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.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application Ser. No. 09/685,432, filed Oct. 10, 2000, which is a continuation-in-part of U.S. patent application Ser. No. 09/444,112, filed Nov. 22, 1999 (pending), which is a continuation-in-part of U.S. patent application Ser. No. 09/098,206, filed Jun. 16, 1998, now U.S. Pat. No. 6,174,673, which is a continuation-in-part of U.S. patent application Ser. No. 08/876,276, filed Jun. 16, 1997 (pending), the contents of which are all incorporated by reference in their entirety herein.
Divisions (1)
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Number |
Date |
Country |
Parent |
09685432 |
Oct 2000 |
US |
Child |
10145314 |
May 2002 |
US |
Continuation in Parts (3)
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Number |
Date |
Country |
Parent |
09444112 |
Nov 1999 |
US |
Child |
09685432 |
Oct 2000 |
US |
Parent |
09098206 |
Jun 1998 |
US |
Child |
09444112 |
Nov 1999 |
US |
Parent |
08876276 |
Jun 1997 |
US |
Child |
09098206 |
Jun 1998 |
US |