Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses

Abstract

Provided are compositions and processes that utilize genomic regions that are differentially methylated between a mother and her fetus to separate, isolate or enrich fetal nucleic acid from a maternal sample. The compositions and processes described herein are particularly useful for non-invasive prenatal diagnostics, including the detection of chromosomal aneuploidies.

What is claimed is: 
     
         1 . A method for determining the amount of fetal nucleic acid in a sample comprising:
 a) contacting nucleic acid from a pregnant female, which nucleic acid comprises fetal nucleic acid and maternal nucleic acid, the combination of the fetal nucleic acid and the maternal nucleic acid comprising total nucleic acid in the sample, with a methylation sensitive restriction enzyme that specifically digests the maternal nucleic acid at a plurality of loci, wherein the plurality of loci comprises:
 i) loci that are hypermethylated in fetal nucleic acid; and 
 ii) one or more loci selected from loci of SEQ ID NOs:90-163, 176, 179, 180, 184, 188, 189, 190, 191, 193, 195, 198, 199, 200, 201, 202, 203, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 221, 223, 225, 226, 231, 232, 233, 235, 239, 241, 257, 258, 259, and 261, thereby enriching the fetal nucleic acid; 
   b) determining the amount of fetal nucleic acid in the sample.   
     
     
         2 . The method of  claim 1 , comprising determining the amount of total nucleic acid present in the sample. 
     
     
         3 . The method of  claim 2 , wherein determining the amount of fetal nucleic acid in the sample comprises comparing the amount of fetal nucleic acid in (b) to the total amount of nucleic acid. 
     
     
         4 . The method of  claim 1 , wherein determining the amount of fetal nucleic acid comprises introducing one or more competitors at known concentrations in an amplification reaction. 
     
     
         5 . The method of  claim 4 , comprising determining the absolute amount of fetal nucleic acid in the sample. 
     
     
         6 . The method of  claim 1 , wherein the plurality of loci comprises a locus of SEQ ID NO:154. 
     
     
         7 . The method of  claim 1 , wherein the plurality of loci comprises a locus of SEQ ID NO:158. 
     
     
         8 . The method of  claim 1 , wherein the plurality of loci comprises a locus of SEQ ID NO:163. 
     
     
         9 . The method of  claim 1 , wherein the plurality of loci further comprises loci selected from loci of SEQ ID NOs:1-59. 
     
     
         10 . The method of  claim 1 , wherein the plurality of loci further comprises loci selected from loci of SEQ ID NO:42 and SEQ ID NO:52. 
     
     
         11 . The method of  claim 1 , wherein the amount of fetal nucleic acid at 3 or more loci is determined. 
     
     
         12 . The method of  claim 11 , wherein the amount of fetal nucleic acid at 10 or more loci is determined. 
     
     
         13 . The method of  claim 1 , further comprising:
 i) determining the digestion efficiency of the methylation sensitive restriction enzyme; and   ii) determining the presence or absence of Y-chromosome nucleic acid in the nucleic acid.   
     
     
         14 . The method of  claim 13 , wherein the amount of Y-chromosome nucleic acid present in the nucleic acid is determined for a male fetus. 
     
     
         15 . The method of  claim 14 , wherein the amount of fetal nucleic acid is compared to the amount of Y-chromosome nucleic acid. 
     
     
         16 . The method of  claim 14 , wherein two or more assays are used to determine the total amount of nucleic acid, one or more assays are used to determine the amount of Y-chromosome nucleic acid for a male fetus, and one or more assays are used to determine the digestion efficiency of the methylation sensitive restriction enzyme. 
     
     
         17 . The method of  claim 1 , wherein determining the amount of fetal nucleic acid in (b) comprises use of a mass spectrometry method. 
     
     
         18 . The method of  claim 1 , wherein determining the amount of fetal nucleic acid in (b) comprises use of a sequencing method. 
     
     
         19 . The method of  claim 18 , wherein the sequencing method comprises sequencing by synthesis.