Patent Application
20080090234
The present invention provides methods of diagnosing a primary immunodeficiency, particularly a TNFRSF6-related syndrome such as, but not limited to, Autoimmune Lymphoproliferative Syndrome (ALPS). The invention provides methods of amplifying a region or regions of the TNFRSF6 gene, and methods of determining the nucleotide sequence of a region or regions of the TNFRSF6 gene. Compositions of the invention include isolated nucleic acid molecules and oligonucleotide pairs useful in amplifying a region or regions of the TNFRSF6 gene, a TNFRSF6 oligonucleotide pair library, as well as kits comprising isolated nucleic acid molecules of the invention. The invention relates to methods of efficiently amplifying multiple regions of the TNFRSF6 gene. The invention allows direct sequencing of the entire coding region and intron/exon boundaries of the TNFRSF6 gene.
TNFRSF6 related syndromes are primary immunodeficiencies characterized by defective function of the Fas system, autoimmunities, chronic non-malignant lymphadenopathy, splenomegaly, and high levels of circulating αβ+CD4−-CD8− lymphocytes. TNFRSF6 related syndromes include, but are not limited to, ALPS, Canale-Smith syndrome, ALPS Ia, splenomegaly, hepatomegaly, immune cytopenias, immune thrombocytopenia, autoimmune hemolytic anemia, neutropenia, immune hepatitis, arthritis, insulin-dependent diabetes mellitus, thyroiditis, rashes, atopy, dyserythropoietic anemia, Guillian-Barre syndrome, immune mediated thrombocytopenic purpura, glomerulonephritis, primary biliary cirrhosis, urticarial rash, lymphoma, leukemia, myeloma, and other syndromes discussed above herein. See for example U.S. Pat. No. 6,972,323, herein incorporated by reference in its entirety. TNFRSF6 related syndromes have been difficult to diagnose clinically. The symptoms associated with TNFRSF6 related syndromes include, but are not limited to, defective lymphocyte homeostasis, lymphadenopathy, (hepato)splenomegaly with or without hypersplenism, autoimmunity, autoimmune cytopenias, life-long increased risk of lymphoma, defective Fas-mediated apoptosis, chronic non-malignant lymphoproliferation, elevated αβ+CD4−-CD8− T cell levels in peripheral blood and/or in tissue specimens, expansion of the red pulp in splenectomized patients, adenopathy, reactive follicular hyperplasia, plasmocytosis, parafollicular DNT expansion, hepatomegaly, steroid responsive recurrent fevers, hypergammaglobulinemia, anemia, thyroiditis, eczema, arthritis, and asthma, among others.
Compositions of the invention include isolated nucleic acid molecules having the nucleotide sequences set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16, or a fragment or variant thereof. Additional compositions of the invention include isolated nucleic acid molecules consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the TNFRSF6-targetting segment is at the 3′ end of the molecule. By “TNFRSF6-targetting segment” is intended a segment of a nucleic acid molecule having a nucleotide sequence that hybridizes to the TNFRSF6 gene having the nucleotide sequence set forth in SEQ ID NO:19. TNFRSF6-targetting segments of particular interest have a nucleotide sequence set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16, or a fragment or variant thereof. The nucleic acid molecules of the invention anneal to the TNFRSF6 gene (SEQ ID NO:19). The invention encompasses isolated or substantially purified nucleic acid compositions. An “isolated” or substantially “purified” nucleic acid molecule, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques or substantially free of chemical precursors or other chemicals when chemically synthesized.
By fragments or variants thereof is intended isolated nucleic acid molecules or TNFRSF6-targetting segments of isolated nucleic acid molecules having a nucleotide sequence that differs by one nucleotide alteration from that set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16, 17, 18, 19, 21, or a nucleotide sequence that hybridizes under stringent conditions to a complement of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. A fragment or variant that differs by one nucleotide alteration from a nucleotide sequence set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 differs from that nucleotide sequence by the addition, insertion, deletion, removal, subtraction, or substitution of one nucleotide. Fragments or variants include isolated nucleic acid molecules or TNFRSF6 targetting segments having a nucleotide sequence that hybridizes under stringent conditions to a complement of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.
