Methods for detecting silent carrier genotypes

Abstract

To provide methods and compositions for the detection of silent carriers of chromosomal deletion alleles in a human subject with spinal muscular atrophy (SMA).SOLUTION: Provided herein are methods and compositions for the detection of silent carriers of chromosomal deletion alleles in a human subject using haploid cells (e.g., sperm cells or egg cells) derived from the subject. The methods provided herein allow for the detection of silent (2+0) carriers of SMA, where the individual has a deletion of the SMN1 gene on one chromosome 5 homolog and two or more copies of the SMN1 gene on other chromosome 5 homolog.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 080,047, filed November 14, 2014, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Spinal muscular atrophy (SMA) is the second most common fatal autosomal recessive disorder after cystic fibrosis, affecting approximately 1 in 6,000–10,000 births. The disorder is characterized by hypotension, proximal muscle weakness, and respiratory distress due to degeneration of spinal motor neurons. SMA is caused by mutations in the survival motor neuron 1 (SMN1) gene, located on chromosome 5 at 5q11.2–13.3. The majority of affected individuals exhibit loss of the SMN1 gene, either by complete gene deletion or through a gene conversion event involving the adjacent SMN2 gene. The SMN2 gene differs from the SMN1 gene by a single nucleotide in exon 7 (840C>T) and is present in cis with the SMN1 gene in the opposite orientation on chromosome 5. At least one copy of the SMN1 gene is essential for normal motor neuron survival. In contrast, although the number of SMN2 copies can alter the clinical phenotype in some cases, approximately 5–10% of normal individuals lack both copies of SMN2, so both copies of the SMN2 gene are not necessary.

[0003] Molecular diagnosis of SMA is typically achieved by detecting a homozygous deletion of SMN1. Over 95% of SMA patients have a homozygous deletion of SMN1 exon 7. However, carrier testing for SMA is particularly challenging for several reasons. Because the SMN1 gene is highly homologous to SMN2, abnormalities in the SMN1 gene can only be detected by carefully designed allele-specific assays. Furthermore, approximately 4% of the carrier population has a chromosomal mutation that places both copies of the SMN1 gene on one chromosome and no copies on the other (i.e., silent carrier or 2+0 genotype). Gene dosage analysis can determine SMN1 copy number to detect carrier status in individuals heterozygous for the absence of SMN1, but is ineffective at detecting the silent carrier genotype, in which two copies of the SMN1 gene are present on only one chromosome. Furthermore, the SMN1 and SMN2 genes are separated by a long distance (800 kb) on the same chromosome, making linkage analysis of chromosomal defects difficult. Summary of the Invention [Means for solving the problem]

[0004] In certain embodiments, methods and compositions are described herein for detecting silent carriers of chromosomal deletion alleles in human subjects using haploid cells derived from the subject. In some embodiments, the haploid cells are gametes (e.g., sperm cells or egg cells). In certain embodiments, the methods provided herein allow for the detection of silent (2+0) carriers of SMA, in which an individual has a deletion of the SMN1 gene on one chromosome 5 homolog and two or more copies of the SMN1 gene on another chromosome 5 homolog.

[0005] In certain embodiments, provided herein are methods for identifying a subject as a silent carrier of a target gene null allele. In some embodiments, the method includes: (a) performing multiple nucleic acid amplification reactions, each comprising a genomic DNA sample obtained from a single haploid cell from the subject, at least one pair of oligonucleotide primers for amplifying a target region of the target gene to generate a target gene amplification product, wherein the region amplified in the target gene amplification product is deleted in the target gene null allele, and at least one pair of oligonucleotide primers for amplifying a target region of a reference gene to generate a reference gene amplification product; (b) detecting the presence or absence of the target gene amplification product; (c) detecting the presence or absence of the reference gene amplification product; and (d) characterizing the subject as a carrier of the target gene null allele if the ratio of the target gene amplification product to the reference gene amplification product is equal to or less than a threshold level. In some embodiments, the threshold level is about 0.5 to about 0.8. For example, in some embodiments, the threshold level is about 0.75 or about 0.8. In some embodiments, the ratio of target gene amplification product to reference gene amplification product in silent carriers of a target gene null allele is about 0.5. The methods provided herein are typically performed on samples obtained from mammalian subjects, particularly human subjects. In certain embodiments, the target gene for amplification is SMN1. In some embodiments, the target gene amplification product comprises exon 7 of SMN1 or a portion thereof. In some embodiments, the reference gene is selected from CFTR, GAPDH, HMBS, B2M, HPRT1, RPL13A, SDHA, TBP, UBC, YWHAZ, PRDX6, ADD1, HLA-A, RAD9A, ARHGEF7, EIF2B2, PSMD7, BCAT2, and ATP5O. In certain embodiments, the reference gene is CFTR. In some embodiments, homozygous deletion of the target gene is associated with a disease or condition. In some embodiments, the disease or condition is spinal muscular atrophy (SMA).

[0006] Exemplary haploid cells for use in this method include naturally occurring gamete cells or induced haploid cells. In some embodiments, the haploid cell is a sperm cell or an egg cell. In some embodiments, when the haploid cell is an induced haploid cell, the haploid cell is derived from an induced pluripotent stem cell (iPSC). In some embodiments, the iPSC is generated from adult stem cells from a subject.

[0007] In some embodiments, at least one oligonucleotide primer of a primer pair for amplifying a target region of a target gene and / or a reference gene is labeled with a detectable moiety, such as a radioactive moiety, a fluorescent moiety, or a dye molecule. In some embodiments, the nucleic acid amplification reaction is a polymerase chain reaction (PCR), or in particular, quantitative PCR. In some embodiments, each nucleic acid amplification reaction is performed in a separate well of a multi-well plate. In some embodiments, the target gene amplification product and / or the reference gene amplification product are detected with a labeled nucleic acid probe specific for the target gene amplification product.

[0008] In some embodiments, provided methods include preparing genomic DNA from a single population of haploid cells. In exemplary methods, preparing genomic DNA from a single population of haploid cells includes (a) sorting the single population of haploid cells into separate reaction vessels at a concentration of one haploid cell per reaction vessel, and (b) contacting each sorted cell with a lysis buffer to release genomic DNA from the cells. In some embodiments, the lysis buffer contains an enzyme that assists in lysing the haploid cells. For example, in some embodiments, the lysis buffer contains a protease. In some embodiments, the lysis buffer contains proteinase K. The preparation of genomic DNA and the nucleic acid amplification reaction can be performed in the same reaction vessel or in separate reaction vessels. Preparing genomic DNA and the nucleic acid amplification reaction in the same reaction vessel minimizes loss of genomic DNA. In some embodiments, the reaction vessel is a well of a microtiter plate, a microchip, or a reaction grid slide.

[0009] In some embodiments, the method comprises droplet digital PCR. In some embodiments, each haploid cell to be analyzed is first encapsulated in a microdroplet. In some embodiments, the microdroplets are dispersed in a water-in-oil emulsion in a single container. In some embodiments, the microdroplets are sorted into individual containers. In some embodiments, each haploid cell is lysed within the microdroplet. In some embodiments, the microdroplets containing the lysed cells are then subjected to a nucleic acid amplification reaction. In some embodiments, the nucleic acid amplification product is detected within the microdroplet. In other embodiments, the amplification product is isolated from the microdroplet and detected.

[0010] In some embodiments, the method further comprises determining the copy number of the target gene in diploid cells from the test subject. In some embodiments, the method further comprises generating a cell line from the diploid cells of the test subject. In some embodiments, the method further comprises sequencing the SMN1 and / or SMN2 genes or portions thereof.

[0011] In some embodiments, the method further comprises generating a report comprising an assessment of the likelihood that the subject is a silent carrier of a target gene null allele.

[0012] Also provided herein are kits for carrying out the methods described herein. In exemplary embodiments, the kits for carrying out the methods provided include: The kit includes: (a) an oligonucleotide primer pair specific to the SMN1 gene for generating a target gene amplification product, wherein the region to be amplified in the SMN1 gene amplification product is deleted in SMN1 silent carriers; (b) an oligonucleotide primer pair specific to a reference gene for generating a reference gene amplification product that is not deleted in SMN1 silent carriers; and (c) one or more reagents for performing a nucleic acid amplification reaction. In some embodiments, the kit includes nucleotide triphosphates, a thermostable polymerase, and / or an appropriate buffer. In some embodiments, the reference gene is selected from CFTR, GAPDH, HMBS, B2M, HPRT1, RPL13A, SDHA, TBP, UBC, YWHAZ, PRDX6, ADD1, HLA-A, RAD9A, ARHGEF7, EIF2B2, PSMD7, BCAT2, and ATP5O. In some embodiments, the target gene amplification product includes exon 7 of SMN1 or a portion thereof.

