Processes and compositions for methylation-based enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
Provided are compositions and processes that utilize genomic regions 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 useful for non-invasive prenatal diagnostics, including the detection of chromosomal aneuploidies.
1. A method for determining the amount of fetal nucleic acid of a target chromosome comprising:
a) contacting nucleic acid from a pregnant female, which nucleic acid comprises differentially methylated fetal nucleic acid and maternal nucleic acid, with an agent that specifically binds methylated nucleotides, wherein the agent that binds methylated nucleotides is a methyl-CpG binding protein or fragment thereof, thereby producing bound nucleic acid;
b) immunoprecipitating the bound nucleic acid of a), thereby enriching for sequences comprising differentially methylated fetal nucleic acid;
c) amplifying the nucleic acids of b) with primers that specifically amplify SEQ ID NO: 33 or a portion thereof, thereby generating amplification products; and
d) determining from the products of c) the amount of fetal nucleic acid for SEQ ID NO: 33, wherein determining the amount of fetal nucleic acid comprises the use of RT-PCR or a sequencing method.
2. The method of claim 1 , wherein the methyl-CpG binding protein or fragment thereof is fused to an Fc fragment of an antibody.
3. The method of claim 1 , wherein the agent that binds methylated nucleotides is a methylation specific antibody.
4. The method of claim 1 , wherein step c) further comprises amplifying the nucleic acids of b) with primers that specifically amplify at least one of SEQ ID NOs: 1-32 and 34-59 or a portion thereof; and step d) further comprises determining from the products of c) the amount of fetal nucleic acid for the at least one of SEQ ID NOs: 1-32 and SEQ ID NOs: 34-59.
5. The method of claim 1 , further comprising determining the amount of control DNA from a euploid region of a chromosome and comparing the amount of fetal DNA as determined in step d) to the amount of the control nucleic acid to determine the presence or absence of fetal aneuploidy.
6. The method of claim 5 , wherein the control nucleic acid is obtained from a female with a normal euploid pregnancy.
7. The method of claim 1 , wherein the amount of fetal nucleic acid at 3 or more loci is determined.
8. The method of claim 1 , wherein the amount of fetal nucleic acid at 10 or more loci is determined.
9. The method of claim 1 , wherein the amount of fetal nucleic acid at 50 or more loci is determined.