By “generic segment” is intended a segment of an isolated nucleic acid molecule varying in length from 1 to 100 nucleotides, preferably 1-51 nucleotides, more preferably 1-30 nucleotides, yet more preferably 1-20 nucleotides. In an embodiment the a portion or all of the nucleotide sequence of the generic segment facilitates determining the sequence of amplified DNA obtained by enzymatic amplification performed with the isolated nucleic acid molecule of interest. In an aspect of the invention, the nucleotide sequence of the generic segment comprises that of a universal sequencing primer. By “universal sequencing primer” is intended a nucleic acid molecule suitable for use in determining the nucleotide sequence of a target sequence; often these sequences anneal to regions commonly found within commercially available plasmids. Universal sequencing primers are known in the art and include, but are not limited to, M13 forward (SEQ ID NO:17), M13 reverse (SEQ ID NO:18), M13f(−41), M13f(−21), M13r (−27), M13r (−48), SP6, T7 promoter, T7 terminator, T3, pBluescript KS, pBluescript SK, XPress forward, TrcHis Forward, TrcHis Reverse, RV3, RV4, GL1, GL2, pGEX5′, and pGEX3′. Any universal sequencing primer known in the art can be used in the compositions and methods of the invention. In an embodiment an isolated nucleic acid molecule of the invention consists of a generic segment and a TNFRSF6-targetting segment and the nucleotide sequence of the generic segment is a M13 universal sequencing primer set forth in SEQ ID NO:17 or SEQ ID NO:18.
By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1 × to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA—DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
Compositions of the invention include oligonucleotide pairs. An oligonucleotide pair of the invention consists of a first isolated nucleic acid molecule of the invention and a second isolated nucleic acid molecule of the invention with different nucleotide sequences. An oligonucleotide pair of the invention is suitable for use as a primer pair or primer set in a PCR reaction. In an embodiment, an oligonucleotide pair comprises a first nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, or a fragment or variant thereof and a second nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, or a fragment or variant thereof. In an embodiment, an oligonucleotide pair comprises a first nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:1 or a fragment or variant thereof and a second nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:2 or a fragment or variant thereof. In an embodiment, an oligonucleotide pair comprises a first nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:3 or a fragment or variant thereof and a second nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:4 or a fragment or variant thereof. In an embodiment, an oligonucleotide pair comprises a first nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:5 or a fragment or variant thereof and a second nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:6 or a fragment or variant thereof. In an embodiment, an oligonucleotide pair comprises a first nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:7 or a fragment or variant thereof and a second nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:8 or a fragment or variant thereof. In an embodiment, an oligonucleotide pair comprises a first nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:9 and a second nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:10 or a fragment or variant thereof. In an embodiment, an oligonucleotide pair comprises a first nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:11 or a fragment or variant thereof and a second nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:12 or a fragment or variant thereof. In an embodiment, an oligonucleotide pair comprises a first nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:13 or a fragment or variant thereof and a second nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:14 or a fragment or variant thereof. In an embodiment, an oligonucleotide pair comprises a first nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:15 or a fragment or variant thereof and a second nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the TNFRSF6-targetting segment is set forth in SEQ ID NO:16 or a fragment or variant thereof. Additional oligonucleotide pairs comprising the isolated nucleic acid molecules of the invention are encompassed by the invention. The first and second nucleic acid molecules of an oligonucleotide pair anneal to opposite strands of the TNFRSF6 gene such that they allow amplification of a region of the TNFRSF6 gene bracketed by the primer pair.
In an embodiment of the invention an oligonucleotide pair of the invention comprises a first isolated nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the generic region is set forth in SEQ ID NO:17. In a further embodiment of the invention, the nucleotide sequence of the generic segment of a first isolated nucleic acid molecule is set forth in SEQ ID NO:17 and the sequence of the adjacent TNFRSF6-targetting segment is set forth in SEQ ID NO:1, 3, 5, 7, 9, 11, 13, or 15, or fragments or variants thereof. In an embodiment of the invention an oligonucleotide pair of the invention comprises a second isolated nucleic acid molecule consisting of a generic segment adjacent to a TNFRSF6-targetting segment wherein the nucleotide sequence of the generic region is set forth in SEQ ID NO:18. In a further embodiment of the invention, the nucleotide sequence of the generic segment of a second isolated nucleic acid molecule is set forth in SEQ ID NO:18 and the sequence of the adjacent TNFRSF6-targetting segment is set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, or 16, or fragments or variants thereof.
