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 copy number of fetal nucleic acid in a maternal plasma sample, which sample comprises extracellular nucleic acid, comprising:
 (a) contacting a sample nucleic acid with one or more methylation sensitive restriction enzymes, which sample nucleic acid comprises differentially methylated 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, thereby generating differentially digested sample nucleic acid;   (b) contacting under amplification conditions the digested sample nucleic acid with:
 (i) a first set of amplification primers that specifically amplify a first region in sample nucleic acid comprising two or more loci that are differentially methylated between the fetal nucleic acid and maternal nucleic acid, wherein at least one of the two or more loci in the first region comprises a nucleotide sequence selected from among SEQ ID NO:42, SEQ ID NO:52, SEQ ID NO:154, SEQ ID NO:158 and SEQ ID NO:163; and 
 (ii) a predetermined copy number of one or more first competitor oligonucleotides that compete with the first region for hybridization of primers of the first amplification primer set, thereby generating fetal nucleic acid amplification products and competitor amplification products; 
   (c) incorporating adaptor oligonucleotides into the amplification products in (b) using a unidirectional ligation process, which adaptor oligonucleotides comprise sample specific index sequences; thereby generating adaptor-modified amplification products;   (d) obtaining nucleotide sequences of the adaptor-modified amplification products in (c) by a sequencing-by-synthesis process, thereby generating sequence reads;   (e) quantifying the sequence reads; and   (f) determining the copy number of fetal nucleic acid in the sample based on a quantification of the sequence reads in (e) and the amount of competitor oligonucleotide used, wherein the copy number of fetal nucleic acid is determined with an R 2  value of 0.97 or greater when compared to a copy number of fetal nucleic acid determined using a mass spectrometry method.