Detecting methylation in a subpopulation of genomic dna

Abstract

This invention provides methods of determining the biological, pathological, genetic, epigenetic or disease status in a biological sample by determining the methylation status of a subpopulation of genomic DNA in the sample.

1 . A method of detecting a biological, pathological, genetic or epigenetic state of a subpopulation of genomic DNA (gDNA) in a sample, the method comprising:
 a) dividing a biological sample comprising gDNA into at least a first portion and a second portion;   b) enriching a subpopulation of gDNA in the first portion; and   c) determining the DNA methylation status at one or more gDNA regions in the first portion and in the second portion, wherein a difference in the extent of DNA methylation in the subpopulation of gDNA in the first portion relative to the extent of DNA methylation in the second portion at the one or more gDNA regions is correlated with the biological, pathological, genetic or epigenetic state in the subpopulation of gDNA.   
     
     
         2 . The method of  claim 1 , further comprising the step of obtaining the biological sample. 
     
     
         3 . The method of  claim 1 , wherein the biological sample is a population of cells. 
     
     
         4 . The method of  claim 3 , wherein the population of cells is treated with a permeabilization agent and a DNA modification agent prior to the enrichment step b). 
     
     
         5 . The method of  claim 3 , wherein the population of cells is in situ. 
     
     
         6 . The method of  claim 1 , wherein the biological sample is a solid tissue sample. 
     
     
         7 . The method of  claim 1 , wherein the enriching step comprises enriching for accessible chromatin. 
     
     
         8 . The method of  claim 1 , wherein the enriching step comprises enriching for inaccessible chromatin. 
     
     
         9 . The method of  claim 1 , further comprising performing chromatin immunoprecipitation (ChIP). 
     
     
         10 . The method of  claim 1 , wherein the second portion comprises total gDNA. 
     
     
         11 . The method of  claim 1 , wherein the second portion is from a biological sample that has been treated with a pharmacological agent. 
     
     
         12 . The method of  claim 1 , wherein the extent of DNA methylation status is determined via methylation-sensing restriction enzyme analysis 
     
     
         13 . The method of  claim 1 , wherein the extent of DNA methylation status is determined by contacting the gDNA with bisulfite and detecting methylation of bisulfite-modified gDNA. 
     
     
         14 . The method of  claim 1 , wherein the extent of DNA methylation status is determined via affinity purification. 
     
     
         15 . The method of  claim 1 , wherein the extent of DNA methylation status is determined via direct nucleic acid sequencing. 
     
     
         16 . The method of  claim 1 , wherein the extent of methylation at the one or more gDNA regions in the first portion is higher than the extent of methylation at the one or more gDNA regions in the second portion. 
     
     
         17 . The method of  claim 1 , wherein the extent of methylation at the one or more gDNA regions in the first portion is lower than the extent of methylation at the one or more gDNA regions in the second portion. 
     
     
         18 . A method of detecting the presence of cancer in a biological sample, the method comprising:
 a) dividing a biological sample comprising gDNA, wherein the biological sample comprises cells suspected of being cancerous, into at least a first portion and a second portion;   b) enriching a subpopulation of gDNA in the first portion; and   c) determining the DNA methylation status at one or more gDNA regions in the first portion and in the second portion, wherein a difference in the extent of DNA methylation in the enriched gDNA in the first portion relative to the extent of DNA methylation in the gDNA in the second portion at the one or more gDNA regions is correlated with the presence of cancer in the biological sample.   
     
     
         19 - 39 . (canceled) 
     
     
         40 . A method of determining genomic imprinting of a preselected gDNA region in a biological sample, the method comprising:
 a) dividing a biological sample comprising gDNA into at least a first portion and a second portion;   b) enriching for inaccessible gDNA in the first portion and retaining total gDNA in the second portion; and   c) determining the DNA methylation status at the preselected gDNA region in the first portion and in the second portion, wherein an extent of DNA methylation in the inaccessible gDNA in the first portion that is about 100% and the extent of DNA methylation in the total gDNA in the second portion is about 50% at the preselected gDNA region is correlated with proper imprinting of the preselected gDNA region, and wherein an extent of DNA methylation in the inaccessible gDNA in the first portion that is less than about 90% and the extent of DNA methylation in the total gDNA in the second portion is about 50% at the preselected gDNA region is correlated with loss of imprinting of the preselected gDNA region.   
     
     
         41 . A method of determining genomic imprinting of a preselected gDNA region in a biological sample, the method comprising:
 a) dividing a biological sample comprising gDNA into at least a first portion and a second portion;   b) enriching for accessible gDNA in the first portion and retaining total gDNA in the second portion; and   c) determining the DNA methylation status at the preselected gDNA region in the first portion and in the second portion, wherein an extent of DNA methylation in the accessible gDNA in the first portion that is about 0% and the extent of DNA methylation in the total gDNA in the second portion that is about 50% at the preselected gDNA region is correlated with proper imprinting of the preselected gDNA region.   
     
     
         42 . A method of detecting a biological, pathological, genetic or epigenetic state of accessible gDNA in a sample, the method comprising:
 a) dividing a biological sample comprising gDNA into at least a first portion and a second portion;   b) enriching for accessible gDNA in the first portion and retaining total gDNA in the second portion; and   c) determining the DNA methylation status at one or more gDNA regions in the first portion and in the second portion, wherein a difference in the extent of DNA methylation in the subpopulation of gDNA in the first portion relative to the extent of DNA methylation in the total gDNA in the second portion at the one or more gDNA regions is correlated with the biological, pathological, genetic or epigenetic state in the subpopulation of gDNA.   
     
     
         43 . (canceled)