Screening methods based on isolating a collection of polynucleotides corresponding to accessible regions of chromatin

• US 7,097,978 B2
• Filed: 05/08/2003
• Issued: 08/29/2006
• Est. Priority Date: 04/28/2000
• Status: Expired due to Term

• Current Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Johnstone, Brian, Guschin, Dmitry, Li, Xiao-Yong, Urnov, Fyodor, Collingwood, Trevor, Wolffe, Alan
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US10/434,947
• Publication Number:
  US 20030190664A1
• Time in Patent Office: 1,209 Days
• Field of Search: 435/6
• US Class Current: 435/6.13
• 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 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• 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 30/10 : Sequence alignment; Homology search
• CPC Class Codes: G16B 50/00 : ICT programming tools or database systems specially adapted for bioinformatics

Abstract

Methods for assessing potential pharmacological effects and/or side effects of an agent, based on the effect of the agent on the distribution and properties of accessible regions in cellular chromatin, are provided.

Claims

1. A method for screening a compound, the method comprising:

(a) exposing a sample of cellular chromatin to the compound;

(b) isolating a first collection of polynucleotides from the sample of cellular chromatin exposed to the compound, wherein the isolated collection of polynucleotides correspond to accessible regions of the cellular chromatin; and

(c) comparing the first collection of isolated polynucleotides to a second collection of isolated polynucleotides corresponding to accessible regions of cellular chromatin from cells that have not been exposed to the compound;

wherein a difference between the first and second collections of isolated polynucleotides is attributable to the compound.

2. The method of claim 1, wherein the cellular chromatin is in a cell and the cell is exposed to the compound.

3. The method of claim 1, wherein step (b) comprises:

(i) providing a cell nucleus comprising the cellular chromatin;

(ii) contacting the nucleus with a first enzyme that reacts with accessible regions of cellular chromatin;

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

(iv) contacting the deproteinized DNA with a second enzyme to generate DNA fragments;

(v) contacting the DNA fragments with a population of vector molecules, wherein each vector molecule comprises 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

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

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

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

6. The method of claim 4, wherein the nuclease is a restriction enzyme.

7. The method of claim 3, wherein the second enzyme is a restriction enzyme.

8. The method of claim 3, wherein the nucleus is embedded in agarose.

9. The method of claim 1, wherein a plurality of different first collections of polynucleotides are prepared and further wherein, for each collection, a different first enzyme is used.

10. The method of claim 9, wherein the different collections are combined.

11. The method of claim 1, further comprising the step of determining the nucleotide sequence of one or more of the polynucleotides in the first collection of isolated polynucleotides.

12. The method of claim 11, wherein the nucleotide sequences are used to populate a first database and further wherein the first database is compared to a second database comprising sequences of previously identified accessible regions of the cellular chromatin.

13. The method of claim 11, wherein the difference between the first and second collections of isolated polynucleotides is indicative of toxicity of the compound.

14. The method of claim 11, wherein the difference between the first and second collections of isolated polynucleotides is indicative of therapeutic effect of the compound.

15. A method for screening a compound, the method comprising:

(a) exposing a sample of cellular chromatin to the compound; and

(b) isolating a collection of polynucleotides from the sample exposed to the compound, wherein the isolated polynucleotides correspond to accessible regions of the cellular chromatin.

16. The method of claim 15, further comprising the step of hybridizing one or more of the isolated polynucleotides to an array of polynucleotides on a solid support.

17. The method of claim 15, further comprising the step of determining the nucleotide sequence of one or more of the polynucleotides in the collection of isolated polynucleotides.