US 10,480,021 B2Granted

Methods for closed chromatin mapping and DNA methylation analysis for single cells

AssigneeYale University

InventorsXinghua Pan, Sherman M. Weissman

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Abstract

Methods of identifying DNase I Hyper-Resistant Sites (DHRS), or in board sense, highly compact chromatin and characterizing the DNA methylation status of DMRs such as CpG islands and CpG island shores are provided. The methods are particularly useful for analysis of genomic DNA from low quantities of cells, for example, less than 1,000 cells, less than 100 cells, less than 10 cells, or even one cell, and can be used to generate chromatin and methylation profiles. The downstream analyses include in parallel massive sequencing, microarray, PCR and Sanger sequencing, hybridization and other platforms. These methods can be used to generate chromatin and DNA methylation profiles in drug development, diagnostics, and therapeutic applications are also provided.

Claims19

Dependent claims

A method of identifying regions of methylated genomic DNA in a double stranded genomic DNA sample, the method comprising sequentially:

(a) digesting a double stranded genomic DNA sample with one or more methylation sensitive restriction endonucleases (MSREs), thereby forming digested, unligated DNA fragments;
(b) amplifying the digested, unligated DNA fragments by multiple displacement amplification (MDA), thereby producing a plurality of heterogeneous amplicons, the MDA comprising denaturing the digested, unligated DNA fragments and forming single stranded DNA fragments, annealing random primers to the single stranded DNA fragments, and extending the primers annealed to the single stranded DNA fragments with a polymerase;
(c) sequencing the amplicons;
and
(d) identifying the regions of methylated genomic DNA in the double stranded genomic DNA sample based on the sequences of the amplicons.

2 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/747,796Filed 23 Jun 2015 · us provisional application
Priority: 23 Jun 2014
Published: US 2015/0368694 A124 Dec 2015
Granted: 19 Nov 2019
Term ends: Not estimatedThe source records don’t support an estimate.

Classification2 codes

C12Q 1/6827
Hybridisation assays: for detection of mutation or polymorphism
C12Q
Measuring or testing processes involving enzymes, nucleic acids or microorganisms

Family

Application62/015,965Us provisional application · filed 23 Jun 2014
US 2015/0368694 A1Pre-grant publication · published 24 Dec 2015
US 10,480,021 B2This patent

From the US filing history. Foreign family members aren’t listed.

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