[0013] Also provided herein is a microtiter plate for carrying out the methods described herein. A typical microtiter plate includes multiple reaction vessels (e.g., wells), one or more of which contain (a) an oligonucleotide primer pair specific to the SMN1 gene for generating a target gene amplification product, where the oligonucleotide primers are deleted in SMN1 silent carriers for the region to be amplified in the SMN1 gene amplification product; (b) an oligonucleotide primer pair specific to a reference gene for generating a reference gene amplification product that is not deleted in SMN1 silent carriers; and (c) one or more reagents for carrying out a nucleic acid amplification reaction. In some embodiments, one or more reaction vessels contain nucleotide triphosphates, a thermostable polymerase, and / or an appropriate buffer. In some embodiments, the reference gene is selected from CFTR, GAPDH, HMBS, B2M, HPRT1, RPL13A, SDHA, TBP, UBC, YWHAZ, PRDX6, ADD1, HLA-A, RAD9A, ARHGEF7, EIF2B2, PSMD7, BCAT2, and ATP5O. In some embodiments, the target gene amplification product comprises exon 7 of SMN1 or a portion thereof. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 shows the organization of the SMN1 and SMN2 gene loci at 5q13 on chromosome 5. Details of the chromosomal locations of the SMN1 and SMN2 gene copies associated with normal, carrier, and silent carrier genotypes are provided. [Figure 2] FIG. 2 shows an exemplary assay workflow for a single sperm cell qPCR assay. [Figure 3] Figure 3 shows data for detecting the SMN1 gene in a single sperm qPCR assay. Mean single-cell qPCR assay ratio values for SMN1 vs. reference gene and both gene targets vs. reference gene are shown. *P<0.01. Standard deviation of the observations is indicated by error bars. [Figure 4] Figure 4 shows the resolution of the identified 2+0 genotype results. (A) Mean single-cell qPCR assay ratio values for SMN1 vs. reference gene and both gene targets vs. reference gene for sample DS11. (B) Non-specific sequencing of SMN1 and SMN2 genes, position +6 of exon 7 c.840C>T highlighted in red box. (C) Specific sequencing of SMN1 qPCR primer and probe sites. *P<0.001. Standard deviation of the observed values is indicated by error bars. DETAILED DESCRIPTION OF THE INVENTION

[0015] Specific Terminology To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0016] As used herein, the singular forms "a," "an," and "the" include plural references unless specifically stated otherwise. Thus, for example, a reference to "an oligonucleotide" includes a plurality of oligonucleotide molecules, a reference to "a label" is a reference to one or more labels, a reference to "a probe" is a reference to one or more probes, and a reference to "a nucleic acid" is a reference to one or more polynucleotides.

[0017] As used herein, unless otherwise indicated, when referring to a numerical value, the term "about" means plus or minus 10% of the recited value.

[0018] As used herein, a "carrier" or "gene carrier" is an individual who has at least one copy of an allele of a genetic determinant responsible for the manifestation of a particular phenotype, such as SMA.

[0019] As used herein, a "silent carrier" refers to a genetic carrier that cannot be detected using copy number-based diagnostic techniques. For example, a "silent carrier" refers to a genetic carrier that has a deletion of all or part of a target gene on one chromosomal homolog and has two or more copies of the target gene on another chromosomal homolog.

[0020] As used herein, an "SMA silent carrier" or "SMA(2+0) carrier" is a genetic carrier who has a deletion of all or part of the SMN1 gene on one chromosome 5 homolog and two or more copies of the SMN1 gene on the other chromosome 5 homolog.

[0021] As used herein, the terms "amplification" or "amplifying" include methods of copying a target nucleic acid, thereby increasing the copy number of a selected nucleic acid sequence. Amplification may be exponential or linear. The target nucleic acid may be either DNA or RNA. The sequence amplified in this manner forms an "amplification product," also known as an "amplicon." The exemplary method described below relates to amplification using the polymerase chain reaction (PCR), although many other methods for amplifying nucleic acids (e.g., isothermal methods, rolling circle methods, etc.) are known in the art. Those skilled in the art will understand that these other methods may be used instead of or in conjunction with PCR. See, e.g., PCR protocols in Saiki, "Amplification of Genomic DNA," Innis et al., eds., Academic Press, San Diego, Calif., 1990, pp. 13-20; Wharam et al., Nucleic Acids Res., 29(11):E54-E54, 2001; Hafner et al., Biotechniques, 30(4):852-56, 858, 860, 2001; Zhong et al., Biotechniques, 30(4):852-6, 858, 860, 2001.

[0022] As used herein, the term "detect" refers to observing a signal from a detectable label to indicate the presence of a target. More specifically, detection is used in the context of detecting a specific sequence.

[0023] As used herein, the terms "complement," "complementary," or "complementarity" with respect to polynucleotides (i.e., sequences of nucleotides such as oligonucleotides or genomic nucleic acids) refer to those related by the base-pairing rules. As used herein, the complement of a nucleic acid sequence refers to an oligonucleotide that "antiparallel associates" when aligned with the nucleic acid sequence such that the 5' end of one sequence pairs with the 3' end of the other. For example, the sequence 5'-AGT-3' is complementary to the sequence 3'-TCA-5'. Certain bases not normally found in natural nucleic acids may be included in the nucleic acids of the present disclosure, including, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes can contain mismatched or mismatched bases. Those skilled in the art of nucleic acid technology can empirically consider many variables, including the length of the oligonucleotide, the base composition and sequence of the oligonucleotide, ionic strength, and the frequency of mismatched base pairs, to determine duplex stability. Complementarity can be "partial," where only some of the nucleic acids' bases match according to the base-pairing rules, or there can be "complete," "total," or "total" complementarity between the nucleic acids.

[0024] As used herein, the term "detectable label" refers to a molecule or compound or group of molecules or compounds associated with a probe and used to identify the probe hybridized to a genomic or reference nucleic acid.

[0025] A "fragment" in the context of a polynucleotide refers to a sequence of nucleotide residues, either double-stranded or single-stranded, that is at least about 2 nucleotides, at least about 5 nucleotides, at least about 10 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, or at least about 100 nucleotides.

[0026] The terms "identity" and "identical" refer to the degree of identity between sequences. There can be partial identity or complete identity. A partially identical sequence is one that is less than 100% identical to another sequence. A partially identical sequence can have an overall identity of at least 70%, or at least 75%, at least 80%, or at least 85%, or at least 90%, or at least 95%.

[0027] As used herein, the terms "isolated," "purified," or "substantially purified" refer to molecules, such as nucleic acids, that have been removed from their natural environment, isolated or separated, and are at least 60%, preferably 75%, and most preferably 90% free from other components with which they are naturally associated. Thus, isolated molecules are substantially purified molecules.

[0028] As used herein, the term "oligonucleotide" or "polynucleotide" refers to a short polymer composed of deoxyribonucleotides, ribonucleotides, or any combination thereof. Oligonucleotides are generally from about 10, 11, 12, 13, 14, 15, 20, 25, or 30 nucleotides (nt) to about 150 nucleotides (nt) in length, and more preferably from about 10, 11, 12, 13, 14, 15, 20, 25, or 30 nucleotides (nt) to about 70 nt.

[0029] As used herein, a "primer" is an oligonucleotide that is complementary to a target nucleotide sequence and that, in the presence of a DNA or RNA polymerase, results in the addition of a nucleotide to the 3' end of the primer. The 3' nucleotide of a primer should generally be identical to the target sequence at the corresponding nucleotide position for optimal extension and / or amplification. The term "primer" includes all forms of primers that can be synthesized, including peptide nucleic acid primers, locked nucleic acid primers, phosphorothioate-modified primers, labeled primers, etc. As used herein, a "forward primer" is a primer that is complementary to the antisense strand of DNA. A "reverse primer" is complementary to the sense strand of DNA.

[0030] An oligonucleotide (e.g., a probe or primer) specific to a target nucleic acid "hybridizes" to the target nucleic acid under appropriate conditions. As used herein, "hybridization" or "hybridize" refers to the process by which an oligonucleotide single strand anneals to a complementary strand through base pairing under defined hybridization conditions. This is a specific, i.e., non-random, interaction between two complementary polynucleotides. Hybridization and the strength of hybridization (i.e., the strength of the association between nucleic acids) are affected by factors such as the degree of complementarity between the nucleic acids, the stringency of the conditions involved, and the Tm of the formed hybrid.

[0031] "Specific hybridization" indicates that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after subsequent washing steps. Permissive conditions for annealing of nucleic acid sequences can be routinely determined by one of skill in the art and can occur, for example, at 65°C in the presence of approximately 6x SSC. Hybridization stringency can be expressed, in part, with reference to the temperature at which the washing step is performed. Such a temperature is typically selected to be approximately 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe (under defined ionic strength and pH). Equations for calculating Tm and conditions for nucleic acid hybridization are known in the art.

[0032] As used herein, an oligonucleotide is "specific" for a nucleic acid if it can hybridize to the intended target and is substantially incapable of hybridizing to nucleic acids other than the intended target. High levels of sequence identity are preferred, including at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and more preferably at least 98% sequence identity. Sequence identity can be determined using commercially available computer programs with default settings that utilize algorithms well known in the art (e.g., BLAST).

[0033] As used herein, the term "region of interest" or "target region" refers to a region of nucleic acid that is to be amplified.

[0034] The term "emulsion droplets" or "emulsion microdroplets" refers to droplets formed when two immiscible liquids are combined. For example, aqueous droplets can be formed when an aqueous liquid is mixed with a non-aqueous liquid. As another example, droplets can be formed by adding a non-aqueous liquid to an aqueous liquid. Droplets can be formed by a variety of methods, including those performed by microfluidic devices or other methods, such as injecting one fluid into another, forcing or drawing multiple liquids through an orifice or opening, or forming droplets by shear forces. Emulsion droplets can have a uniform or heterogeneous distribution. Any emulsion disclosed herein can be monodisperse (composed of droplets at least generally of a single size) or polydisperse (composed of droplets of various sizes). If monodisperse, emulsion droplets can vary in volume with a standard deviation of less than about plus or minus 100%, 50%, 20%, 10%, 5%, 2%, or 1% of the average droplet volume. The droplets generated from the orifice can be monodisperse or polydisperse. The emulsion can have any suitable composition. The emulsion can be characterized by the predominant liquid compound or type of liquid compound used. The predominant liquid compounds in the emulsion can be water and oil. "Oil" refers to any liquid compound or mixture of liquid compounds that is immiscible with water and has a high carbon content. In some examples, the oil can also have a high content of, among other elements, hydrogen, fluorine, silicon, oxygen, or any combination thereof. For example, any emulsion disclosed herein can be a water-in-oil (W / O) emulsion (i.e., aqueous droplets in a continuous oil phase). The oil can be or include, among other elements, at least one silicone oil, mineral oil, fluorocarbon oil, vegetable oil, or a combination thereof. Any other suitable components, such as at least one surfactant, reagent, sample (i.e., a fraction thereof), buffer, salt, ionic element, other additive, label, particle, or any combination thereof, can be present in any of the emulsion phases.