By “oligonucleotide pair library” is intended a molecular library consisting of a plurality of oligonucleotide pairs of predetermined sequence. By “TNFRSF6 oligonucleotide pair library” is intended an oligonucleotide pair library consisting of oligonucleotide pairs capable of amplifying a region of the TNFRSF6 gene set forth in SEQ ID NO:19.
By “amplification” is intended an increase in the amount of nucleic acid molecules in a sample. Amplifying DNA increases the amount of acid precipitable nucleic acid molecules. The amount of acid precipitable material increases by 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, or more. Methods of quantifying nucleic acid molecules are known in the art and include, but are not limited to, UV absorption spectra, radiolabel incorporation, agarose gel electrophoresis, and ethidium bromide staining. See, for example, Ausubel et al., eds. (2003)Current Protocols in Molecular Biology, (John Wiley & Sons, New York) and Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y., herein incorporated by reference.
Enzymatic amplification is the process of using enzymes to perform amplification. A common type of enzymatic amplification is the polymerase chain reaction (PCR). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like. Known methods of PCR include, but are not limited to, methods using DNA polymerases from extremophiles and engineered DNA polymerases. It is recognized that it is preferable to use high fidelity PCR reaction conditions in the methods of the invention. See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).
Methods of performing enzymatic amplification, particularly PCR, are known in the art and discussed elsewhere herein. Known PCR methods include but are not limited to a series of incubation periods or cycles at predetermined temperatures. Various methods of controlling the duration and temperature of each incubation period are known in the art and include, but are not limited to, the use of thermocyclers and transfer between baths at predetermined temperatures. It is envisioned that any means of controlling the PCR reaction incubation conditions may be used in the practice of the invention. In an embodiment a thermocycler is used to regulate the PCR incubation conditions of the enzymatic amplification. By “incubation conditions” is intended the temperature and duration of each incubation period in the enzymatic amplification. In an embodiment the invention provides oligonucleotide pairs that will amplify multiple regions of the TNFRSF6 gene in multiple reactions that are performed under similar incubation conditions. By using similar incubation conditions multiple enzymatic amplification reactions can be performed in one thermocycle chamber simultaneously. The invention reduces the thermocycler resources required for enzymatic amplification of the TNFRSF6 gene. Further, by amplifying the exons and exon-intron boundaries of TNFRSF6 with only eight oligonucleotide pairs the compositions and methods of the invention facilitate automated amplification and sequencing of the gene.
In PCR, typically multiple cycles of a series of incubation periods are performed. Often a single incubation period precedes the cycled incubation period series. The cycle incubation conditions of the incubation period series are designed to facilitate denaturation of the template DNA, annealing of the primer to the template DNA, and extension of the template bound primer. Various factors such as, but not limited to, the primer nucleotide sequence, the template nucleotide sequence, and the type of DNA polymerase can be used to predict suitable temperatures and durations of the incubation periods. An example of incubation conditions suitable for use with the oligonucleotide pairs of the invention is described elsewhere herein. It is recognized that multiple suitable incubation temperatures and durations exist and that the invention is not limited to the precise incubation descriptions described elsewhere herein, particularly with regards to the denaturing incubation temperature and the extension incubation temperature.
The compositions of the invention allow for simultaneous amplification of all TNFRSF6 exons and exon-intron boundaries. The simultaneous amplification requires a carefully controlled temperature for the annealing incubation. The temperature for the annealing incubations of the methods of the invention ranges from 54° C. to 57.9° C., particularly 55° C. to 57.9° C., more particularly 56° C. to 57.5° C., yet more particularly 56.5° C. to 57.5° C. It is recognized that an annealing incubation temperature of 57° C. provides adequate yields of all eight amplicons. It is further recognized that as the primary annealing temperature is varied from 57° C., the ability of the methods of the invention to amplify all TNFRSF6 exons and exon-intron boundaries diminishes. It is also recognized that transitions from the denaturing incubation temperature to the annealing incubation temperature or from the annealing incubation temperature to the extension incubation temperature are not instantaneous and that the enzymatic amplification reaction mixture will pass through other temperature ranges as the incubation temperature shifts from the denaturing incubation temperature to the annealing incubation temperature and from the annealing incubation temperature to the extension temperature or to the storage temperature.
