HIGH THROUGHPUT METHOD FOR DISCOVERY OF GENE CLUSTERS
First Claim
1. A method for detecting genes which act together in a coordinated manner and are clustered together in a genome, said method comprising the steps of:
- a) preparing, from isolated genomic DNA, a large-insert library of DNA fragments;
b) determining the DNA sequence of at least part of some of the fragments in the large-insert library to form a plurality of Gene Sequence Tags (GSTs);
c) comparing, under computer control, the DNA sequence of the GST with sequences in a database containing genes, gene fragments, DNA sequences or amino acid sequences known to be part of a cluster of genes that act together in a coordinated manner and that are clustered together on a chromosome to identify a GST that has similar structure to a gene, gene fragment, DNA sequence or amino acid sequence known to be part of a cluster of genes that act together in a coordinated manner; and
d) using the GST having similar structure to a gene, gene fragment, DNA sequence or amino acid sequence known to be part of a cluster of genes that act together in a coordinated manner to detect a DNA fragment from the large-insert library, which DNA fragment from the large insert library contains the GST and genes which act together in a coordinated manner and are clustered together on a chromosome.
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Abstract
A method for identifying gene cluster is disclosed. The method may be used for identifying gene clusters involved in the biosynthesis of natural products. A small insert library of DNA fragments of genomic DNA and a large insert library of DNA fragments of genomic DNA are prepared. Fragments in the small insert library are sequenced and compared by homology comparison under computer control to a database containing genes, gene fragments or proteins known to be involved in the biosynthesis of microbial natural products. Fragments having similar structure to genes, gene fragments or proteins known to be involved in the biosynthesis of naturally occurring metabolites are used as probes to screen the large insert library of genomic DNA to detect gene clusters involved in the biosynthesis of microbial natural products.
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Citations
25 Claims
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1. A method for detecting genes which act together in a coordinated manner and are clustered together in a genome, said method comprising the steps of:
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a) preparing, from isolated genomic DNA, a large-insert library of DNA fragments; b) determining the DNA sequence of at least part of some of the fragments in the large-insert library to form a plurality of Gene Sequence Tags (GSTs); c) comparing, under computer control, the DNA sequence of the GST with sequences in a database containing genes, gene fragments, DNA sequences or amino acid sequences known to be part of a cluster of genes that act together in a coordinated manner and that are clustered together on a chromosome to identify a GST that has similar structure to a gene, gene fragment, DNA sequence or amino acid sequence known to be part of a cluster of genes that act together in a coordinated manner; and d) using the GST having similar structure to a gene, gene fragment, DNA sequence or amino acid sequence known to be part of a cluster of genes that act together in a coordinated manner to detect a DNA fragment from the large-insert library, which DNA fragment from the large insert library contains the GST and genes which act together in a coordinated manner and are clustered together on a chromosome.
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2. A method for detecting genes which act together in a coordinated manner and are clustered together in a genome, said method comprising the steps of:
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a) preparing, from isolated genomic DNA, a small insert library of DNA fragments of the genomic DNA and a large insert library of DNA fragments of the genomic DNA; b) determining the DNA sequence of at least part of some of the fragments in the small insert library to form a plurality of Gene Sequence Tags (GSTs); c) comparing, under computer control, the DNA sequence of the GSTs or the amino acid sequence corresponding to the DNA sequence of the GSTs with sequences in a database containing genes, gene fragments, DNA, or amino acid sequences known to be part of a cluster of genes that act together in a coordinated manner and are clustered together on a chromosome to identify a GST that has similar structure to a gene, gene fragment, DNA or amino acid sequence known to be part of a cluster of genes that act together in a coordinated manner; and d) using the GST having similar structure to a gene, gene fragment, DNA or amino acid sequence known to be part of a cluster of genes that act together in a coordinated manner to detect a DNA fragment from the large insert library, which DNA fragment from the large insert library contains the GST and genes which act together in a coordinated manner and are clustered together on a chromosome. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A high-throughput method for identifying a gene or gene cluster involved in the biosynthesis of a microbial natural product comprising:
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a) preparing, from isolated genomic DNA, a large insert library of DNA fragments of about 30 kbp to about 300 kbp; b) determining the DNA sequence of a least part of some of the fragments in the small insert library to form a plurality of Gene Sequence Tags (GSTs); c) comparing, under computer control, the DNA sequence of the GSTs or the amino acid sequence corresponding to the GSTs with sequences in a database containing genes, gene fragments, DNA sequences or amino acid sequences known to be involved in the biosynthesis of microbial natural products to identify a GST that has a similar structure to a gene, gene fragment, DNA sequence or amino acid sequence known to be involved in the biosynthesis of microbial natural products; and d) using the GST having similar structure to a gene, gene fragment, DNA or amino acid sequence known to be involved in the biosynthesis of microbial natural products, or portions thereof, to identify a DNA fragment from the large insert library, which DNA fragment contains the GST and a gene or gene cluster involved in the biosynthesis of a microbial natural product.