[0035] As used herein, the term "droplet" refers to a small amount of liquid, typically spherical or as a slug filling the diameter of a microchannel surrounded by an immiscible fluid. The volume of a droplet in an emulsion and / or the average volume of the droplets may be less than about 1 microliter (i.e., a "microdroplet") (or between about 1 microliter and 1 nanoliter, or between about 1 microliter and 1 picoliter), less than about 1 nanoliter (or between about 1 nanoliter and 1 picoliter), or less than about 1 picoliter (or between about 1 picoliter and 1 femtoliter). The droplets may have a diameter (or average diameter) of less than about 1000, 100, or 10 micrometers, or between about 1000 and 10 micrometers, among others. The droplets may be spherical or non-spherical. In some embodiments, the droplets have a volume and diameter large enough to encapsulate cells. In some embodiments, the droplets have a volume and diameter large enough to encapsulate haploid cells. In some embodiments, the droplets have a volume and diameter large enough to encapsulate sperm cells.

[0036] The terms "bulk sequencing," "next-generation sequencing," or "massively parallel sequencing" refer to any high-throughput sequencing technology that parallelizes the DNA sequencing process. For example, bulk sequencing can typically generate more than one million polynucleic acid amplicons in a single assay. The terms "bulk sequencing," "massively parallel sequencing," and "next-generation sequencing" refer only to general methods and do not necessarily refer to obtaining more than one million sequence tags in a single run. For example, any bulk sequencing method, such as reversible terminator chemistry (e.g., Illumina), pyrosequencing using polony emulsion droplets (e.g., Roche), ion semiconductor sequencing (IonTorrent), single-molecule sequencing (e.g., Pacific Biosciences), or massively parallel code sequencing, can be implemented in the present invention.

[0037] As used herein, the term "subject" refers to a mammal, such as a human or non-human primate, but may also be another animal, such as a domestic animal (e.g., dog, cat, etc.), an agricultural animal (e.g., cow, sheep, pig, horse, etc.), or a laboratory animal (e.g., monkey, rat, mouse, rabbit, guinea pig, etc.). The term "patient" refers to a "subject" that has or is suspected of having a genetic polymorphism of interest.

[0038] Silent Carrier Genotyping Assay Overview Provided herein is a method for genotyping a disorder in which at least one chromosome of a homologous pair lacks a gene of interest. In certain instances, the method involves determining the carrier status of a gene that is prone to chromosomal rearrangements that can nullify one chromosome for the gene. In certain instances, the method involves detecting the presence or absence of a target gene of interest in haploid cells, such as gamete cells (e.g., sperm or egg cells). Because gametes are naturally haploid, the presence of a chromosome that is null for the target gene can be detected by single-cell genomic analysis methods, such as nucleic acid amplification. In some embodiments, artificial haploid cells (e.g., by induced germ cell differentiation of pluripotent stem cells) can also be used.

[0039] Traditional genotyping is performed using diploid cells, most commonly lymphocytes (white blood cells) isolated from whole blood. Such methods are ineffective for determining the carrier status of 2+0 silent carrier genotypes because carriers have two copies of the normal gene. Testing for genetic disorders using a single haploid cell as a source eliminates the complexity of having two copies of every autosomal gene as a potential template for analysis.

[0040] In an exemplary method, a single haploid cell is delivered to a reaction vessel and processed (e.g., lysed) to make the cell's genomic DNA available for performing a genetic assay (e.g., PCR). In another exemplary method, a single haploid cell is encapsulated in a microdroplet (i.e., one cell per droplet), for example, in a water-in-oil emulsion, agarose-in-oil emulsion, or embedded in an alginate microsphere (see, e.g., Clausell-Tormos et al. (2008) Chem. Biol. 15:427-437). In such an embodiment, the cell is processed (e.g., lysed) within the microdroplet to make the cell's genomic DNA available for performing a genetic assay (e.g., PCR).

[0041] In some embodiments, the assay involves nucleic acid amplification of a target gene and a reference gene (e.g., a housekeeping gene) present in cells in the same reaction vessel or microdroplet and analysis of the resulting amplification products. In some embodiments, a single reaction vessel contains multiple microdroplets, each containing a single cell, and nucleic acid amplification is performed in each microdroplet. The presence or absence of the target gene relative to a reference gene that should always be present is determined from multiple reactions, each of which represents the genetic state of a single haploid cell. Statistics can be used to analyze replication reactions to determine carrier status. In some embodiments, the amplification products are labeled. In some embodiments, the amplification products are labeled using a primer pair for amplification, in which at least one primer of the primer pair is labeled with a detectable moiety. In some embodiments, the target gene amplification product and the reference gene amplification product are labeled with different detectable moieties.

[0042] In certain embodiments, a plurality of haploid cells (e.g., sperm cells) are obtained from a test subject. The haploid cells are delivered to a reaction vessel at a concentration of one cell per vessel. Once sorted, each cell is processed individually to make the cellular genomic DNA available for performing a genetic assay. In some embodiments, the genetic assay involves nucleic acid amplification of a target gene and a reference gene (e.g., a housekeeping gene) present in cells in the same reaction vessel. Statistics can be used to analyze replicate reactions for the presence or absence of the target gene relative to the reference gene, which should always be present. In such embodiments, when a plurality of haploid cells are assayed, the absence of the target gene in approximately 50% of cells from an individual indicates that the individual is a carrier of a null deletion mutation.

[0043] In an alternative embodiment, a plurality of haploid cells (e.g., sperm cells) are obtained from a test subject and the haploid cells are encapsulated in microdroplets at a concentration of one cell per microdroplet. The microdroplets can be sorted into reaction vessels at a concentration of one cell per vessel, or multiple microdroplets can be contained in one or more vessels in a water-in-oil emulsion. In some embodiments, the microdroplets are sorted based on whether they contain cells to enrich for microdroplets containing cells. In some embodiments, the cells are labeled. The microdroplets, each containing a haploid cell, are processed to make the genomic DNA of the cell available for performing a genetic assay. In some embodiments, the genetic assay involves nucleic acid amplification of a target gene and a reference gene (e.g., a housekeeping gene) present in the cells in the same microdroplet. The amplification products can be analyzed by detecting the amplification products in each microdroplet. In some embodiments, a microfluidic detection device is used to scan the droplets for target gene and reference gene amplification products. Statistics can be used to analyze the replication response for the presence or absence of the target gene relative to a reference gene that should always be present. In such an embodiment, when multiple haploid cells are assayed, the absence of the target gene in about 50% of the cells from an individual indicates that the individual is a carrier of a null deletion mutant. Exemplary methods for microdroplet-based emulsion amplification and detection from single cells are known and can be used in combination with the haploid cell amplification methods provided herein (see, e.g., U.S. Pat. Nos. 8,338,166, 8,454,906; Novak et al. (2011) Angew Chem Int Ed Engl. 50(2):390-395; Clausell-Tormos et al. (2008) Chem. Biol. 15:427-437; Novake et al. (2010) Anal Chem. 82(8):3183-90; and Solvas et al. (2001) J. Vis. Exp. (58):e3437).

[0044] As described herein, the provided methods are useful for detecting the SMN1 silent carrier genotype of SMA, in which two copies of the SMN1 gene are located on a single chromosome 5 and no copies of the gene are located on its chromosome 5 homolog. In certain embodiments, a plurality of haploid cells are obtained from a test subject suspected of having SMA. In such cases, a single haploid cell obtained from the subject is delivered to a reaction vessel at a concentration of one cell per vessel. Each cell is individually processed to make the cellular genomic DNA available for performing a genetic assay for detecting the SMN1 gene. In some embodiments, the genetic assay involves nucleic acid amplification of a target region of the SMN1 gene and a target region of a reference gene present in cells (e.g., a housekeeping gene) in the same reaction vessel. Statistics can be used to analyze replicate reactions for the presence or absence of the SMN1 gene relative to the reference gene, which should always be present. In such embodiments, when a plurality of haploid cells are assayed, the absence of the SMN1 gene in approximately 50% of cells from an individual indicates that the individual is a carrier of an SMN1 null deletion variant.

[0045] In an alternative embodiment for detecting an individual as a carrier of an SMN1 null deletion mutant, a plurality of haploid cells (e.g., sperm cells) are obtained from a test subject, and the haploid cells are encapsulated in microdroplets at a concentration of one cell per microdroplet. The microdroplets can be sorted into reaction vessels at a concentration of one cell per vessel, or multiple microdroplets can be contained in one or more vessels in a water-in-oil emulsion. In some embodiments, the microdroplets are sorted based on whether they contain cells to enrich for microdroplets containing cells. In some embodiments, the cells are labeled. Microdroplets, each containing a haploid cell, are processed to make the genomic DNA of the cell available for performing a genetic assay for detecting the SMN1 gene. In some embodiments, the genetic assay includes nucleic acid amplification of a target region of the SMN1 gene and a reference gene present in the cells (e.g., a housekeeping gene) in the same microdroplet. The amplification products can be analyzed by detecting the amplification products in each microdroplet. In some embodiments, a microfluidic detection device is used to scan the droplets for target gene and reference gene amplification products. Statistics can be used to analyze the replication response for the presence or absence of the SMN1 gene relative to the reference gene, which should always be present. In such embodiments, when a plurality of haploid cells are assayed, the absence of the SMN1 gene in about 50% of the cells from an individual indicates that the individual is a carrier of a null deletion mutation.Exemplary methods of microdroplet-based emulsion amplification and detection from single cells are known and can be used in combination with the haploid cell amplification methods provided herein for detection of SMN1 null deletion mutants and silent carrier states (e.g., U.S. Pat. Nos. 8,338,166, 8,454,906; Novak et al. (2011) Angew Chem Int Ed Engl. 50(2):390-395; Clausell-Tormos et al. (2008) Chem. Biol. 15:427-437; Novake et al. (2010) Anal Chem. 82(8):3183-90; and Solvas et al. (2001) J. Vis. Exp. (58):e3437).