The compositions of the invention allow for simultaneous amplification of all TNFRSF6 exons and exon-intron boundaries in eight amplification reactions that can be performed simultaneously in the same incubation chamber or thermocycler. The methods and compositions of the invention were designed so that eight or fewer amplification reactions can be performed simultaneously. The use of eight or fewer oligonucleotide pairs and thus eight or fewer amplification reactions facilitates high through-put screening, automation, and even robotic use as many of the commercially available reagents such as, but not limited to, plasticware, thermocyclers, multiple pipettors, and robots are currently optimized to work with sets comprising a multiple of eight. These commercially available tools include but are not limited to, 8 well plastic tube strips, 96-well plastic reaction trays, 8-, 16-, 24-, 48-, or 96-well thermocycler devices, 96 well formats for PCR product purification and other molecular biology products. The use of this format also facilitates automated sequencing.
The human TNFRSF6 gene is located on the long arm of chromosome 10 at q24.1. The human genomic sequence (SEQ ID NO:19) spans 28339 nucleotides in 9 exons. The TNFRSF6 family of proteins appears to be involved in Fas mediated apoptosis. Mutations in the TNFRSF6 gene result in ALPS which is characterized by chronic non-malignant lymphadenopathy, splenomegaly, elevated αβCD4−-CD8− T cell levels and defective Fas-mediated apoptosis. Considerable clinical variation exists among ALPS patients, and mutation detection is considered essential for accurate diagnosis and prognostic determinations. The oligonucleotide pairs of the invention allow amplification of the exons and exon-intron boundaries of the TNFRSF6 gene.
By “region” is intended a portion of the entire nucleotide sequence of interest, such as the genomic TNFRSF6 gene. A region or fragment of the entire nucleotide sequence of interest, such as the genomic TNFRSF6 gene may range from 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 349, 350, 360, 370, 380, 390, 400, 410, 420, 421, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 604, 610, 620, 630, 640, 650, 660, 670, 680, 690, 698, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 834, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1611, 1650, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 10000, 10100, 10200, 10300, 10400, 10500, 10600, 10700, 10800, 10900, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 20100, 20200, 20300, 20400, 20500, 20600, 20700, 20800, 20900, 21000, 21100, 21200, 21300, 21400, 21500, 21600, 21700, 21800, 21900, 22000, 22100, 22200, 22300, 22400, 22500, 22600, 22700, 22800, 22900, 23000, 24000, 24500, 25000, 25500, 26000, 26500, 27000, 27500, 28000, 28, 339 nucleotides, or up to the total number of nucleotides on chromosome 10 q24.1. Such a region contains nucleotide sequence from one or more TNFRSF6 gene exons, one or more TNFRSF6 gene introns, or the regulatory control region operably linked to the TNFRSF6 gene. The oligonucleotide pairs of the invention amplify various regions of the TNFRSF6 gene (See
By “biological sample” is intended a sample collected from a subject including, but not limited to, tissues, cells, mucosa, fluid, scrapings, hairs, cell lysates, and secretions. Biological samples such as blood samples can be obtained by any method known to one skilled in the art.
The invention further provides methods of determining the nucleotide sequence of a region or regions of the TNFRSF6 gene. The method involves obtaining a biological sample from a human subject and performing enzymatic amplification of a region of the TNFRSF6 gene using at least one oligonucleotide pair of the invention, providing amplified DNA of a region of the TNFRSF6 gene, and determining the nucleotide sequence of the amplified DNA. It is recognized that an embodiment of the invention involves obtaining a biological sample from a human subject, performing enzymatic amplification of a region of the TNFRSF6 gene, providing amplified DNA of a region of the TNFRSF6 gene, and using microarray sequence analysis to determine the nucleotide sequence of a region or regions of the TNFRSF6 gene.
By “amplified DNA” is intended the product of enzymatic amplification.
Any method of DNA sequencing known in the art can be used in the methods of the invention. Methods of sequencing DNA are known in the art and are described in Graham & Hill eds. (2001) DNA Sequencing Protocols (Humana Press, Totowa N.J.), Kieleczawa ed (2004) DNA Sequencing: Optimizing the Process and Analysis (Jones & Bartlett Publishers, Ontario), Ausubel et al., eds. (2003) Current Protocols in Molecular Biology, (John Wiley & Sons, New York) and Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (3d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y., herein incorporated by reference in their entirety). Any method of DNA sequencing known in the art that utilizes a resequencing microarray can be used in the methods of the invention. Methods of microarray sequencing DNA are known in the art and are described in Warrington et al. (2002) Hum Mutat 19:402-409 and Cutler et al. (2001) Genome Res 11: 1913-1925, herein incorporated by reference in their entirety.
