Method of Targeted and Comprehensive Sequencing Using High-Density Oligonucleotide Array
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Abstract
A method for targeted and comprehensive sequencing using high-density oligonucleotide array, comprising the steps of hybridizing nucleic acid from an investigative species with high-density oligonucleotide arrays of a related species, identifying the oligonucleotide probes that generate high hybridization signals, using the probes sequences to make PCR primers, amplifying heterologous genes by PCR with the gene specific PCR primers and an anchoring primer, and sequencing the PCR products.
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Citations
31 Claims
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1-10. -10. (canceled)
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11. A method of selecting a primer suitable for PCR amplification of a heterologous nucleic acid comprising the steps of:
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a) providing a nucleic acid corresponding to an expressed sequence from a species of interest; b) hybridizing the nucleic acid to a microarray of one or more oligonucleotides derived from expressed sequences of a species different from the species of interest; c) detecting hybridization of the nucleic acid to the one or more oligonucleotides on the microarray; d) identifying and selecting those oligonucleotides which hybridize to the nucleic acid at a level suitable to act as PCR primers. - View Dependent Claims (12, 13, 14, 15, 17, 18, 19)
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16. The method of step 15, further comprising the step of fragmenting said cRNA.
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20. A method for PCR amplification of a heterologous nucleic acid comprising the steps of:
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a) providing a nucleic acid corresponding to an expressed gene sequence from a species of interest; b) hybridizing the nucleic acid to a microarray of two or more oligonucleotides derived from expressed gene sequences of a species different from the species of interest; c) detecting hybridization of the nucleic acid to the two or more oligonucleotides on the micro array; d) identifying and selecting those oligonucleotides which have hybridized to the nucleic acid to levels suitable to act as PCR primers; and e) amplifying the nucleic acid using one of the identified oligonucleotides or a portion thereof as a first primer to perform PCR using DNA derived from the species of interest as template. - View Dependent Claims (21, 22, 23, 24, 26, 27, 28, 29)
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25. The method of step 24, further comprising the step of fragmenting said cRNA.
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30. A method for determining the sequence of a heterologous nucleic acid comprising the steps of:
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a) providing a nucleic acid corresponding to an expressed sequence from a species of interest; b) hybridizing the nucleic acid to a microarray of one or more oligonucleotides derived from expressed sequences of a species different from the species of interest; c) detecting hybridization of the nucleic acid to the one or more oligonucleotides on the microarray; d) identifying and selecting those oligonucleotides which have hybridized to the nucleic acid to levels suitable to act as PCR primers; e) amplifying the nucleic acid using one of the identified oligonucleotides or a portion thereof as a first primer to perform PCR using DNA derived from the species of interest as template; and f) determining the sequence of the amplified nucleic acid.
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31-45. -45. (canceled)
Specification