[0046] In some embodiments, the assay further comprises analyzing gene copy number in diploid cells of the test subject. In some embodiments, genetic analysis of a single diploid cell from the test subject is performed to determine the copy number of the SMN1 and / or SMN2 gene. In some embodiments, genetic analysis of a single diploid cell from the test subject is performed to confirm that the subject has two copies of the SMN1 gene. In some embodiments, the assay for determining gene copy number of the SMN1 gene is performed as described in Curet et al. (2007) Neurogenetics 8:271-278. In some embodiments, the diploid cell is a blood cell.

[0047] In some embodiments, the assay further comprises generating a cell line from the test subject's diploid cells (e.g., derived from lymphocytes, fibroblasts, stem cells, epithelial cells, etc.). In some embodiments, the cell line is generated using standard techniques for cell transformation (e.g., Hahn (2002) Mol. Cells 13(3):351-361; Stabley et al. (2015) Mol. Gen Genomic Med. 3(4) 248-257). In some embodiments, the transformed cell line is used for gene copy number analysis of the SMN1 and / or SMN2 genes. In some embodiments, genetic analysis of the transformed cell line is performed to confirm that the subject has two copies of the SMN1 gene.

[0048] In some embodiments, the assay further comprises generating a cell line from the diploid cells of a test subject identified as having the SMN1 gene silent carrier genotype. The generation of the cell line provides a long-term record of individuals with this rare genotype. The immortal cell line provides an unlimited amount of samples from such individuals without any additional sampling. Such cell lines can be used to identify sequence markers specific to the "silent carrier" founder allele.

[0049] In some embodiments, the method further comprises sequencing a target gene, such as the SMN1 and SMN2 genes, or one or more portions thereof. In some embodiments, the target gene amplification products are sequenced. In some embodiments, the target gene amplification products generated by PCR in oil-water microdroplets are sequenced. Any suitable method for sequencing nucleic acids can be used. In some embodiments, next-generation sequencing is employed.

[0050] In some embodiments, the method further comprises generating a report based on the results of the assay. In some embodiments, the method further comprises determining the risk of producing offspring with SMA based on the results of the assay.

[0051] Target and reference genes The method described herein can be used to detect null deletions in target genes. In certain embodiments, the target gene is one in which at least one copy of the gene is deleted on one chromosome in silent carriers, and multiple copies (e.g., 2, 3, 4 or more) are present on homologous chromosomes or other locations. In certain embodiments, the target gene is the SMN1 gene.

[0052] For the practice of the methods provided herein, the absence of a target gene is determined by the absence of a nucleic acid amplification product relative to a reference nucleic acid amplification product from a selected reference gene, wherein the target gene and the reference gene are amplified in the same reaction vessel. Exemplary reference genes for use in the provided methods include cystic fibrosis transmembrane transregulator (CFTR), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), succinate dehydrogenase complex, subunit A (SDHA), TATA box-binding protein (TBP), ubiquitin C (UBC), and tyrosine 3 binding to phosphorylated activated protein. Examples of genes that may be used as reference genes include, but are not limited to, tryptophan 5-monooxygenase, zeta polypeptide (YWHAZ), peroxiredoxin-6 (PRDX6), alpha-adducin (ADD1), major histocompatibility complex class IA (HLA-A), RAD9A, Rho guanine nucleotide exchange factor (GEF) 7 (ARHGEF7), eukaryotic translation initiation factor 2B subunit 2 beta (EIF2B2), 26S proteasome non-ATPase regulatory subunit 7 (PSMD7), branched-chain amino acid transaminase 2 (BCAT2), and ATP synthase subunit O (ATP5O). In certain embodiments, the reference gene for use in the methods provided herein is the CFTR gene.

[0053] Subject and sample acquisition for testing Generally, the methods provided herein are used to determine the silent carrier status in mammals (e.g., primates, rabbits, dogs, cats, sheep, and pigs). In certain embodiments, the subject is a human patient.

[0054] In certain instances, selection of a subject for testing for silent carrier status for a particular target gene is based on multiple factors. In some embodiments, a subject is selected for testing based on the prevalence of the deletion in the general population or a particular ethnic group. In some embodiments, a subject is selected for testing based on a confirmed or suspected family history of a disease associated with the target gene. In some embodiments, a subject is selected for testing based on a confirmed or suspected family history of SMA. In certain instances, a subject is selected for testing for silent carrier status based on the recommendation of a licensed physician or as part of a genetic counseling procedure or program.

[0055] In certain instances, subjects selected for testing are suspected of having a deletion of the SMN1 gene on one chromosome 5 homolog and two or more copies of the SMN1 gene on another chromosome 5 homolog.

[0056] In certain embodiments, naturally occurring haploid cells are used in the assay. In such instances, standard methods for obtaining appropriate male or female gametes for a particular subject can be used.

[0057] In certain embodiments, induced haploid cells derived from adult stem cells are used in the assay. In such instances, any source of stem cells from a subject can be used. Adult stem cells can be obtained from various organs and tissues, including, but not limited to, the brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, intestine, liver, ovarian epithelium, and testis. In certain instances, adult stem cells are treated to induce stem cell pluripotency (e.g., to generate induced pluripotent stem cells (iPSCs)). For example, in certain instances, adult stem cells can be modified to express one or more genes that induce pluripotency, such as Oct4, Sox2, cMyc, and / or Klf4. Once pluripotent cells are generated, the cells can be treated to induce meiosis to generate artificial haploid cells (see, e.g., Eguizabal et al. (2011) Stem Cells 29:1186-1195).

[0058] Single cell sorting method In the provided methods, any suitable method for sorting individual haploid cells into separate reaction vessels can be used for analysis. Exemplary cell sorting methods include, but are not limited to, dilution sorting, droplet-based microfluidics, flow cytometry, fluorescence-activated cell sorting (FACS), magnetically activated cell sorting (MACS), laser-assisted cell picking, micropatterning of controlled patches of extracellular matrix (ECM) or other ligands, or microfluidic chip sorting. In some embodiments, cells are seeded into reaction vessels at a concentration of one cell per well. Methods for sorting single cells and seeding single cells into various reaction vessels for genomic analysis are known in the art, including, for example, those described in U.S. Patent No. 6,673,542 and U.S. Patent Application Publication No. 2011 / 0237445.

[0059] In certain embodiments, to aid in haploid cell sorting methods, haploid cells are labeled with an appropriate dye (e.g., Hoechst 33342). In such methods, haploid cells are contacted with the dye for a predetermined length of time to allow labeling of the haploid cells. The labeled haploid cells are then subjected to a selected cell sorting method.

[0060] Cells can be sorted into any suitable reaction vessel suitable for carrying out the methods provided herein. In some embodiments, cells are sorted into a suitable reaction vessel suitable for hybridization of gene-specific probes. In some embodiments, cells are sorted into a suitable reaction vessel suitable for nucleic acid amplification. Exemplary reaction vessels include, but are not limited to, multiwell plates, microtiter plates, reaction grid slides (e.g., AmpliGrid slides and chemically structured glass slides containing hydrophilic anchor spots, each framed by a hydrophobic ring), and PCR tubes. In certain embodiments, single cells are sorted into multiwell plates, such as 96-, 384-, 1536-, or larger multiwell plates. In some embodiments, the placement of single cells in the reaction vessel is confirmed by visual or automated inspection under a microscope. In some embodiments, the placement of single cells in the reaction vessel is confirmed by the addition of a cell-specific dye or probe. In some embodiments, single haploid cells are labeled prior to cell sorting, and confirmation of cell sorting is confirmed by detection of labeled cells. For example, in some embodiments, detection of the intensity of a signal, such as a fluorescent signal, is indicative of the number of cells per well.

[0061] In some embodiments, cells are seeded into a reaction vessel at a concentration of 1 cell per reaction vessel for cell lysis, and a nucleic acid amplification reaction is carried out in the same reaction vessel. In some embodiments, cells are seeded into a reaction vessel at a concentration of 1 cell per reaction vessel for cell lysis, and a nucleic acid amplification reaction is carried out in a different reaction vessel (i.e., the genomic DNA sample is transferred to a new reaction vessel for the nucleic acid amplification reaction).

[0062] In some embodiments, cells are individually encapsulated in microdroplets. Microdroplets generally contain a quantity of a first sample fluid in a second carrier fluid. Any technique known in the art for forming droplets may be used with the methods of the present invention. An exemplary method includes flowing a stream of sample liquid containing target material (e.g., haploid cells) across two opposing streams of flowing carrier liquid. The carrier liquid is immiscible with the sample liquid. The crossing of the sample liquid with the two opposing streams of flowing carrier liquid results in the sample liquid splitting into individual sample droplets containing the target material. The carrier liquid may be any liquid immiscible with the sample liquid. An exemplary carrier liquid is oil. In certain embodiments, the carrier liquid includes a surfactant.