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22. A method for scanning the genome of a microorganism to identify a gene cluster involved in the biosynthesis of a lipopeptide, said method comprising:
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a) providing genomic DNA from a microorganism; b) preparing a randomly generated small insert library of DNA fragments of about 1.5 kbp to about 10 kbp of the genomic DNA, and a randomly generated large insert library of DNA fragments of the genomic DNA of about 10 kbp to about 300 kbp; c) sequencing at least part of some of the fragments in the small insert library to form a plurality of Gene Sequence Tags (GSTs) of about 300 base pairs (bp) to about 700 bp, translating the DNA sequences of the GSTs into the corresponding amino acid sequence and providing the amino acid sequence of the GSTs in computer readable form; d) comparing, under computer control, the amino acid sequences of the GSTs with sequences in a database containing amino acid sequences known to be involved in the biosynthesis of lipopeptides to identify a GST that has a similar structure to an amino acid sequence known to be involved in the biosynthesis of lipopeptides; and e) using the GST of step d) as a hybridization probe to screen the large insert library of genomic DNA to detect a DNA fragment containing a gene cluster involved in the biosynthesis of a lipopeptide.
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23. A method for scanning the genome of a microorganism to identify a gene cluster involved in the biosynthesis of an enediyne, said method comprising:
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a) providing genomic DNA from a microorganism; b) preparing a randomly generated small insert library of DNA fragments of about 1.5 kbp to about 10 kbp of the genomic DNA, and a randomly generated large insert library of DNA fragments of the genomic DNA of about 10 kbp to about 300 kbp; c) sequencing at least part of some of the fragments in the small insert library to form a plurality of gene sequence tags (GSTs) of about 300 bp to about 700 bp, translating the DNA sequence of the GSTs into the corresponding amino acid sequence and providing the amino acid sequences of the GSTs in computer readable form; d) comparing, under computer control, the amino acid sequences of the GSTs with sequences in a database containing amino acid sequences known to be involved in the biosynthesis of enediynes to identify a GST that has a similar structure to an amino acid sequence known to be involved in the biosynthesis of enediynes; and e) using the GST of step d) as a hybridization probe to screen the large insert library of genomic DNA to detect a DNA fragment containing a gene cluster involved in the biosynthesis of an enediyne.
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24. A method for scanning the genome of a microorganism to identify a gene cluster involved in the biosynthesis of an orthosomycin, said method comprising:
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a) providing genomic DNA from a microorganism; b) preparing a randomly generated small insert library of DNA fragments of about 1.5 kbp to about 10 kbp of the genomic DNA, and a randomly generated large insert library of DNA fragments of the genomic DNA of about 10 kbp to about 300 kbp; c) sequencing at least part of the fragments in the small insert library to form a plurality of gene sequence tags (GSTs) of about 300 bp to about 700 bp, translating the DNA sequence of the GSTs into the corresponding amino acid sequence and providing the amino acid sequences of the GSTs in computer readable form; and d) comparing, under computer control, the amino acid sequences of the GSTs with sequences in a database containing amino acid sequences known to be involved in the biosynthesis of orthosomycins to identify a GST that has a similar structure to an amino acid sequences known to be involved in the biosynthesis of orthosomycins; and
e) using the GST of step d) as a hybridization probe to screen the large insert library of genomic DNA to detect a DNA fragment containing a gene cluster involved in the biosynthesis of an orthosomycin.
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25. A method for scanning the genome of a microorganism to identify a polyketide synthase gene or a gene cluster including a polyketide synthase gene, said method comprising:
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a) providing genomic DNA from a microorganism; b) preparing a randomly generated small insert library of DNA fragments of about 1.5 kbp to about 10 kbp of the genomic DNA, and a randomly generated large insert library of DNA fragments of the genomic DNA of about 10 kbp to about 300 kbp; c) sequencing at least part of some of the fragments in the small insert library to form a plurality of Gene Sequence Tags (GSTs) of about 300 base pairs (bp) to about 700 bp, translating the DNA sequences of the GSTs into the corresponding amino acid sequence and providing the amino acid sequence of the GSTs in computer readable form; d) comparing, under computer control, the amino acid sequences of the GSTs with sequences in a database containing amino acid sequences known to be associated with a polyketide synthase to identify a GST that has a similar structure to an amino acid sequence known to be associated with a polyketide synthase; and e) using the GST of step d) as a hybridization probe to screen the large insert library of genomic DNA to detect a DNA fragment containing a polyketide synthase gene or a gene cluster including polyketide synthase gene.
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Specification