Quantification of a minority nucleic acid species

• US 8,450,061 B2
• Filed: 04/27/2012
• Issued: 05/28/2013
• Est. Priority Date: 04/29/2011
• Status: Active Grant

• Current Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Nygren, Anders
• Primary Examiner(s): Babic, Christopher M.
• Application Number:
  US13/457,978
• Publication Number:
  US 20120276542A1
• Time in Patent Office: 396 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2537/16 Assays for determining copy number or wherein the copy number is of special importance
• CPC Class Codes: C12Q 2545/107 with a competitive internal standard/control
• CPC Class Codes: C12Q 2600/118 Prognosis of disease development
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: C12Q 2600/166 Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

The technology relates in part to quantification of a minority nucleic acid species from a nucleic acid sample. In some embodiments, methods for determining the amount of fetal nucleic acid (e.g. absolute amount, relative amount) in a maternal sample are provided.

Claims

1. A method for determining the amount of fetal nucleic acid in a sample comprising:

(a) contacting under amplification conditions a nucleic acid sample from a pregnant human subject, which sample nucleic acid contains extracellular nucleic acid comprising fetal nucleic acid and maternal nucleic acid, the combination of the fetal species and the maternal species comprising total nucleic acid in the sample, with;

(i) a first set of amplification primers that specifically amplify a first region in sample nucleic acid comprising a feature that (1) is present in the fetal nucleic acid and is not present in the maternal nucleic acid, or (2) is not present in the fetal nucleic acid and is present in the maternal nucleic acid,

(ii) a second set of amplification primers that amplify a second region in the sample nucleic acid allowing for a determination of total nucleic acid in the sample, wherein the first region and the second region are different, and

(iii) one or more inhibitory oligonucleotides, each comprising a nucleotide sequence complementary to a nucleotide sequence in the second region, wherein the one or more inhibitory oligonucleotides (1) hybridize to the second region but are not extended and (2) reduce the amplification of the second region;

thereby generating fetal nucleic acid amplification products and total nucleic acid amplification products, wherein the total nucleic acid amplification products are reduced relative to total amplification products that would be generated if no inhibitory oligonucleotide was present, whereby the total nucleic acid amplification products do not dominate the fetal nucleic acid amplification products;

(b) separating the fetal nucleic acid amplification products and total nucleic acid amplification products, thereby generating separated fetal nucleic acid amplification products and total nucleic acid amplification products; and

(c) determining the fraction of the fetal nucleic acid in the sample based on the amount of each of the separated fetal nucleic acid amplification products and total nucleic acid amplification products.

2. The method of claim 1, wherein the fraction of the fetal nucleic acid in the sample is relative to the total amount of the nucleic acid in the sample.

3. The method of claim 1, wherein the feature that is present in the fetal nucleic acid and not present in the maternal nucleic acid is methylation.

4. The method of claim 3, wherein the first region is methylated.

5. The method of claim 4, wherein the second region is unmethylated.

6. The method of claim 1, further comprising contacting the nucleic acid sample with one or more restriction enzymes prior to (a).

7. The method of claim 6, wherein the one or more restriction enzymes are methylation sensitive.

8. The method of claim 1, further comprising contacting under amplification conditions the nucleic acid sample with a third set of amplification primers that amplify a third region in the sample nucleic acid allowing for a determination of the presence or absence of fetal specific nucleic acid.

9. The method of claim 8, further comprising contacting under amplification conditions the nucleic acid sample with a fourth set of amplification primers that amplify a fourth region in the sample nucleic acid allowing for a determination of the amount of digested or undigested nucleic acid, as an indicator of digestion efficiency.

10. The method of claim 9, wherein the first, second, third and fourth regions each comprise one or more genomic loci.

11. The method of claim 10, wherein the first region comprises one or more loci that are differentially methylated between the fetal and maternal species.

12. The method of claim 10, wherein the second region comprises one or more loci which do not contain a restriction site for a methylation-sensitive restriction enzyme.

13. The method of claim 9, wherein the first, second, third and fourth amplification primer sets comprise one or more primer pairs comprising a 5′

tail having a distinct length for each amplification primer set.

14. The method of claim 1, wherein the inhibitory oligonucleotide and a primer in the second set of amplification primers are complementary to the same nucleotide sequence in the second region.

15. The method of claim 1, wherein the inhibitory oligonucleotide comprises one or more 3′

mismatched nucleotides.

16. The method of claim 1, further comprising contacting under amplification conditions the nucleic acid sample with one or more first competitor oligonucleotides that compete with the first region for hybridization of primers of the first amplification primer set.

17. The method of claim 1, further comprising contacting under amplification conditions the nucleic acid sample with one or more second competitor oligonucleotides that compete with the second region for hybridization of primers of the second amplification primer set.

18. The method of claim 8, further comprising contacting under amplification conditions the nucleic acid sample with one or more third competitor oligonucleotides that compete with the third region for hybridization of primers of the third amplification primer set.

19. The method of claim 9, further comprising contacting under amplification conditions the nucleic acid sample with one or more fourth competitor oligonucleotides that compete with the fourth region for hybridization of primers of the fourth amplification primer set.

20. The method of claim 19, wherein the competitor oligonucleotides comprise a stuffer sequence comprising a nucleotide sequence from a non-human genome.

21. The method of claim 19, wherein the competitor oligonucleotides comprise a stuffer sequence having a constant length for one or more of the first competitor oligonucleotides, second competitor oligonucleotides, third competitor oligonucleotides and fourth competitor oligonucleotides.

22. The method of claim 19, wherein the competitor oligonucleotides comprise a stuffer sequence having a variable length for one or more of the first competitor oligonucleotides, second competitor oligonucleotides, third competitor oligonucleotides and fourth competitor oligonucleotides.

23. The method of claim 19, wherein one or more competitor oligonucleotides comprise a fluorophore.

24. The method of claim 23, wherein the fluorophore is different for each competitor oligonucleotide.

25. The method of claim 16, further comprising determining the copy number of the fetal nucleic acid based on a predetermined copy number of competitor oligonucleotide used.

26. The method of claim 1, wherein the sample nucleic acid is from plasma or serum.

27. The method of claim 1, wherein the amplification is in a single reaction vessel.

28. The method of claim 1, wherein two or more of the amplification products are different lengths.

29. The method of claim 1, wherein the separation of amplification products is performed using electrophoresis.

30. The method of claim 1, wherein the amount of fetal nucleic acid amplification products and the amount of total nucleic acid amplification products are similar or about the same.