[0063] In some embodiments, a microfluidic device is used to generate single-cell emulsion droplets. The microfluidic device ejects single cells in an aqueous reaction buffer into a hydrophobic oil mixture. The device can create thousands of emulsion microdroplets per minute. After the emulsion microdroplets are created, the device ejects the emulsion mixture into a trough. The mixture can be pipetted or collected into standard reaction tubes for dissolution and / or thermal cycling. In some embodiments, the microdroplets are seeded into individual reaction vessels (e.g., microtiter plates, microchips, or reaction grid slides).

[0064] In some embodiments, the microdroplets are sorted to enrich for microdroplets carrying single haploid cells. In some embodiments, the microdroplets carrying single haploid cells are sorted from empty microdroplets and / or microdroplets carrying two or more haploid cells based on the difference in light refractory properties of the microdroplets carrying single haploid cells. In some embodiments, the haploid cells are labeled with a suitable dye (e.g., Hoechst 33342) to aid in the cell sorting method. In such methods, the haploid cells are contacted with the dye for a predetermined time to allow labeling of the haploid cells. The microdroplets carrying the labeled haploid cells are then subjected to a selected cell sorting method.

[0065] Droplets having an average diameter of, less than, or greater than about, or at least about 0.001, 0.01, 0.05, 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150, 160, 180, 200, 300, 400, or 500 microns can be produced. The droplets can have an average diameter of about 0.001 to about 500, about 0.01 to about 500, about 0.1 to about 500, about 0.1 to about 100, about 0.01 to 100, or about 1 to about 100 microns. Microfluidic methods for producing emulsion droplets using microchannel cross-flow focusing or physical agitation are known to produce either monodisperse or polydisperse emulsions. The droplets may be monodisperse. Droplets can be generated such that the size of the droplets does not vary by more than plus or minus 5% of the average size of the droplets. In some cases, droplets are generated such that the size of the droplets does not vary by more than plus or minus 2% of the average size of the droplets. The droplet generator can generate a population of droplets from a single sample such that no droplets vary in size by more than plus or minus about 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of the average size of the total droplet population.

[0066] Microfluidic systems and devices have been described in a variety of contexts, typically in the context of miniaturized laboratory (e.g., clinical) analysis. Other applications have been described as well, for example, in International Patent Application Publication Nos. WO 01 / 89788, WO 2006 / 040551, WO 2006 / 040554, WO 2004 / 002627, WO 2008 / 063227, WO 2004 / 091763, WO 2005 / 021151, WO 2006 / 096571, WO 2007 / 089541, WO 2007 / 081385, and WO 2008 / 063227.

[0067] Custom microfluidic devices for single-cell analysis are routinely fabricated in academic and commercial laboratories (Kintses et al. (2010) Current Opinion in Chemical Biology 14:548-555). For example, chips may be fabricated from polydimethylsiloxane (PDMS), plastic, glass, or quartz. In some embodiments, fluids are passed through the chip by pressure or the action of a syringe pump. Single cells can even be manipulated on programmable microfluidic chips using custom dielectrophoresis devices (Hunt et al. (2008) Lab Chip 8:81-87). In one embodiment, a pressure-based PDMS chip consisting of a flow-focusing geometry fabricated with soft lithography techniques is used (Dolomite Microfluidics, Royston, UK) (Anna et al. (2003) Applied Physics Letters 82:364-366). Stock designs are typically capable of generating 10,000 water-in-oil microdroplets per second, with sizes ranging from 10 to 150 μm in diameter. In some embodiments, the hydrophobic phase will consist of a fluorinated oil containing an ammonium salt of a carboxy-perfluoropolyether, ensuring optimal conditions for molecular biology and reducing the probability of droplet fusion (Johnston et al. (1996) Science 271:624-626). To measure the periodicity of cell and droplet flow, images can be recorded at 50,000 frames per second using standard techniques such as a Phantom V7 camera or Fastec InLine (Abate et al. (2009) Lab Chip 9:2628-31).

[0068] Microfluidic systems can optimize droplet size, input cell density, chip design, and cell loading parameters to ensure that >98% of droplets contain a single cell. Three common methods for achieving this are (i) limiting dilution of the cell solution, (ii) fluorescent selection of droplets containing a single cell, and (iii) optimization of cell input periodicity. For each method, metrics for success include (i) encapsulation rate (i.e., the number of droplets containing exactly one cell), (ii) yield (i.e., the proportion of the original cell population that end up in droplets containing exactly one cell), (iii) multi-hit rate (i.e., the proportion of droplets containing more than one cell), (iv) negative rate (i.e., the proportion of droplets containing no cells), and (v) encapsulation rate per second (i.e., the number of droplets containing a single cell formed per second).

[0069] In some embodiments, single-cell emulsions are generated by limiting cell dilution: Under random conditions, the probability that a microdroplet contains k cells is given by the Poisson distribution as follows: f(k;λ)=(λ k e -λ ) / k!

[0070] where e is the natural logarithm and the expected number of occurrences in the interval is λ. Thus, if P(k=1)≈0.98, then λ≈0.04, and the cell solution must be extremely dilute so that only 3.84% of all droplets contain a single cell.

[0071] In some embodiments, a simple microfluidic chip with a droplet generation junction is used, where a stream of water flows through a 10 μm square nozzle to dispense a water-in-oil emulsion mixture into a reservoir. The emulsion mixture can then be pipetted from the reservoir and thermally cycled in a standard reaction tube, microtiter plate, microchip, or reaction grid slide. This method predictably yields high encapsulation rates and low multi-hit rates, but the encapsulation rate per second is low. A design capable of achieving a 1000 Hz filled droplet throughput has been shown to generate 10 droplets in less than 17 minutes. 6 It is possible to separate up to 100 cells.

[0072] In some embodiments, fluorescence techniques can be used to sort microdroplets with specific emission characteristics (Baroud et al. (2007) Lab Chip 7:1029-1033, Kintses et al. (2010) Current Opinion in Chemical Biology 14:548-555). In these studies, cells are stained using chemical methods. In some embodiments, autofluorescence is used to select microdroplets containing cells. Fluorescence detectors reduce the negative rate due to limiting cell dilution. The microfluidic device can also include a laser directed at a "Y" sorting junction below the cell encapsulation junction. The Y junction has a "keep" and a "waste" channel. A photomultiplier tube is used to collect the fluorescence of each droplet as it passes through the laser. A voltage difference is calibrated between an empty droplet and a droplet containing at least one cell. The device then detects droplets containing at least one cell, and electrodes at the Y-sorting junction generate an electric field gradient via dielectrophoresis (Hunt et al. (2008) Lab on a Chip 8:81-87), pushing the droplets containing the cells into the keep channel. The microfluidic device controls the multiple hit rate and fluorescent cell sorting using limiting cell dilution to reduce the negative rate.

[0073] In some embodiments, the input cell flow is matched to the droplet formation periodicity so that >98% of droplets contain single cells (Edd et al. (2008) Lab Chip 8:1262-1264, Abate et al. (2009) Lab Chip 9:2628-31). In these microfluidic devices, a dense suspension of cells is passed through a high aspect ratio channel such that the cell diameter is a large percentage of the channel width. The chip is designed with a 27 μm × 52 μm rectangular microchannel that channels cells into microdroplets at >104 / min (Edd et al. (2008) Lab Chip 8:1262-1264). Multiple input channel widths and flow rates are tested to arrive at an optimal solution.

[0074] In some embodiments, cells with different morphologies behave differently in the microchannel flow of a microfluidic device, confounding the optimization of the technique when applied to clinical biological samples. To address this issue, in some embodiments, a perpendicular electric field gradient is induced in the microchannel by dielectrophoresis. Dielectrophoresis draws cells to one side of the microchannel, creating order within the channel that is independent of cell morphology. Because this method requires substantial optimization of charge and flow rate and more complex chip and device design, it may be necessary when existing methodologies are not feasible for a particular cell type.

[0075] In some embodiments, the methods of the present invention use single cells in reaction vessels rather than emulsion droplets. Examples of such reaction vessels include 96-well plates, 0.2 mL tubes, 0.5 mL tubes, 1.5 mL tubes, 384-well plates, 1536-well plates, etc.

[0076] Preparation of genomic DNA The preparation of genomic DNA samples from haploid cells for genetic assays such as nucleic acid amplification typically involves lysing cells to expose genomic DNA.Any suitable lysis buffer can be used for the preparation of genomic DNA from cells.In certain instances, certain haploid cells, such as sperm cells, are resistant to conventional lysis techniques.Therefore, in certain embodiments, the preparation method of genomic DNA samples involves lysing cells using one or more suitable enzymes (for example, proteases such as proteinase K).

[0077] In some embodiments, haploid cells are lysed in an alkaline lysis solution (e.g., potassium hydroxide alkaline lysis solution) or a detergent solution, such as Tween 20. In some embodiments, the lysis solution includes an enzyme to aid in lysis, such as a protease (e.g., proteinase K). In some embodiments, the lysis solution also includes one or more additional components, such as a redox stabilizing reagent (e.g., dithiothreitol (DDT)), a chelating agent (e.g., EDTA), or a buffering agent.

[0078] In some embodiments, to prevent premature cell rupture when encapsulating haploid cells in microdroplets, a co-flow droplet generator is used to introduce lysis buffer during cell encapsulation.

[0079] Nucleic Acid Amplification and Detection After preparing genomic DNA, a genetic assay is performed to detect the target gene null allele. In certain embodiments of the provided method, the target gene null allele is detected by nucleic acid amplification, for example, by polymerase chain reaction (PCR). In some embodiments, a region overlapping with or including the deletion in the target gene is amplified from a genomic DNA sample derived from a single haploid cell from a subject. In samples with deletions, the target gene region is not amplified. To ensure that the genomic DNA sample is present in the reaction vessel and the conditions for nucleic acid amplification are appropriate, a reference gene is also amplified in the same amplification reaction as the target gene. Thus, the success or failure of target gene amplification is evaluated by comparing it with the amplification of the reference gene.

