in the wells. 4. Briefly spin the plate or strip tube in a centrifuge or mini-plate spinner. 5. Put the plate or strip tube in a magnetic separator to collect the beads. Wait until the solution is clear, then remove and discard the supernatant. 6. Resuspend the beads in 200 μL of SureSelect Wash Buffer 1. Mix by pipetting up and down until beads are fully resuspended. 7. Incubate the samples for 15 minutes at room temperature.
8. Briefly spin in a centrifuge or mini-plate spinner. 9. Put the plate or strip tube in the magnetic separator. Wait for the solution to clear, then remove and discard the supernatant. 10. Wash the beads with SureSelect Wash Buffer 2: a. Resuspend the beads in 200 μL of 65°C prewarmed Wash Buffer 2. Pipette up and down until beads are fully resuspended. b. Cap the wells, then incubate the sample plate or strip tube for 10minutes at 65°C on the thermal cycler. c. Put the plate or strip tube in the magnetic separator. Wait for the solution to clear, then remove and discard the supernatant. d. Repeat step a through step c for a total of 3 washes. Make sure all the wash buffer has been removed during the final wash. 11. Add 25 μL of nuclease-free water to each sample well. Pipette up and down to resuspend the beads. Keep the samples on ice. Post-Capture Sample Processing for Multiplexed Sequencing Step 1. Amplify the captured libraries In this step, the SureSelect-enriched DNA libraries are PCR amplified. Before you begin, thaw the reagents listed below and keep on ice. Preprogram a SureCycler 8800 thermal cycler (with the heated lid ON) with the program in Table 6. Start the program, then immediately press the Pause button, allowing the heated lid to reach temperature while you set up the reactions. Table 6 Post-capture PCR thermal Cycler program
Table 7 Post-capture PCR cycle number recommendations
Table 8 Preparation of Post-capture PCR Reaction Mix (XT-HS reagents)
1. Prepare the appropriate volume of PCR reaction mix, as described in Table 8, on ice. Mix well on a vortex mixer. 2. Add 25 μl of the PCR reaction mix prepared in Table 8 to each sample well containing 25 μl of bead-bound target-enriched DNA (prepared on page 4 and held on ice). 3. Mix the PCR reactions well by pipetting up and down until the bead suspension is homogeneous. Avoid splashing samples onto well walls; do not spin the samples at this step. 4. Place the plate or strip tube in the SureCycler 8800 thermal cycler. Press the Play button to resume the thermal cycling program in Table 6. 5. When the PCR amplification program is complete, spin the plate or strip tube briefly. Remove the streptavidin-coated beads by placing the plate or strip tube on the magnetic stand at room temperature. Wait 2 minutes for the solution to clear, then remove each supernatant (approximately 50 μl) to wells of a fresh plate or strip tube. The beads can be discarded at this time. Step 2. Purify the amplified captured libraries using AMPure XP beads 1. Let the AMPure XP beads come to room temperature for at least 30 minutes. Do not freeze the beads at any time. 2. Prepare 400 μl of fresh 70% ethanol per sample, plus excess, for use in step 8.
3. Mix the AMPure XP bead suspension well so that the suspension appears homogeneous and consistent in color. 4. Add 50 μl of the homogeneous AMPure XP bead suspension to each amplified DNA sample (approximately 50 μl) in the PCR plate or strip tube. Mix well by pipetting up and down 15–20 times. Check that the beads are in a homogeneous suspension in the sample wells. Each well should have a uniform color with no layers of beads or clear liquid present. 5. Incubate samples for 5 minutes at room temperature. 6. Put the plate or strip tube on the magnetic stand at room temperature. Wait for the solution to clear (approximately 3 to 5 minutes). 7. While keeping the plate or tubes in the magnetic stand, carefully remove and discard the cleared solution from each well. Do not disturb the beads while removing the solution. 8. Continue to keep the plate or tubes in the magnetic stand while you dispense 200 μl of freshly-prepared 70% ethanol in each sample well. 9. Wait for 1 minute to allow any disturbed beads to settle, then remove the ethanol. 10. Repeat step 8 and step 9 once for a total of two washes. Make sure to remove all of the ethanol at each wash step. 11. Seal the wells with strip caps, then briefly spin to collect the residual ethanol. Return the plate or strip tube to the magnetic stand for 30 seconds. Remove the residual ethanol with a P20 pipette. 12. Dry the samples by placing the unsealed plate or strip tube on the thermal cycler, set to hold samples at 37°C, until the residual ethanol has just evaporated (typically 1–2 minutes). 13. Add 25 μl of nuclease-free water to each sample well. 14. Seal the sample wells, then mix well on a vortex mixer and briefly spin to collect the liquid without pelleting the beads. 15. Incubate for 2 minutes at room temperature. 16. Put the plate or strip tube in the magnetic stand and leave for 2 minutes or until the solution is clear. 17. Remove the cleared supernatant (approximately 25 μl) to a fresh well. You can discard the beads at this time. [00178] The foregoing description, for purpose of explanation, has been described with reference to specific embodiments. However, the illustrative discussions above are not intended to be exhaustive or to limit the scope of claims to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. The embodiments were chosen and described in order to best explain the principles of the various described embodiments and their practical applications, to thereby enable others skilled in the art to best utilize the principles and the various described embodiments with various modifications as are suited to the particular use contemplated.
