High throughput screening for sequences of interest

• US 20030092022A1
• Filed: 05/13/2002
• Published: 05/15/2003
• Est. Priority Date: 06/16/1997
• Status: Abandoned Application

• Current Assignee: Diversa Corporation (BASF SE)
• Original Assignee: Diversa Corporation (BASF SE)
• Inventors: Keller, Martin, Short, Jay M.
• Application Number:
  US10/145,314
• Publication Number:
  US 20030092022A1
• Time in Patent Office: Days
• US Class Current: 435/6.18
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12N 11/04 entrapped within the carrier, e.g. gel or hollow fibres
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms
• CPC Class Codes: G01N 33/554 the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
• CPC Class Codes: G01N 33/569 for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/56911 Bacteria

Abstract

Provided is a method of screening or enriching a sample containing polynucleotides from a mixed population of organisms. The method includes creating a DNA library from a plurality of nucleic acid sequences of a mixed population of organisms and separating clones containing a polynucleotide sequence of interest on a fluorescence analyzer based upon hybridization of a fluorescence labeled oligonucleotide probe to a target sequence in a clone. The separated or enrich library can then be further process by activity based screening or sequence based screening. In addition, the enriched sequence can be compared to a database and to identify sequences in the database which have homology to a clone in the library thereby obtaining a nucleic acid profile of the mixed population of organisms.

Claims

1. A method for identifying a polynucleotide encoding a polypeptide of interest comprising:

co-encapsulating in a microenvironment a plurality of library clones containing DNA obtained from a mixed population of organisms, with a mixture of oligonucleotide probes comprising a fluorescence marker and at least a portion of a polynucleotide sequence encoding a polypeptide of interest having a specified bioactivity under such conditions and for such time as to allow interaction of complementary sequences; and

identifying clones containing a complement to the oligonucleotide probe encoding the polypeptide of interest by separating clones with a fluorescent analyzer that detects fluorescence.

2. A method for high throughput screening of a polynucleotide library for a polynucleotide of interest that encodes a molecule of interest, comprising:

(a) contacting a library containing a plurality of clones comprising polynucleotides derived from a mixed population of organisms with a plurality of oligonucleotide probes labeled with a fluorescence molecule; and

(b) separating clones with a fluorescent analyzer that detects fluorescence.

3. The method of claim 2, further comprising:

(a) contacting the separated clones with a reporter system that identifies a polynucleotide encoding the molecule of interest; and

(b) identifying clones capable of modulating expression or activity of the reporter system thereby identifying a polynucleotide of interest.

4. The method of claim 2, wherein the library is an expression library.

5. The method of claim 2, wherein the mixed population of organisms is from an environmental sample.

6. The method of claim 2, wherein the mixed population of organisms comprises microorganisms.

7. The method of claim 6, wherein the environmental sample contains extremophiles.

8. The method of claim 7, wherein the extremophiles are selected from the group consisting of hyperthermophiles, psychrophiles, halophiles, psychrotrophs, alkalophiles, and acidophiles.

9. The method of claim 3, wherein the reporter system is a bioactive substrate.

10. The method of claim 9, wherein the bioactive substrate comprises C12FDG.

11. The method of claim 10, wherein the bioactive substrate further comprises a lipophilic tail.

12. The method of claim 2, further comprising prior to (a):

(i) obtaining polynucleotides from a mixed population of organisms; and

(ii) generating a polynucleotide library.

13. The method of claim 12, further comprising normalizing the polynucleotides prior to generating the library.

14. The method of claim 2, wherein the clones are encapsulated in a gel microdrop.

15. The method of claim 2, wherein the polynucleotide of interest encodes an enzyme.

16. The method of claim 15, wherein the enzyme is selected from the group consisting of lipases, esterases, proteases, glycosidases, glycosyl transferases, phosphatases, kinases, mono- and dioxygenases, haloperoxidases, lignin peroxidases, diarylpropane peroxidases, eposize hydrolases, nitrile hydratases, nitrilases, transaminases, amidases, and acylases.

17. The method of claim 3, wherein the reporter system comprises a detectable label.

18. The method of claim 2, wherein the reporter system comprises a first test protein linked to a DNA binding moiety and a second test protein linked to a transcriptional activation moiety, wherein modulation of the interaction of the first test protein linked to a DNA binding moiety with the second test protein linked to a transcription activation moiety results in a change in the expression of a detectable protein.

19. The method of claim 2, wherein the polynucleotide of interest encodes a small molecule.

20. The method of claim 2, wherein the polynucleotide of interest, or fragments thereof, comprise one or more operons, or portions thereof.

21. The method of claim 20, wherein the operons, or portions thereof, encodes a complete or partial metabolic pathway.

22. The method of claim 21, wherein the operons or portions thereof encoding a complete or partial metabolic pathway encodes polyketide syntheses.

23. The method of claim 2, wherein the fluorescent analyzer is a fluorescence activated cell sorting (FACS) apparatus.

24. The method of claim 2, wherein the plurality of oligonucleotide probes have different nucleic acid sequences.

25. The method of claim 24, wherein the sequences are portions of a polynucleotide encoding a molecule of interest.

26. The method of claim 2, wherein the plurality of oligonucleotide probes have the same nucleic acid sequence.