[0080] For any particular subject who is a silent carrier of a null allele of a target gene, approximately 50% of the haploid cells produced by the subject will have the null allele.Therefore, 50% of the haploid cells tested from the subject using the method provided herein will be unable to amplify the target gene compared to the reference gene, which indicates that the target gene has a deletion in the haploid cells.Therefore, to confirm the existence of a deletion, multiple amplification reactions are carried out (i.e., multiple haploid cells from the tested subject).In some embodiments, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000, 5000 or more amplification reactions are carried out, where each amplification reaction represents a single haploid cell.

[0081] Exemplary reaction vessels for nucleic acid amplification include, but are not limited to, multiwell plates, microtiter plates, microchips, reaction grid slides (e.g., AmpliGrid slides and chemically structured glass slides containing hydrophilic anchor spots each framed by a hydrophobic ring), and PCR tubes.

[0082] An exemplary nucleic acid amplification reaction mixture for carrying out the method includes a DNA template (e.g., a genomic DNA sample obtained from a single haploid cell), at least one oligonucleotide primer set specific for a target gene (e.g., for amplification of a target region of the target gene), at least one oligonucleotide primer set specific for a reference gene (e.g., for amplification of a target region of the reference gene), a thermostable DNA polymerase, deoxynucleoside triphosphates (dNTPs), Mg 2+ , and an appropriate buffer.

[0083] In exemplary embodiments, the amplification reaction mixture comprises about, more than, or less than 1, 5, 10, 15, 20, 30, 50, 100, or 200 mM Tris. In some embodiments, the amplification reaction mixture comprises about, more than, or less than 10, 20, 30, 40, 50, 60, 80, 100, or 200 mM potassium chloride. In some embodiments, the amplification reaction mixture comprises about 15 mM Tris and 50 mM KCl. In some embodiments, the amplification reaction mixture comprises deoxyribonucleotide triphosphate molecules, including dATP, dCTP, dGTP, and dTTP, each at a concentration of about, more than, or less than 50, 100, 200, 300, 400, 500, 600, or 700 μM. In some embodiments, magnesium chloride or magnesium acetate (MgCl) is added to the amplification reaction mixture at a concentration of about 1.0, 2.0, 3.0, 4.0, or 5.0 mM, or greater, or less. In some embodiments, the amplification reaction mixture contains MgCl at a concentration of about 3.2 mM. In some embodiments, the amplification reaction mixture contains magnesium acetate, or magnesium is used. In some embodiments, magnesium sulfate. In some embodiments, the amplification reaction mixture contains a nonspecific blocking agent, such as BSA or gelatin derived from bovine skin, present in a concentration range of about 0.1-0.9% w / v. Other potential blocking agents may include beta-lactoglobulin, casein, powdered milk, or other common blocking agents. In some cases, the preferred concentration of BSA and gelatin is about 0.1% w / v.

[0084] Exemplary polymerase enzymes for nucleic acid amplification include Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopalitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNA polymerase, Thermus eggertssonii (Teg) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, and DEEPVENT™.) DNA polymerase, Pyrococcus woosii (Pwo) DNA polymerase, Pyrococcus sp. KDD2 (KOD) DNA polymerase, Bacillus sterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bea) DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus (Tfl / Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus Examples of suitable polymerases include, but are not limited to, thermostable DNA polymerases such as B. brockianus (DYNAZYME) DNA polymerase, Methanobacterium thermoautotrophicum (Mth) DNA polymerase, Mycobacterium DNA polymerase (Mtb, Mlep), or mutants, variants, or derivatives thereof. In some embodiments, the polymerase is a hot-start polymerase, such as hot-start Taq polymerase. In some embodiments, the polymerase is a chemically modified hot-start polymerase or an antibody-modified hot-start polymerase.

[0085] Standard methods for nucleic acid amplification of nucleic acids from genomic DNA obtained from a single cell are known in the art and can be used in the methods provided herein (see, e.g., U.S. Patent Application Publication No. 2011 / 0237445).In a typical protocol, nucleic acid amplification generally involves the steps of: (a) contacting each nucleic acid strand template with four different nucleotide triphosphates and a pair of oligonucleotide primers, one for each different specific sequence to be amplified, wherein each primer of the primer pair is selected to be substantially complementary to a different strand of each specific sequence, such that the extension product synthesized from one primer, when separated from its complement, can serve as a template for synthesis of an extension product of the other primer, and the contacting is performed at a temperature that promotes hybridization of each primer to its complementary nucleic acid strand; (b) contacting each nucleic acid strand, simultaneously with or after step (a), with a thermostable DNA polymerase, such as Thermus aquaticus, that enables the combination of the nucleotide triphosphates to form primer extension products complementary to each strand of each nucleic acid; and (c) promoting the activity of the enzyme and synthesizing extension products of each primer complementary to each nucleic acid strand template for each different sequence to be amplified. (d) maintaining the mixture from step (c) at a temperature and for a time effective to separate the primer extension products from the template from which they were synthesized to produce single-stranded molecules, but not so high as to irreversibly denature the enzyme; (e) heating the mixture from step (d) at a temperature and for a time effective to promote hybridization of each primer to each single-stranded molecule produced in step (d). (f) maintaining the mixture from step (e) for a time and at an effective temperature effective to promote activity of the enzyme and to synthesize, for each different sequence to be amplified, an extension product of each primer that is complementary to each nucleic acid strand template produced in step (d), but not so high as to separate each extension product from its complementary strand template, wherein the effective time and effective temperature for steps (e) and (f) can be coincident (steps (e) and (f) performed simultaneously) or can be separate.Steps (d) through (f) may be repeated until the desired level of sequence amplification is achieved.

[0086] In some embodiments where lysed haploid cells are encapsulated in microdroplets, the amplification reaction mixture is introduced by dilution of the microdroplets by droplet fusion and / or droplet pico-injection of amplification reagents.

[0087] In some embodiments, the amplification reaction is carried out in microdroplets by performing digital PCR, such as microfluidic-based digital PCR or droplet digital PCR. In some embodiments, thermal cycling is accomplished in a single vessel (e.g., a tube, microtiter well, microchip, or reaction grid slide) containing multiple microdroplets, or as a continuous flow of microdroplets via a microfluidic channel through defined temperature zones (see, e.g., U.S. Patent Application Publication No. 2009 / 0042737).

[0088] In some cases, the target region for amplification is, exceeds, or is less than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 bases or base pairs in length. In some cases, the amplification target is about 10 to about 100, about 100 to about 200, about 100 to about 300, about 100 to about 400, about 100 to about 500, about 100 to about 600, about 100 to about 700, about 100 to about 800, about 100 to about 900, about 100 to about 1000, about 1000 to about 2000, about 1000 to about 5000, or about 1000 to about 10,000 bases or base pairs in length.

[0089] The length of the forward primer and the reverse primer can depend on the sequence of the target polynucleotide and the target locus. For example, the length and / or Tm of the forward primer and the reverse primer can be optimized. In some cases, the primer has a length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides, or more, or less. In some cases, the primers are about 15 to about 20, 15 to about 25, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 15 to about 55, about 15 to about 60, about 20 to about 25, about 20 to about 30, about 20 to about 35, about 20 to about 40, about 20 to about 45, about 20 to about 50, about 20 to about 55, or about 20 to about 60 nucleotides in length.

[0090] In some embodiments, the primers for amplification in the amplification reaction mixture can have a concentration of, greater than, or less than about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, or 2.0 μM. The primer concentration in the aqueous phase can be about 0.05 to about 2, about 0.1 to about 1.0, about 0.2 to about 1.0, about 0.3 to about 1.0, about 0.4 to about 1.0, or about 0.5 to about 1.0 μM. The primer concentration can be about 0.5 μM. The acceptable range of target nucleic acid concentration in PCR is about 1 pg to about 500 ng.

[0091] In an exemplary assay according to the methods provided herein, the nucleic acid amplification reaction includes an oligonucleotide primer pair, which is an oligonucleotide primer set for amplifying a target region of a target gene (e.g., exon 7 or a portion thereof of SMN1) and an oligonucleotide primer set specific to a reference gene. Exemplary primers for amplifying a target gene (e.g., an SMN1 target gene) and a reference gene (e.g., a CFTR reference gene) are provided. In some embodiments, for amplification of the target region of SMN1, at least one of the primers in the primer pair discriminates between SMN1 and SMN2. SMN1 and SMN2 differ by a single nucleotide difference (C / T) at position 6 in exon 7 of SMN1 and SMN2. In some embodiments, the allele-specific primer is a forward primer that discriminates between SMN1 and SMN2, with a C or T at the 3' end of the primer corresponding to position 6 in exon 7 of SMN1 and SMN2, respectively. In some embodiments, a mismatch T→G was also added at position -3 from the 3' end of both the SMN1 and SMN2 forward primers to aid in allele specificity.

[0092] In some embodiments, the forward primer for amplifying the target region of SMN1 has the sequence of SEQ ID NO: 1. In some embodiments, the reverse primer for amplifying the target region of SMN1 has the sequence of SEQ ID NO: 3. In some embodiments, the forward primer for amplifying the target region of SMN2 has the sequence of SEQ ID NO: 2. In some embodiments, the reverse primer for amplifying the target region of SMN2 has the sequence of SEQ ID NO: 3.

[0093] In some embodiments, the forward primer for amplification of the CFTR reference gene has the sequence of SEQ ID NO: 4. In some embodiments, the reverse primer for amplification of the CFTR reference gene has the sequence of SEQ ID NO: 5.

