Quantification of a minority nucleic acid species

• US 8,460,872 B2
• Filed: 04/27/2012
• Issued: 06/11/2013
• 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:
  US13/458,341
• Publication Number:
  US 20120322072A1
• Time in Patent Office: 410 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 a minority nucleic acid species in a sample comprising:

(a) contacting under amplification conditions a nucleic acid sample comprising a minority species and a majority species, the combination of the minority species and the majority 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 minority nucleic acid species and is not present in the majority nucleic acid species, or (2) is not present in the minority nucleic acid species and is present in the majority nucleic acid species,

(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 minority 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 minority nucleic acid amplification products;

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

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

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

3. The method of claim 1, wherein the first and second regions each comprise one or more genomic loci, wherein the first region comprises one or more loci that are differentially methylated between the minority species and majority species and the second region comprises one or more loci which do not contain a restriction site for a methylation-sensitive restriction enzyme.

4. The method of claim 1, wherein the first and second amplification primer sets comprise one or more primer pairs comprising a 5′

tail having a distinct length for each amplification primer set.

5. 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.

6. The method of claim 5, 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.

7. The method of claim 6, wherein the competitor oligonucleotides comprise a stuffer sequence comprising a nucleotide sequence from a non-human genome.

8. The method of claim 5, further comprising determining the copy number of the minority nucleic acid based on a predetermined copy number of competitor oligonucleotide used.

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

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

11. The method of claim 1, wherein the sample nucleic acid is extracellular nucleic acid.

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

mismatched nucleotides.

12. A method for determining the copy number of a minority nucleic acid species in a sample comprising:

(a) contacting under amplification conditions a nucleic acid sample comprising a minority species and a majority species, the combination of the minority species and the majority 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 minority nucleic acid species and is not present in the majority nucleic acid species, or (2) is not present in the minority nucleic acid species and is present in the majority nucleic acid species,

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

(iii) one or more first competitor oligonucleotides that compete with the first region for hybridization of primers of the first amplification primer set, and

(iv) one or more second competitor oligonucleotides that compete with the second region for hybridization of primers of the second amplification primer set, thereby generating minority nucleic acid amplification products, total nucleic acid amplification products, first competitor amplification products and second competitor amplification products, wherein each of the minority nucleic acid, total nucleic acid, first competitor and second competitor amplification products are of different lengths;

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

(c) determining the copy number of the minority nucleic acid species in the sample based on the separated amplification products.

13. The method of claim 12, further comprising contacting the nucleic acid sample with one or more methylation sensitive restriction enzymes prior to (a).

14. The method of claim 12, wherein the first and second regions each comprise one or more genomic loci, wherein the first region comprises one or more loci that are differentially methylated between the minority species and majority species and the second region comprises one or more loci which do not contain a restriction site for a methylation-sensitive restriction enzyme.

15. The method of claim 12, wherein the first and second amplification primer sets comprise one or more primer pairs comprising a 5′

tail having a distinct length for each amplification primer set.

16. The method of claim 12, further comprising contacting under amplification conditions the nucleic acid sample with one or more inhibitory oligonucleotides that reduce the amplification of the second region.

17. The method of claim 12, further comprising determining the fraction of the minority nucleic acid in the sample based on the amount of each of the separated minority and total nucleic acid amplification products.

18. The method of claim 12, wherein the competitor oligonucleotides comprise a stuffer sequence comprising a nucleotide sequence from a non-human genome.

19. The method of claim 12, wherein the amplification is in a single reaction vessel.

20. The method of claim 12, wherein the separation of amplification products is performed using electrophoresis.

21. The method of claim 12, wherein the sample nucleic acid is extracellular nucleic acid.

23. The method of claim 16, wherein the inhibitory oligonucleotide comprises one or more 3′

mismatched nucleotides.