METHODS TO CAPTURE AND SEQUENCE LARGE FRAGMENTS OF DNA AND DIAGNOSTIC METHODS FOR NEUROMUSCULAR DISEASE
First Claim
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1. A method of sequencing a large fragment of DNA, the method comprising:
- a) isolating genomic DNA from a biological sample;
b) hybridizing the genomic DNA with a set of probes to form genomic DNA-probe complexes, wherein the set of probes targets sequences across the large fragment of DNA at intervals;
c) purifying the genomic DNA from the complexes with affinity chromatography;
d) shearing the genomic DNA to produce small fragments of DNA, wherein the small fragments of DNA comprise coding and non-coding sequences from the large fragment of DNA; and
e) sequencing the small fragments of DNA with Next Generation Sequencing (NGS) to obtain the sequence of the large fragment of DNA.
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Abstract
The present invention provides methods of sequencing a large fragment of DNA by hybridizing a set of specifically designed probes to the DNA, shearing the DNA, and sequencing the DNA with Next Generation Sequencing. The probes are designed to target genes of interest at intervals to allow the capture of relatively large DNA fragments. The present invention also provides methods of diagnosing a neuromuscular disease (NMD) comprising detecting mutations in one or more of SCML2, CHRND, OFD1, DYNC1H1, COL6A3, EMD, ARHGAP4, FLNA, MID1IP1, MID1, and CFP.
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Citations
22 Claims
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1. A method of sequencing a large fragment of DNA, the method comprising:
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a) isolating genomic DNA from a biological sample; b) hybridizing the genomic DNA with a set of probes to form genomic DNA-probe complexes, wherein the set of probes targets sequences across the large fragment of DNA at intervals; c) purifying the genomic DNA from the complexes with affinity chromatography; d) shearing the genomic DNA to produce small fragments of DNA, wherein the small fragments of DNA comprise coding and non-coding sequences from the large fragment of DNA; and e) sequencing the small fragments of DNA with Next Generation Sequencing (NGS) to obtain the sequence of the large fragment of DNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)
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15. A method of sequencing a large fragment of DNA, the method comprising:
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a) isolating genomic DNA from a biological sample; b) hybridizing the genomic DNA with a first set of probes to form genomic DNA-probe complexes with a portion of the genomic DNA encoding a pseudogene, wherein the set of probes targets sequences across the pseudogene at intervals; c) removing the portion of the genomic DNA encoding the pseudogene with affinity chromatography; d) hybridizing the genomic DNA with a second set of probes to form genomic DNA-probe complexes, wherein the second set of probes targets sequences across the large fragment of DNA at intervals; e) purifying the genomic DNA from the complexes with affinity chromatography; f) shearing the genomic DNA to produce small fragments of DNA, wherein the small fragments of DNA comprise coding and non-coding sequences from the large fragment of DNA; and g) sequencing the small fragments of DNA with Next Generation Sequencing (NGS) to obtain the sequence of the large fragment of DNA. - View Dependent Claims (16, 17, 18, 19, 20)
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21. A method of diagnosing a neuromuscular disease (NMD) in a subject, the method comprising:
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a) obtaining a biological sample from the subject; b) isolating genomic DNA from the biological sample; c) sequencing in the genomic DNA at least one gene selected from the group consisting of SCML2, CHRND, OFD1, DYNC1H1, COL6A3, EMD, ARHGAP4, FLNA, MID1IP1, MID1, and CFP; and d) diagnosing NMD in the subject if there is a mutation in the at least one gene. - View Dependent Claims (22)
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Specification