[0094] In some embodiments, one or more oligonucleotide primers in a nucleic acid amplification reaction are labeled. In some embodiments, the oligonucleotide primers are labeled with a detectable moiety, such as a radioactive moiety, a fluorescent moiety, or a dye molecule. In some embodiments, the composition comprises a dual-labeled fluorescence energy transfer (FRET) probe. In certain embodiments, at least one primer in the oligonucleotide primer set specific to the target gene and at least one primer in the oligonucleotide primer set specific to the reference gene are labeled.

[0095] In some embodiments, the presence or absence of target gene and reference gene amplification products is detected after the nucleic acid amplification reaction. Any suitable method for detecting the amplification products can be used, such as gel electrophoresis or labeled nucleic acid probes.

[0096] In some embodiments, quantitative PCR is used to monitor the amplification of the target gene and the reference gene in each nucleic acid reaction (e.g., comparative Ct method). In such methods, at least one oligonucleotide primer of each primer pair specific to the target gene or the reference gene is labeled with a different detectable moiety, which allows detection of different amplification products in the same reaction vessel.

[0097] In certain embodiments, the method further comprises determining the gene copy number in the test subject. In some embodiments, the gene copy number is determined by nucleic acid amplification from a genomic DNA sample isolated from a plurality of diploid cells or a plurality of haploid cells from the subject using quantitative PCR analysis (e.g., RT-PCR). In such methods, the copy number is determined by comparative Ct to a reference gene.

[0098] In some embodiments, one or more oligonucleotide primers in a nucleic acid amplification reaction comprise additional nucleic acid sequences to aid in the identification and / or subsequent manipulation or analysis of the amplified product. For example, in some embodiments, one or more oligonucleotide primers in a nucleic acid amplification reaction comprise a unique nucleic acid barcode. In some embodiments, one or more oligonucleotide primers in a nucleic acid amplification reaction comprise an adapter sequence for annealing of a sequencing primer for further amplification, which immobilizes the amplified product to a solid support such as a microbead. In some embodiments, one or more oligonucleotide primers are linked to a solid support such as a microbead.

[0099] Data analysis After nucleic acid amplification and detection of the amplified products, the number of samples containing each amplification product, reference gene amplification product, and / or target gene amplification product is counted. The silent carrier status is determined based on the absence of target gene amplification product in approximately 50% of the samples containing reference gene amplification product. In some embodiments, the ratio of target gene amplification product to reference gene amplification product is determined. For example, a ratio of about 0.5 of target gene amplification product to reference gene amplification product indicates a silent carrier.

[0100] For individuals who are not silent carriers of the SMN1 gene deletion and who contain two copies of the SMN1 gene, the ratio of target gene amplification product to reference gene amplification product is expected to be about 1. In some embodiments, if the ratio of target gene amplification product to reference gene amplification product significantly deviates from about 1, the sample is selected for further analysis. In some embodiments, a significant deviation of the ratio of target gene amplification product to reference gene amplification product from about 1 indicates that the individual is a silent carrier of the SMN1 null allele. Thus, in some embodiments, if the ratio of target gene amplification product to reference gene amplification product is below a threshold level, a potential silent carrier is selected. The threshold level can be determined by an appropriate statistical method, such as a single-value t-test. In some embodiments, the threshold level is 0.8 or less. In some embodiments, the threshold level is 0.75 or less.

[0101] It is understood that the methods provided herein can be performed with the aid of one or more automated devices or computer modules. For example, the steps of cell seeding, genomic DNA preparation, dispensing, mixing, removing, and / or transferring reagents to or from reaction vessels, thermal cycling for nucleic acid amplification, detection and quantification of amplification products, analysis of data, and generation of reports can be partially or fully automated with the aid of one or more automated devices or computer modules.

[0102] kit In some embodiments, kits are provided for carrying out the methods provided herein. In some embodiments, the kits include one or more reagents for carrying out amplification reactions for target genes and reference genes, and optionally instructions for use. In some embodiments, they include reaction vessels such as microtiter plates, microchips, or reaction grid slides, and / or other vessels suitable for carrying out the methods provided herein.

[0103] In some embodiments, a microtiter plate is provided that contains one or more reagents for performing amplification reactions of a target gene and a reference gene. In some embodiments, one or more reagents are lyophilized in the microtiter plate. In some embodiments, the lyophilized reagents are reconstituted in an appropriate buffer before use. For example, the lyophilized reagents are reconstituted in an appropriate buffer before addition of a genomic DNA sample. In some embodiments, the microtiter plate contains a buffer. In some embodiments, the buffer is selected from Tris, MOPS, HEPES, TAPS, bicine, tricine, TES, PIPES, and MES. In some embodiments, the buffer is Tris. In some embodiments, the microtiter plate contains a polymerase and a polymerase stabilizer, such as a non-ionic surfactant, a zwitterionic compound, a cationic ester compound, a polymer, BSA, or a polysaccharide. In some embodiments, the microtiter plate contains at least one dNTP. In some embodiments, the microtiter plate contains an oligonucleotide primer set for amplifying a reference gene, a target gene, or both a reference gene and a target gene. [Example]

[0104] [Example 1] method: Frozen human semen samples (Bioreclamation IVT) were thawed and counted using a TC20 automated cell counter (BioRad). Prior to counting, samples were incubated at 37°C for 30 minutes, vortexed for a minimum of 30 seconds, and diluted 1:1 with TE to ensure a single-cell suspension. The resulting counts were then used for genotyping and single-sperm assays. For SMN1 and SMN2 genotyping assays, DNA from sperm samples was extracted using a modified Puregene manual extraction protocol (Qiagen). For single-cell sperm assays, each sample was counted individually on a TC20 cell counter and diluted to final concentrations of 0.8 and 0.4 cells / μl.

[0105] Sperm lysis was performed in a 96-well PCR plate, in which 1 μl of diluted donor sperm sample was added to 5 μl of lysis buffer (0.1 M DDT, 10 mM EDTA, 0.4 M KOH, and 10% Roche recombinant PCR-grade proteinase K) for a total volume of 6 μl per well. Each sample had 48 replicate wells for final concentrations of 0.8 and 0.4 cells / μl. Once lysis was complete, each plate was prepared for quantitative PCR assays using a slight modification of the standard operating procedure, with a total reaction volume of 50 μl. SMN1 probe and primers (100 μM concentration) were added to TaqMan Fast Advanced Master Mix (Life Technologies) and run for 60 cycles as a comparative CT experiment on a ViiA 7 real-time PCR system. Samples were analyzed using ViiA 7 software, in which SMN1 and reference control probe-positive targets were identified and quantified. Each sperm sample was diluted to 0.8 and 0.4 cells per well and tested for SMN1 along with a reference gene to confirm the presence of single cells. Each well was counted and the values for the reference gene and SMN1 were compared.

[0106] For amplification of SMN1 and SMN2, oligonucleotide primers were designed to amplify exon 7 of each gene as described in Curet et al. (2007) Neurogenetics 8:271-278. The SMN forward primer distinguishes SMN1 from SMN2 by terminating at a nucleotide difference (C / T) at position 6 of exon 7. A mismatch T→G added at position −3 from the 3′ end of both the SMN1 and SMN2 forward primers aids in allele specificity.

[0107] -ex7F-3g: 5'-TTCCTTTATTTTCCTTACAGGGTGTC-3' (SEQ ID NO: 1)

[0108] SMN2-ex7F-3g: 5'-TTCCTTTATTTTCCTTACAGGGTGTT-3' (SEQ ID NO: 2)

[0109] SMN-ex7R: 5'-GCTGGCAGACTTACTCCTTAATTTAA-3' (SEQ ID NO: 3)

[0110] CFTR-F: 5'-TAGGAAGTCACCAAAGCAGTACAGC-3' (SEQ ID NO: 4)

[0111] CFTR-R: 5'-AGCTATTCTCATCTGCATTCCAATG-3' (SEQ ID NO: 5)

[0112] result: A total of 46 African American men were screened for SMA carrier status using the single sperm qPCR assay (Figure 2). Low-quality specimens, including low counts and contamination, resulted in discrepancies in cell counts and a significant failure of the qPCR reactions [7 / 46 (15%)]. All reliable specimens had at least two copies of the SMN1 gene, as detected by a conventional dose assay using DNA extracted from the same semen specimen (data not shown). No conventional SMA carriers (single copy of SMN1) were identified in this initial dataset.

[0113] A single sample (DS11) showed statistical variation from the expected SMN1 to reference gene ratio by 0.729 ± SD 0.66 for the mean ratio of two-copy individuals (single-valued t-test p-value = 0.0014) (Figure 3). An SMN1 to reference gene ratio of approximately 0.5 indicates a carrier sample in which half of the sperm cells are null for SMN1, carrying a 50% risk of missing the disease allele. The observed genotype of two copies of SMN1 and 50% null sperm indicates a 2+0 genotype or silent carrier.

[0114] The DS11 sample was retested by qPCR to determine the dosage of the SMN1 and SMN2 genes and plated in two complete plates (n=192 total wells) at two different dilutions in a single cell assay. The genotype of sample DS11 was confirmed to have two copies of SMN1 and two copies of SMN2. The resulting ratio of SMN1 to the reference genes was 0.589 ± SD 0.024 (single-valued t-test p-value=2.2 × 10 -4 ), which confirmed the 2+0 genotype and the initial silent carrier result (Figure 4A). The specimen was sequenced by Sanger sequencing for SMN1 and by nonspecific sequencing for SMN1 and its homolog, SMN2. Nonspecific sequencing of the two homologous genes revealed an approximately 50 / 50 (C / T) ratio at position +6 of exon 7, indicating equal numbers of copies of the two genes (Figure 4B). Specific sequencing of the SMN1 gene revealed no sequence variants under the qPCR probe or primer sites that could result in missing alleles or reduced probe affinity (Figure 4C).