Claims
WHAT IS CLAIMED IS: 1. A method for preparing nucleic acid fractions useful for analyzing genetic loci associated with a risk of a cancer, comprising: obtaining nucleic acid from a subject; fragmenting the nucleic acid to obtain nucleic acid fractions; mixing the nucleic acid fractions with one or more capture probes in a solution, a respective capture probe coupled with a capture moiety, for forming one or more hybrids between the one or more capture probes and the nucleic acid fractions; and separating the one or more hybrids from the solution by using capture moieties of the one or more capture probes.
2. The method of claim 1, further comprising: analyzing the genetic loci in one or more nucleic acid fractions in the one or more hybrids.
3. The method of claim 2, wherein: analyzing the genetic loci includes identifying an insertion or deletion at a respective genetic locus of the genetic loci.
4. The method of claim 3, wherein: the insertion or deletion at the respective genetic locus of the genetic loci is identified by sequencing.
5. The method of any of claims 1-4, further comprising: subsequent to separating the one or more hybrids from the solution, separating one or more nucleic acid fractions from the one or more hybrids.
6. The method of any of claims 1-4, wherein: a capture probe of the one or more capture probes comprises a nucleic acid sequence having at least 80% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150.
7. The method of claim 6, wherein: the capture probe has 100% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150.
8. The method of any of claims 1-4, including: mixing the nucleic acid fractions with two or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
9. The method of any of claims 1-4, including: mixing the nucleic acid fractions with two or more capture probes, a respective capture probe of the two or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
10. The method of any of claims 1-4, including: mixing the nucleic acid fractions with one hundred or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
11. The method of any of claims 1-4, including: mixing the nucleic acid fractions with one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
12. The method of any of claims 1-4, including: mixing the nucleic acid fractions with one thousand or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
13. The method of any of claims 1-4, including: mixing the nucleic acid fractions with one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
14. The method of any of claims 1-4, wherein:
the capture moiety is selected from a group consisting of avidin, streptavidin, and biotin.
15. The method of any of claims 1-4, wherein: the subject is a human subject.
16. The method of any of claims 1-4, wherein: the nucleic acid is a genomic deoxyribonucleic acid (gDNA).
17. A reagent kit, comprising: one or more capture probes, a respective capture probe coupled with a capture moiety and having at least 80% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150.
18. The reagent kit of claim 17, comprising: two or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
19. The reagent kit of claim 17, including: two or more capture probes, a respective capture probe of the two or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
20. The reagent kit of claim 17, including: one hundred or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
21. The reagent kit of claim 17, including: one hundred or more capture probes, a respective capture probe of the one hundred or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
22. The reagent kit of claim 17, including: one thousand or more capture probes comprising nucleic acid sequences selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
23. The reagent kit of claim 17, including: one thousand or more capture probes, a respective capture probe of the one thousand or more capture probes comprising a nucleic acid sequence having at least 80% sequence identity to a distinct nucleic acid sequence selected from the group of nucleic acid sequences selected from SEQ ID NOs: 1 through 116150.
24. The reagent kit of any of claims 17-23, wherein: the capture moiety is selected from a group consisting of avidin, streptavidin, and biotin.
25. A capture probe coupled with a capture moiety and having at least 80% sequence identity to any one of the sequences selected from SEQ ID NOs: 1 through 116150.
26. The capture probe of claim 25, wherein the capture moiety is selected from a group consisting of avidin, streptavidin, and biotin.
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