ISOLATION OF FACTORS THAT ASSOCIATE DIRECTLY OR INDIRECTLY WITH CHROMATIN
First Claim
1. A method for isolating one or more non-coding nucleic acids associated with a target DNA sequence that is comprised within chromatin, comprising the steps of:
- (a) obtaining a sample that comprises a target DNA sequence as well as one or more non-coding nucleic acids that are associated with the target DNA sequence;
(b) contacting the sample with at least one oligonucleotide probe that comprises a sequence that is complimentary to and capable of hybridising with at least a portion of the target DNA sequence, wherein the oligonucleotide probe comprises at least one modified nucleotide analogue and wherein the oligonucleotide probe further comprises at least one affinity label;
(c) allowing the at least one oligonucleotide probe and the target DNA sequence to hybridise with each other so as to form a probe-target hybrid;
(d) isolating the probe-target hybrid from the sample by immobilizing the probe-target hybrid through a molecule that binds to the at least one affinity label; and
(e) eluting the one or more non-coding nucleic acids that are associated with the target DNA sequence.
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Accused Products
Abstract
Methods for isolating non-coding nucleic acids that are associated with chromatin at a target genomic locus are provided. The methods comprise the steps of obtaining a sample that comprises a target genomic DNA sequence and one or more non-coding nucleic acids associated with that DNA sequence; contacting the sample with at least one oligonucleotide probe that comprises a sequence that is complimentary to and capable of hybridising with at least a portion of the target DNA sequence, wherein the oligonucleotide probe comprises at least one modified nucleotide analogue and wherein the oligonucleotide probe further comprises at least one affinity label; allowing the at least one oligonucleotide probe and the target DNA sequence to hybridise with each other so as to form a probe-target hybrid; isolating the probe-target hybrid from the sample by immobilizing the probe-target hybrid through a molecule that binds to the at least one affinity label; and eluting the one or more non-coding nucleic acids that are associated with the target genomic DNA sequence. Also provided are probes suitable for use in the methods of the invention. The methods and probes of the invention are suited to identification of non-coding RNAs including microRNAs and snoRNAs that are associated with chromatin remodelling.
11 Citations
36 Claims
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1. A method for isolating one or more non-coding nucleic acids associated with a target DNA sequence that is comprised within chromatin, comprising the steps of:
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(a) obtaining a sample that comprises a target DNA sequence as well as one or more non-coding nucleic acids that are associated with the target DNA sequence; (b) contacting the sample with at least one oligonucleotide probe that comprises a sequence that is complimentary to and capable of hybridising with at least a portion of the target DNA sequence, wherein the oligonucleotide probe comprises at least one modified nucleotide analogue and wherein the oligonucleotide probe further comprises at least one affinity label; (c) allowing the at least one oligonucleotide probe and the target DNA sequence to hybridise with each other so as to form a probe-target hybrid; (d) isolating the probe-target hybrid from the sample by immobilizing the probe-target hybrid through a molecule that binds to the at least one affinity label; and (e) eluting the one or more non-coding nucleic acids that are associated with the target DNA sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. (canceled)
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25. A nucleic acid analogue probe, suitable for use in PICh/RICh, as set out in formula I below:
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A—
[C]n—
X
Iwherein A includes one or more affinity labels tethered to a nucleotide analogue X by a spacer group C of n atoms in length;
A comprises a hapten or an immuno-tag; and
wherein the nucleotide analogue X is selected from a peptide nucleic acid (PNA);
a 2′
modified ribonucleotide analogue, including 2′
-O—
R sugar modifications, wherein R is selected from the group consisting of;
methyl;
ethyl;
C1 to C5 alkyl; and
aryl; anda 2′
substituted ribonucleotide analogue, including 2′
-C and 2′
-deoxy-2′
-halogeno, suitably 2′
-deoxy-2′
-fluoro ribonucleotides; and
a morpholino nucleotide.
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26. A nucleic acid analogue oligonucleotide probe, suitable for use in PICh/RICh, conforming to general formula II, set out below:
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B—
[C]n—
Y
IIwherein B is an affinity label that is tethered to oligonucleotide sequence Y via a spacer group C comprising a linear chain of n atoms;
the oligonucleotide sequence Y comprising at least 10 nucleotides of which no less than 10% are nucleotide analogues;
typically at least 25% of the nucleotides are nucleotide analogues; and
optionally up to 100% of the nucleotides are nucleotide analogues.
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27. (canceled)
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30. (canceled)
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31. (canceled)
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32. (canceled)
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36. (canceled)
Specification