High throughput method for discovery of gene clusters
First Claim
1. A high throughput method for identifying a gene or gene cluster involved in the biosynthesis of a prokaryotic microbial secondary metabolite natural product comprising:
- a) preparing, from isolated genomic DNA, a random small insert library comprised of DNA fragments of the genomic DNA and a random large insert library comprised of DNA fragments of the genomic DNA, wherein said small insert library has inserts smaller than said large insert library;
b) determining the DNA sequence of at least part of a plurality 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 encoded by the DNA sequence of the GSTs with sequences in a database containing genes, gene fragments, DNA or amino acid sequences known to be involved in the biosynthesis of microbial secondary metabolite natural products to identify a GST that has sequence homology, as evidenced by an E value of 10−
5 or lower, to a gene, gene fragment, DNA or amino acid sequence known to be involved in the biosynthesis of microbial secondary metabolite natural products; and
d) hybridizing a nucleic acid probe comprising a sequence generated using the sequence of said GST identified in (c) to said large insert library 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 prokaryotic microbial secondary metabolite natural product.
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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.
16 Citations
19 Claims
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1. A high throughput method for identifying a gene or gene cluster involved in the biosynthesis of a prokaryotic microbial secondary metabolite natural product comprising:
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a) preparing, from isolated genomic DNA, a random small insert library comprised of DNA fragments of the genomic DNA and a random large insert library comprised of DNA fragments of the genomic DNA, wherein said small insert library has inserts smaller than said large insert library; b) determining the DNA sequence of at least part of a plurality 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 encoded by the DNA sequence of the GSTs with sequences in a database containing genes, gene fragments, DNA or amino acid sequences known to be involved in the biosynthesis of microbial secondary metabolite natural products to identify a GST that has sequence homology, as evidenced by an E value of 10−
5 or lower, to a gene, gene fragment, DNA or amino acid sequence known to be involved in the biosynthesis of microbial secondary metabolite natural products; andd) hybridizing a nucleic acid probe comprising a sequence generated using the sequence of said GST identified in (c) to said large insert library 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 prokaryotic microbial secondary metabolite natural product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification
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- Resources
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Current AssigneeThallion Pharmaceuticals Inc. (Sun Pharmaceutical Industries Limited)
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Original AssigneeThallion Pharmaceuticals Inc. (Sun Pharmaceutical Industries Limited)
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InventorsZazopoulos, Emmanuel, Farnet, Chris M., Staffa, Alfredo
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Primary Examiner(s)Brusca; John S.
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Assistant Examiner(s)NEGIN, RUSSELL SCOTT
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Application NumberUS10/232,370Publication NumberTime in Patent Office1,806 DaysField of Search702/19, 702/22US Class Current706/13CPC Class CodesC07K 14/36 from Actinomyces; from Stre...C07K 14/365 from Actinoplanes (G)C12N 15/52 Genes encoding for enzymes ...C12P 19/62 the hetero ring having eigh...