A method of the invention involves the step of comparing the nucleotide sequence of a region of a subject's TNFRSF6 gene with a standard sequence profile. Such comparison may be performed by any means known in the art including, but not limited to, manually, electronically, and automatically. It is recognized that the comparison may be performed by a person or machine.
By “standard sequence profile” is intended a listing of the consensus nucleotide sequence of at least one nucleotide sequence of interest. A standard sequence profile may contain additional information including but not limited to, a correlation of at least one nucleotide sequence variation with a disease, syndrome, prognosis, or treatment protocol. A standard sequence profile may exist in printed or electronic form, a remotely accessible form, in a database, or in any other means known in the art. In an embodiment a standard sequence profile is a TNFRSF6 standard sequence profile. For instance, an exemplary TNFRSF6 standard sequence profile correlates certain nucleotide alterations in the TNFRSF6 gene with a TNFRSF6 related syndrome.
In an embodiment, the invention provides kits for performing the methods of the invention. Such kits comprise an isolated nucleic acid molecule of the invention, particularly multiple isolated nucleic acid molecules of the invention, and an oligonucleotide pair of the invention, particularly multiple oligonucleotide pairs of the invention, yet more particularly eight oligonucleotide pairs of the invention. A kit of the invention may further comprise a description of an enzymatic amplification protocol suitable for use with an oligonucleotide pair of the invention, particularly a description of an enzymatic amplification protocol suitable for use with multiple oligonucleotide pairs of the invention, more particularly a description of an enzymatic amplification protocol suitable for use with eight oligonucleotide pairs of the invention. Additionally a kit of the invention may comprise a listing of the consensus TNFRSF6 gene nucleotide sequence in paper or electronic form or a means of accessing a consensus TNFRSF6 gene nucleotide sequence. A kit of the invention may comprise a resequencing microarray chip comprising segments of the TNFRSF6 gene nucleotide sequence.
A kit of the invention may comprise an enzymatic amplification reaction component. By “reaction component” is intended any substance that facilitates the indicated reaction. Reaction components that facilitate the reaction may or may not participate in the chemical processes of the reaction. Reaction components include, but are not limited to, vessels, such as microfuge tubes and multiwell plates; measuring devices, such as micropipette tips and capillary tubes; filters; separation devices such as microfuge tube filter inserts, vacuum apparati, purification resins, magnetic beads, and columns; reagents; compounds; solutions; molecules; buffers; inhibitors; chelating agents; ions; terminators; stabilizers; precipitants; solubilizers; acids; bases; salts; reducing agents; oxidizing agents; enzymes; catalysts; and denaturants. In an embodiment of the invention, concentrated reaction components are provided in kits of the invention. The concentration of the reaction components provided in a kit of the invention may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more fold concentrated than the desired concentration of the component in the reaction. Reagents and reaction level concentrations of various reagents are discussed elsewhere herein. In an embodiment a kit of the invention may comprise eight enzymatic amplification reaction mixtures each containing an oligonucleotide pair of the invention for use with a subject's DNA.
The following examples are offered by way of illustration and not limitation.
Puregene DNA isolation kit (Gentra Systems, Minneapolis, Minn., USA) was used for genomic DNA isolation from blood and other tissues. All reagents were provided by the kit. Whole blood was collected from a patient. The blood was collected in EDTA tubes to prevent the blood from clotting and to reduce DNA degradation. Fresh blood samples were stored at 2-8° C. for up to 7 days. In some instances whole blood was frozen immediately and stored frozen until use.
Enucleated red blood cells were removed from the whole blood sample by lysis. Red blood cell lysis solution (9 ml) was placed in a 15 ml tube. Two to three ml of whole blood were added to the red blood cell lysis solution. The tube contents were mixed by inversion and incubated for 10 minutes at room temperature. The tube was centrifuged for 10 minutes at 2,400 rpm.
The supernatant was removed leaving a visible white pellet containing white blood cells and residual fluid. The pellet was resuspended in the residual fluid by vigorous vortexing.
Cell lysis solution (3 ml) was added to the resuspended cells. The tube was inverted 15-20 times. When cell clumps remained, the tubes were placed in a 37° C. water bath overnight. If cell clumps persisted, 15 μl proteinase K was added and the tube was incubated at 55° C. for one hour to overnight. Samples were stored in cell lysis solution for up to 18 months at room temperature.
