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Method for isolation of biosynthesis genes for bioactive molecules

Inactive Publication Date: 2002-09-12
CUBIST PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, much of the genetic diversity potentially available in soil microorganisms is unavailable through traditional techniques.

Method used

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Examples

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Effect test

example 1

[0033] Total DNA was extracted from soil samples by a direct lysis procedure as described by Barns et al. (1994). The high molecular weight DNA (>20 kb) in the extract was separated on a Sephadex G200 column (Pharmacia, Uppsala, Sweden) as described by Tsai and Olson, Appl. Environ. Microbiol. 58: 2292-2295 (1992).

[0034] The DNA extract (10-50 ng template DNA) was added to an amplification mixture (total volume 100 .mu.l) containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM MgCl.sub.2, 200 .mu.M of each deoxynucleotide triphosphate, 25 pmol of each Type I polyketide primer (Seq ID Nos 1 and 2) and 5.0 units of Taq polymerase (BRL Life Technologies, Gaithersburg, Md.). The mixture was then thermally cycled for 30 cycles in a MJ Research PTC-100 thermocycler using the following program:

7 denaturation 93.degree. C. 60 seconds annealing 60.degree. C. 30 seconds extension 72.degree. C. 90 seconds

[0035] The PCR products were then electrophoresed in 1% agarose gels and stained with ethidium...

example 2

[0037] The experiment of Example 1 was repeated using isopenicillin N synthase gene primers (Seq ID Nos. 7 and 8). The thermal cycling program was changed to include 60 second extension periods at 72.degree. C., but otherwise the experimental conditions were the same. Twelve clones were picked at random and yielded one unique sequence that was not identical to published sequences. The complete sequence of this clone (Clone ipnsfs) is shown in Seq. ID. No. 15 and the translated amino acid sequence in Seq. ID No. 16. The BLAST search indicated greatest homology for this sequence with isopenicillin N synthases.

example 3

[0038] The experiment of Example 1 was repeated using peptide synthetase primers (Seq. ID Nos 9 and 10). The amplification mixture was changed to a 50 .mu.l volume containing 10 to 50 ng of template DNA, 20 mM (NH.sub.4).sub.2SO.sub.4, 74 mM Tris-HCl (pH 8.8), 1.5 mM MgCl.sub.2, 0.01% Tween 20, 200 .mu.M of each deoxynucleotide triphosphate, 25 pmol of each primer, 0.25% skim milk and 0.4 units of Ultra Therm DNA Polymerase (Bio / Can Scientific, Mississauga, Ontario). The mixture was thermocycled for 30 cycles using the following program:

8 denaturation 95.degree. C. 60 seconds annealing 52.degree. C. 60 seconds extension 72.degree. C. 120 seconds..sup.

[0039] Thirty clones containing a 1.2 kb insert have been partially sequenced. The BLAST analysis of the 30 clones pointed to 28 unique sequences that were not identical to each other or to published sequences. Varying degrees of homology to known peptide synthase genes were seen. Seq. ID No. 17 shows the complete DNA sequence of repres...

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Abstract

Degenerate primers which hybridize with various classes of antibiotic biosynthesis genes were used to amplify fragments of DNA from soil and lichen extracts. Cloning and sequencing of the amplified products showed that these products included a variety of novel and previously uncharacterized antibiotic biosynthesis gene sequences, the products of which have the potential to be active as antibiotics, immunosuppressors, antitumor agents, etc. Thus, antibiotic biosynthesis genes can be recovered from soil or lichens by a combining a sample with a pair of amplification primers under conditions suitable for polymerase chain reaction amplification, wherein the primer set is a degenerate primer set selected to hybridize with conserved regions of known antibiotic biosynthetic pathway genes, for example Type I and Type II polyketide synthase genes, isopenicillin N synthase genes, and peptide synthetase genes; cycling the combined sample through a plurality of amplification cycles to amplify DNA complementary to the primer set; and isolating the amplified DNA.

Description

BACKGROUND OF THE INVENTION[0001] This application relates to a method for the isolation of biosynthesis genes for antibiotics and other bioactive molecules from complex natural sources such as humus, soil and lichens.[0002] Antibiotics play an important role in man's efforts to combat disease and other economically detrimental effects of microorganisms. Traditionally, antibiotics have been identified by screening microorganisms, especially those found naturally in soil, for their ability to produce an antimicrobial substance. In some cases, the gene or genes responsible for antibiotic synthesis have then been identified and cloned into producer organisms which produce the antibiotic in an unregulated manner for commercial applications. However, it has been estimated that less than 1% of the microorganisms present in soil are culturable. Torsvik et al., Appl. Environ. Microbiol. 56: 782-787 (1990). Thus, much of the genetic diversity potentially available in soil microorganisms is u...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/689C12Q1/6895
Inventor WATERS, BARBARA KATHLEENMIAO, VIVIAN P.W.HO, YAP WAITONG, SEOW KAH
Owner CUBIST PHARMA INC
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