[0115] The results of this study support the use of a single-cell sperm qPCR assay to identify silent SMA carriers in men, eliminating the residual risk of traditional gene dosage methods. In combination with specific sequencing of the SMN1 gene, this novel assay can identify 100% of all male SMA carriers resulting from deletions or mutations in the SMN1 locus.

[0116] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes suggested to those skilled in the art are within the spirit and scope of this application and the appended claims. The present invention includes the following embodiments. [1] A method for identifying a subject as a silent carrier of a null allele of a target gene, comprising: (a) performing a plurality of nucleic acid amplification reactions, each nucleic acid amplification reaction comprising: (i) a genomic DNA sample obtained from a single haploid cell from the subject; (ii) at least one pair of oligonucleotide primers for amplifying a target region of a target gene, the target region being absent in a null allele of the target gene; and (iii) at least one pair of oligonucleotide primers for amplifying a target region of a reference gene; (b) detecting the presence or absence of a target gene amplification product; (c) detecting the presence or absence of a reference gene amplification product; and (d) characterizing the subject as a carrier of a null allele of said target gene if the ratio of target gene amplification product to reference gene amplification product is equal to or less than a threshold level of about 0.5 to about 0.8. A method comprising: [2] The method of embodiment 1, wherein the threshold level is about 0.75. [3] The method of any one of embodiments 1 to 2, wherein the ratio of target gene amplification product to reference gene amplification product in silent carriers of the target gene null allele is about 0.5. [4] The method according to any one of embodiments 1 to 4, wherein the haploid cell is a naturally occurring gamete cell or an artificial haploid cell. [5] The method of embodiment 4, wherein the haploid cells are sperm cells. [6] The method of embodiment 4, wherein the induced haploid cells are derived from induced pluripotent stem cells (iPSCs). [7] The method of any one of embodiments 1 to 6, wherein the subject is a human subject. [8] The method of any one of embodiments 1 to 7, wherein the reference genes are selected from CFTR, GAPDH, HMBS, B2M, HPRT1, RPL13A, SDHA, TBP, UBC, YWHAZ, PRDX6, ADD1, HLA-A, RAD9A, ARHGEF7, EIF2B2, PSMD7, BCAT2, and ATP5O. [9] The method of any one of embodiments 1 to 8, wherein the target gene is SMN1.

[10] A method according to any one of embodiments 1 to 9, wherein the target gene amplification product comprises exon 7 of SMN1 or a part thereof.

[11] The method of any one of embodiments 1 to 10, wherein homozygous deletion of the target gene is associated with a disease or condition.

[12] The method of embodiment 11, wherein the disease or condition is spinal muscular atrophy (SMA).

[13] The method of any of embodiments 1 to 12, wherein at least one oligonucleotide primer for amplifying the target gene is labeled with a detectable moiety.

[14] The method of any one of embodiments 1 to 13, wherein at least one oligonucleotide primer for amplification of the reference gene is labeled with a detectable moiety.

[15] The method of embodiment 13 or embodiment 14, wherein the detectable moiety is a fluorescent moiety.

[16] The method according to any one of embodiments 1 to 15, wherein the nucleic acid amplification reaction is a polymerase chain reaction (PCR).

[17] The method described in any one of embodiments 1 to 16, wherein the nucleic acid amplification reaction is quantitative PCR.

[18] The method of any one of embodiments 1 to 17, wherein each nucleic acid amplification reaction is performed in a separate well of a multi-well plate.

[19] The method of any one of embodiments 1 to 17, wherein each haploid cell is encapsulated in a microdroplet.

[20] The method of embodiment 19, wherein the microdroplets are dispersed in a water-in-oil emulsion.

[21] The method of embodiment 19 or 20, wherein the nucleic acid amplification reaction is digital droplet PCR.

[22] The method of any one of embodiments 1 to 21, wherein the target gene amplification product is detected with a labeled nucleic acid probe specific to the target gene amplification product.

[23] The method of any one of embodiments 1 to 22, wherein the reference gene amplification product is detected with a labeled nucleic acid probe specific to the reference gene amplification product.

[24] The method described in any one of embodiments 1 to 23, further comprising determining the copy number of the target gene in diploid cells from the subject.

[25] The method of any one of embodiments 1 to 24, further comprising generating a report including an assessment of the likelihood that the subject is a silent carrier of a null allele of the target gene.

[26] The method of any one of embodiments 1 to 25, comprising preparing genomic DNA from a single haploid cell population.

[27] The step of preparing genomic DNA from a single haploid cell population comprises: (a) sorting the single haploid cell population into separate reaction vessels at a concentration of one haploid cell per reaction vessel; and (b) contacting each sorted cell with a lysis buffer to release genomic DNA from the cell; 27. The method of embodiment 26, comprising:

[28] The method of embodiment 27, wherein the lysis buffer comprises an enzyme.

[29] The method of embodiment 28, wherein the lysis buffer comprises proteinase K.

[30] A method according to any one of embodiments 27 to 29, wherein the preparation of the genomic DNA and the nucleic acid amplification reaction are carried out in the same reaction vessel.

[31] A method according to any one of embodiments 27 to 30, wherein the preparation of the genomic DNA and the nucleic acid amplification reaction are carried out in separate reaction vessels.

[32] The method of any one of embodiments 27 to 31, wherein the reaction vessel is a microtiter plate.

[33] The step of preparing genomic DNA from a single haploid cell population comprises: (a) encapsulating a single population of haploid cells into aqueous microdroplets at a concentration of one haploid cell per reaction vessel; and (b) contacting each single haploid cell in each microdroplet with a lysis buffer to release genomic DNA from the cell. 27. The method of embodiment 26, comprising:

[34] (a) a pair of oligonucleotide primers for amplifying a target region of the SMN1 gene, wherein the target region is deleted in a target gene null allele of an SMN1 silent carrier; and (b) an oligonucleotide primer pair for amplification of a target region of a reference gene that is not deleted in SMN1 silent carriers; and (c) one or more reagents for performing a nucleic acid amplification reaction Kit including:

[35] The kit of embodiment 34, comprising nucleotide triphosphates, a thermostable polymerase, and / or a suitable buffer.

[36] The kit of embodiment 34, wherein the reference genes are selected from CFTR, GAPDH, HMBS, B2M, HPRT1, RPL13A, SDHA, TBP, UBC, YWHAZ, PRDX6, ADD1, HLA-A, RAD9A, ARHGEF7, EIF2B2, PSMD7, BCAT2, and ATP5O.

[37] The kit of embodiment 34, wherein the target gene amplification product comprises exon 7 of SMN1 or a portion thereof.

[0117] The present disclosure includes the following sequence information: SEQUENCE LISTING <110> ATHENA DIAGNOSTICS, INC. <120> METHODS TO DETECT A SILENT CARRIER GENOTYPE <130> PA24-229 <140> JP 2024-075545 <141> 2015-11-13 <150> US 62 / 080,047 <151> 2014-11-14 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 1 ttcctttatt ttccttacag ggtgtc 26 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 2 ttcctttatt ttccttacag ggtgtt 26 <210> 3 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 3 gctggcagac ttactcctta atttaa 26 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 4 taggaagtca ccaaagcagt acagc 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic primer <400> 5 agctattctc atctgcattc caatg 25 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 6 tacagggttt yagacaaaat c 21 <210> 7 <211> 104 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 7 tttttttaac ttcctttatt ttccttacag ggtttcagac aaaatcaaaa agaaggaagg 60 tgctcacatt ccttaaatta aggagtaagt ctgccagcat tatg 104

Claims

1. A method for identifying a subject as a silent carrier of a null allele of a target gene, comprising: (a) performing a plurality of quantitative nucleic acid amplification reactions, each of which comprises: (i) a genomic DNA sample obtained from a single cell from a subject suspected of being a silent carrier of a null allele of a target gene; (ii) at least one pair of oligonucleotide primers for amplifying a target region of a target gene that is not present in a null allele of the target gene, wherein one oligonucleotide primer of the pair of oligonucleotide primers comprises a unique nucleic acid barcode; and (iii) at least one pair of oligonucleotide primers for amplification of a target region of a reference gene; (b) determining the amount of the target gene amplification product; (c) determining the amount of the reference gene amplification product; (d) determining the ratio of the amount of the target gene amplification product to the amount of the reference gene amplification product; and (e) characterizing the subject as a silent carrier of the target gene null allele based on the ratio of the target gene amplification product to the reference gene amplification product. for use in a method for identifying said subject as a silent carrier of a null allele of a target gene, comprising: (A) sorting cells into separate reaction vessels at a concentration of one cell per reaction vessel; and (B) contacting each sorted cell with a lysis buffer to release genomic DNA from the cell; A method for preparing a sample from a subject suspected of being a silent carrier of a null allele of a target gene, comprising:

2. 2. The method of claim 1, wherein the subject is suspected of having a deletion of the SMN1 gene on one chromosome 5 homolog and two or more copies of the SMN1 gene on another chromosome 5 homolog.

3. The method of claim 1 , wherein the cells are haploid cells.

4. The method of claim 1 , wherein the cell is a diploid cell.

5. The method of claim 4 , wherein the cells are blood cells.

6. The method of claim 1 , wherein the lysis buffer comprises an enzyme.

7. The method of claim 6 , wherein the lysis buffer contains proteinase K.

8. Preparing genomic DNA from a single haploid cell (a) encapsulating single haploid cells in aqueous microdroplets at a concentration of one cell per reaction vessel; and (b) contacting each single haploid cell in each microdroplet with a lysis buffer to release genomic DNA from the cell; The method of claim 1 , comprising:

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