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

• US 10,612,086 B2
• Filed: 02/09/2017
• Issued: 04/07/2020
• Est. Priority Date: 09/16/2008
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Ehrich, Mathias, Nygren, Anders Olof Herman, Jensen, Tyler Jacob
• Primary Examiner(s): Sitton, Jehanne S
• Application Number:
  US15/428,659
• Publication Number:
  US 20170314071A1
• Time in Patent Office: 1,153 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

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.

Claims

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 digests nucleic acid at non-methylated sites, thereby enriching methylated nucleic acid;

b) amplifying in a single reaction vessel nucleic acid not digested in (a) at loci consisting of a locus comprising a polynucleotide of SEQ ID NO;

42 or a portion thereof and a locus comprising a polynucleotide of SEQ ID NO;

52 or a portion thereof, thereby generating amplification products; and

c) determining the amount of the fetal nucleic acid in the sample based on the amplification products of SEQ ID NO;

42 or a portion thereof and SEQ ID NO;

52 or a portion thereof in (b).

2. The method of claim 1, wherein determining the amount of fetal nucleic acid comprises use of a PCR-based method.

3. The method of claim 2, wherein the PCR based method is real time PCR.

4. The method of claim 1, wherein determining the amount of fetal nucleic acid comprises use of one or more processes selected from mass spectrometry methods, RT-PCR, digital PCR, array-based methods, sequencing methods, nanopore-based methods, nucleic acid-bound head-based counting methods, competitor-based methods and methods for separating nucleic acid using agents that bind nucleic acid based on methylation status.

5. The method of claim 1, wherein determining the amount of fetal nucleic acid comprises introducing one or more competitors at known concentrations in (b).

6. The method of claim 5, comprising determining the absolute amount of fetal nucleic add in the sample.

7. The method of claim 1, further comprising determining the amount of total nucleic acid present in the sample.

8. The method of claim 7, comprising determining the concentration of fetal nucleic acid in the sample by comparing the amount of fetal nucleic acid to the total amount of nucleic acid.

9. The method of claim 8, wherein the concentration of fetal nucleic acid is used in conjunction with a diagnostic method to determine a fetal trait, wherein the diagnostic method requires a concentration of fetal nucleic acid to meet certain clinical sensitivity or specificity requirements.

10. 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 and if present, determining the amount of Y-chromosome nucleic acid for a male fetus.

11. 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 digests nucleic acid at non-methylated sites, thereby enriching methylated nucleic acid;

b) amplifying in a single reaction vessel nucleic acid not digested in (a) at loci consisting of a locus comprising a polynucleotide of SEQ ID NO;

42 or a portion thereof;

a locus comprising a polynucleotide of SEQ ID NO;

52 or a portion thereof and a locus comprising a polynucleotide of SEQ ID NO;

163 or a portion thereof, thereby generating amplification products; and

c) determining the amount of the fetal nucleic acid in the sample based on the amplification products of SEQ ID NO;

42 or a portion thereof, SEQ ID NO;

52 or a portion thereof, and SEQ ID NO;

163 or a portion thereof in (b).

12. The method of claim 11, wherein determining the amount of fetal nucleic acid comprises use of a PCR-based method.

13. The method of claim 12, wherein the PCR-based method is real time PCR.

14. The method of claim 11, wherein determining the amount of fetal nucleic acid comprises use of one or more processes selected from mass spectrometry methods, RT-PCR, digital PCR, array-based methods, sequencing methods, nanopore-based methods, nucleic acid-bound bead-based counting methods, competitor-based methods and methods for separating nucleic acid using agents that bind nucleic acid based on methylation status.

15. The method of claim 11, wherein determining the amount of fetal nucleic acid comprises introducing one or more competitors at known concentrations in (b).

16. The method of claim 15, comprising determining the absolute amount of fetal nucleic acid in the sample.

17. The method of claim 11, further comprising determining the amount of total nucleic acid present in the sample.

18. The method of claim 17, comprising determining the concentration of fetal nucleic acid in the sample by comparing the amount of fetal nucleic acid to the total amount of nucleic acid.

19. The method of claim 18, wherein the concentration of fetal nucleic acid is used in conjunction with a diagnostic method to determine a fetal trait, wherein the diagnostic method requires a concentration of fetal nucleic acid to meet certain clinical sensitivity or specificity requirements.

20. The method of claim 11, 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; and

if present, determining the amount of Y-chromosome nucleic acid for a male fetus.