Databases of regulatory sequences; methods of making and using same

• US 7,217,509 B2
• Filed: 04/27/2001
• Issued: 05/15/2007
• Est. Priority Date: 04/28/2000
• Status: Expired due to Fees

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Urnov, Fyodor, Wolffe, Alan
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/844,501
• Publication Number:
  US 20020081603A1
• Time in Patent Office: 2,209 Days
• Field of Search: 435/6, 435/91.2, 435/18, 536/23.1, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/24 containing a MBP (maltose binding protein)-tag
• CPC Class Codes: C07K 2319/43 containing a FLAG-tag
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: G16B 30/00 : ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 50/00 : ICT programming tools or database systems specially adapted for bioinformatics
• CPC Class Codes: Y02A 90/10 : Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Abstract

Methods and compositions for the identification, isolation and characterization of regulatory DNA sequences in a cell of interest are provided. Also provided are libraries of regulatory sequences obtained according to the methods, and databases comprising collections of regulatory sequences for a particular cell of interest. In addition, various uses for the regulatory sequences so obtained, and uses for the databases of regulatory sequences, are provided. Also disclosed are computer systems and computer program products for utilizing the databases to conduct various genetic analyses, and uses of accessible regulatory sequences in the design of vectors bearing transgenes.

Claims

1. A method for preparing a library of regulatory DNA sequences from a cell, the method comprising:

(a) providing a cell nucleus, wherein the nucleus comprises cellular chromatin;

(b) contacting the nucleus with a first enzyme, wherein the first enzyme reacts with accessible regions of cellular chromatin;

(c) deproteinizing the cellular chromatin to generate deproteinized DNA;

(d) contacting the deproteinized DNA with a second enzyme to generate a plurality of different DNA fragments;

(e) contacting the DNA fragments obtained in step (d) with a population of vector molecules, wherein the vector molecules comprise a first end that is compatible with the first enzyme and a second end that is compatible with the second enzyme, under conditions favorable to ligation of compatible ends; and

(f) selecting polynucleotides comprising a DNA fragment ligated to a vector molecule.

2. The method of claim 1, wherein the cell is selected from the group consisting of animal cells, plant cells and microbial cells.

3. The method of claim 1, wherein the first enzyme is a nuclease.

4. The method of claim 3, wherein the nuclease is DNase I.

5. The method of claim 3, wherein the nuclease is a restriction enzyme.

6. The method of claim 1, wherein the second enzyme is a restriction enzyme.

7. The method of claim 6, wherein the restriction enzyme is Sau3A I.

8. The method of claim 7, wherein the second end of the vector molecule is generated by digestion with BamH I.

9. The method of claim 4, wherein, subsequent to step (b), the DNase I ends are converted to blunt ends.

10. The method of claim 9, wherein the first end of the vector molecule is a blunt end.

11. The method of claim 10, wherein the first end of the vector molecule is generated by digestion with EcoRV or SmaI.

12. The method of claim 1 wherein, during steps (b)–

(d), the nucleus is embedded in agarose.

13. The method of claim 1, wherein a plurality of different libraries of regulatory DNA sequences are prepared, wherein each library is obtained from a different cell.

14. The method of claim 13 wherein, in step (a), nuclei are obtained from cells at different stages of development.

15. The method of claim 13 wherein, in step (a), nuclei are obtained from cells in different tissues.

16. The method of claim 13 wherein, in step (a), nuclei are obtained from diseased cells and counterpart normal cells.

17. The method of claim 13 wherein, in step (a), nuclei are obtained from infected cells and counterpart uninfected cells.

18. The method of claim 13 wherein, in step (a), nuclei are obtained from cells that express a gene of interest at different levels.

19. The method of claim 1, wherein a plurality of different libraries of regulatory DNA sequences are prepared and, for each library, a different first enzyme is used.

20. The method of claim 19, wherein the different libraries are combined.

21. A method for isolating a collection of polynucleotides comprising cellular regulatory sequences, wherein the method comprises:

(a) contacting cellular chromatin with a probe, wherein the probe reacts with accessible regions of cellular chromatin;

(b) subsequently fragmenting the cellular chromatin to generate a plurality of different polynucleotide fragments; and

(c) selectively cloning polynucleotide fragments of step (b) comprising a site of probe reaction.

22. The method of claim 21, wherein reaction of the probe with cellular chromatin results in polynucleotide cleavage at the site of reaction.

23. The method of claim 21, wherein the cellular chromatin in present in an isolated nucleus.

24. The method of claim 23 wherein, in steps (a) and (b), the isolated nucleus is embedded in agarose.

25. The method of claim 21, wherein the probe is an enzyme.

26. The method of claim 25, wherein the enzyme is a nuclease.

27. The method of claim 26, wherein the nuclease is a restriction enzyme.

28. The method of claim 26, wherein the nuclease is DNase I.

29. The method of claim 21 wherein, in step (b), cellular chromatin is fragmented by restriction enzyme digestion.

30. The method of claim 29, wherein the restriction enzyme is Sau3A1.