Libraries of regulatory sequences, methods of making and using same

• US 7,923,542 B2
• Filed: 10/24/2001
• Issued: 04/12/2011
• Est. Priority Date: 04/28/2000
• Status: Expired due to Fees

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Wolffe, Alan P., Urnov, Fyodor
• Primary Examiner(s): Zhou; Shubo (Joe)
• Application Number:
  US10/083,682
• Publication Number:
  US 20030129603A1
• Time in Patent Office: 3,457 Days
• Field of Search: 435/6, 435/91.2, 435/18, 536/23.1, 536/24.3
• US Class Current: 536/23.1
• 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 15/62 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. In particular, libraries of regulatory sequences are provided, in which each member of the library comprises a polynucleotide comprising sequences from an accessible region of cellular chromatin.

Claims

1. A library comprising a plurality of polynucleotides, each polynucleotide of the library comprising a vector and an insert, wherein each of the insert sequences consist essentially of accessible regions of cellular chromatin, wherein there are at least two different insert sequences and further wherein the library is obtained according to the method of:

(a) contacting cellular chromatin with a probe, wherein reaction of the probe with cellular chromatin results in polynucleotide cleavage at accessible regions of cellular chromatin;

(b) deproteinizing the cleaved chromatin of step (a);

(c) digesting the deproteinized chromatin of step (b) with a nuclease to generate a collection of polynucleotide fragments; and

(d) selectively cloning polynucleotide fragments comprising one end generated by probe cleavage.

2. A library according to claim 1, wherein each insert sequence consists of an accessible region of cellular chromatin.

3. The library of claim 1, wherein the cellular chromatin is obtained from cells at a particular stage of development.

4. The library of claim 1, wherein the cellular chromatin is obtained from cells of a particular tissue.

5. The library of claim 1, wherein the cellular chromatin is obtained from diseased cells.

6. The library of claim 1, wherein the cellular chromatin is obtained from infected cells.

7. The library of claim, 1, wherein, in step (a), the probe is a nuclease.

8. The library of claim 7, wherein the nuclease is a restriction enzyme.

9. The library of claim 8, wherein the probe comprises a plurality of restriction enzymes.

10. The library of claim 1, wherein, in step (c), the nuclease is a restriction enzyme.