Proteins were precipitated by the addition of protein precipitation solution (1 ml). After addition of the protein precipitation solution, the solutions were mixed by vortexing. Proteins were pelleted by centrifugation at 2,400 rpm for 10 minutes. The supernatant (containing the DNA) was transferred into a 15 ml tube containing 3 ml of isopropanol. The solutions were mixed by gentle inversion approximately 50 times. The tubes were spun at 2,400 r.p.m. for three minutes. The pellet size was observed and used to determine the appropriate amount of DNA hydration solution.
The supernatant was removed and the pellet was washed with 3 ml of 70% ethanol. The tubes were spun at 2,400 r.p.m. for 1 minute. The ethanol was removed. The pellet was resuspended in 1 ml 70% ethanol and transferred to a microfuge tube. The microfuge tube was centrifuged for 2 minutes. The supernatant was removed and the pellet was allowed to air dry for 10 minutes.
DNA Hydration Solution was added to the DNA pellet. The DNA pellets were rehydrated overnight at room temperature or by incubation at 65° C. for one hour. The resuspended DNA was stored at 4° C. DNA concentrations were determined by spectrophotometric analysis of a diluted aliquot of the purified DNA.
Oligonucleotides having a generic segment set forth in either SEQ ID NO:17 or SEQ ID NO:18 and a TNFRSF6 targeting segment having the nucleotide sequences set forth in SEQ ID NOS:1-16 were designed. The oligonucleotides were synthesized by a commercial source. The synthetic oligonucleotides were resuspended at a concentration of 100 μM.
PCR amplification is a process sensitive to contamination. Therefore PCR appropriate procedures were used to minimize the risks of contamination.
Master mixes for the PCR reactions were prepared. Master mixes contained 1.5 mM MgCl2, 1 X PCR buffer (Life Technologies Inc. Rockville, Md., USA), 0.2 mM dNTPs, 1.5 units Taq polymerase, 0.4 μM 5′ primer, and 0.4 μM 3′, primer. The master mix was prepared for the desired number of reactions plus two. Separate master mixes for each oligonucleotide pair were prepared.
PCR tubes were placed on ice. Aliquots of the master mix were transferred into the PCR tubes. Template DNA at 0.5 μg/reaction was added to each tube. The reaction tubes were placed in a thermocycler and the power was turned on. The PCR incubation conditions for the incubation periods were: 95° C., for 5 minutes; 35 cycles of 95° C., for 45 seconds, 57° C., for 45 seconds, and 72° C. for 1 minute, 45 seconds; and one incubation at 72° C. for 10 minutes. Following the 10 minute incubation at 72° C., the reactions were stored at 4° C. PCR product quality was assessed by agarose gel electrophoresis of an aliquot of the PCR reaction. All eight oligonucleotide pairs anneal at 57° C.
The amplified products of the PCR reaction were purified using the Qiaquick PCR Purification kit.
Purified amplified DNA was obtained using the above described processes. The M13F (SEQ ID NO:17) and M13R (SEQ ID NO:18) universal sequencing primers were diluted to 3.3 μM.
Sequencing reaction mixtures were prepared. Each sequencing reaction contained 7 μl distilled, deionized H2O. Two μl of the appropriate primer was added to each sequencing reaction tube. Three μl of purified PCR product obtained according to the methods described elsewhere above were added to each sequencing reaction tube. The samples were sequenced by a sequencing core facility (CHMC Sequencing Core). When the sequence data was obtained, the sequence information was compared with the TNFRSF6 gene consensus nucleotide sequence. The TNFRSF6 gene consensus nucleotide sequence (SEQ ID NO:19) is available in the NCBI database (AY450925) as is the human Fas antigen nucleotide sequence (SEQ ID NO:20; M67454). Discrepancies between the patient's nucleotide sequence and the consensus sequence of the TNFRSF6 gene indicate a mutation.
All publications, patents, and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications, patents, and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually incorporated by reference.
Having described the invention with reference to the exemplary embodiments, it is to be understood that it is not intended that any limitations or elements describing the exemplary embodiment set forth herein are to be incorporated into the meanings of the patent claims unless such limitations or elements are explicitly listed in the claims. Likewise, it is to be understood that it is not necessary to meet any or all of the identified advantages or objects of the invention disclosed herein in order to fall within the scope of any claims, since the invention is defined by the claims and since inherent and/or unforeseen advantages of the present invention may exist even though they may not be explicitly discussed herein.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
