Multiplexed sequential ligation-based detection of genetic variants

• US 9,624,490 B2
• Filed: 12/07/2015
• Issued: 04/18/2017
• Est. Priority Date: 07/19/2012
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Ariosa Diagnostics Incorporated (Roche Holding AG)
• Inventors: Zahn, Jacob, Oliphant, Arnold, Mohseni, Morassa
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US14/961,561
• Publication Number:
  US 20160083721A1
• Time in Patent Office: 498 Days
• Field of Search: 435 612, 435 912
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2531/137 Ligase Chain Reaction [LCR]
• CPC Class Codes: C12Q 2537/149 Sequential reactions

Abstract

The present invention provides multiplexed sequential ligation-based analysis of genetic variants in a mixed sample, including copy number variations and single nucleotide polymorphisms. The invention employs the techniques of sequential ligation and amplification.

Claims

1. A method for identifying a genomic region of interest from a single source in a sample comprising DNA from two different sources, comprising the steps of:

providing a sample comprising DNA from two different sources;

introducing to the sample a first set of oligonucleotide probes comprising a first fixed sequence oligonucleotide complementary to a 3′

region in a genomic region of interest and a second fixed sequence oligonucleotide complementary to a 5′

region in the genomic region of interest;

hybridizing the first set of oligonucleotide probes to the genomic region of interest in the sample;

ligating the hybridized oligonucleotides of the first set of oligonucleotide probes to create first ligation products complementary to the genomic region of interest;

introducing to the first ligation products a second set of oligonucleotide probes comprising a first fixed sequence oligonucleotide complementary to a 3′

region in the first ligation product and a second fixed sequence oligonucleotide complementary to a 5′

region in the first ligation product;

hybridizing the second set of oligonucleotide probes to the first ligation products;

ligating the hybridized oligonucleotides of the second set of oligonucleotide probes to create second ligation products complementary to the first ligation products, wherein junctions for ligation for the second set of oligonucleotides probes are different than junctions for ligation for the first set of oligonucleotides probes;

amplifying the second ligation products to create amplification products; and

analyzing the amplification products, wherein the analysis of the amplification products identifies the genomic region of interest from the single source in the sample.

2. The method of claim 1, wherein at least one fixed sequence oligonucleotide of the second set of oligonucleotide probes comprises a complementary region that overlaps a ligation junction of the first ligation products.

3. The method of claim 1, wherein both the first and second fixed sequence oligonucleotides of the second set of oligonucleotide probes comprise a complementary region that overlaps a ligation junction of the first ligation products.

4. The method of claim 1, wherein at least one fixed sequence oligonucleotide of the second set of oligonucleotide probes comprises a region that is complementary to the genomic region of interest.

5. The method of claim 1, wherein both fixed sequence oligonucleotides of the second set of oligonucleotide probes comprise a region that is complementary to the genomic region of interest.

6. The method of claim 1, further comprising a step of amplifying the first ligation products after the first ligating step and before the second introducing step.

7. The method of claim 6, wherein the amplification of the first ligation products is linear or exponential.

8. The method of claim 1, wherein the method is performed for two or more genomic regions of interest from the single source.

9. The method of claim 8, wherein (a) at least 24 different regions of interest are interrogated;

(b) at least 46 different regions of interest are interrogated;

or (c) at least 92 different regions of interest are interrogated.

10. The method of claim 1, further comprising introducing to the sample (a) one or more bridging oligonucleotides that hybridize to the genomic region of interest between and adjacent to the first and second fixed sequence oligonucleotides of the first set of oligonucleotide probes;

and/or (b) one or more bridging oligonucleotides that hybridize to the first ligation product between and adjacent to the first and second fixed sequence oligonucleotides of the second set of oligonucleotide probes.

11. The method of claim 1, wherein the amplification of the second ligation products is linear or exponential.

12. A method for detecting a nucleic acid associated with active or latent infection in an individual, comprising the steps of:

introducing to a sample provided by the individual a first set of oligonucleotide probes comprising a first fixed sequence oligonucleotide complementary to a 3′

region in a nucleic acid associated with active or latent infection and a second fixed sequence oligonucleotide complementary to a 5′

region in a nucleic acid associated with active or latent infection;

hybridizing the first set of oligonucleotide probes to the nucleic acid associated with active or latent infection in the sample;

ligating the hybridized oligonucleotides of the first set of oligonucleotide probes to create first ligation products complementary to the nucleic acid associated with active or latent infection;

introducing to the first ligation products a second set of oligonucleotide probes comprising a first fixed sequence oligonucleotide complementary to a 3′

region in the first ligation product and a second fixed sequence oligonucleotide complementary to a 5′

region in the first ligation product;

hybridizing the second set of oligonucleotide probes to the first ligation products;

ligating the hybridized oligonucleotides of the second set of oligonucleotide probes to create second ligation products complementary to the first ligation products;

amplifying the second ligation products to create amplification products; and

analyzing the amplification products, wherein the analysis of the amplification products identifies the nucleic acid associated with active or latent infection in the sample.

13. The method of claim 12, wherein at least one fixed sequence oligonucleotide of the second set of oligonucleotide probes comprises a complementary region that overlaps a ligation junction of the first ligation products.

14. The method of claim 12, wherein both the first and second fixed sequence oligonucleotides of the second set of oligonucleotide probes comprise a complementary region that overlaps a ligation junction of the first ligation products.

15. The method of claim 12, wherein at least one fixed sequence oligonucleotide of the second set of oligonucleotide probes comprises a region that is complementary the nucleic acid associated with active or latent infection.

16. The method of claim 12, wherein both fixed sequence oligonucleotides of the second set of oligonucleotide probes comprise a region that is complementary to the nucleic acid associated with active or latent infection.

17. The method of claim 12, further comprising a step of amplifying the first ligation products after the first ligating step and before the second introducing step.

18. The method of claim 17, wherein the amplification of the first ligation products is linear or exponential.

19. The method of claim 12, wherein the method is performed for two or more nucleic acids associated with active or latent infection.

20. The method of claim 12, further comprising introducing to the sample (a) one or more bridging oligonucleotides that hybridize to the nucleic acid associated with active or latent infection between and adjacent to the first and second fixed sequence oligonucleotides of the first set of oligonucleotide probes;

and/or (b) one or more bridging oligonucleotides that hybridize to the first ligation product between and adjacent to the first and second fixed sequence oligonucleotides of the second set of oligonucleotide probes.

21. The method of claim 12, wherein the active or latent infection causes a sexually transmitted disease.

22. The method of claim 21, wherein the sexually transmitted disease is caused by an infection of an agent selected from the group consisting of herpes simplex virus, gonorrhea, chlamydia, and human papilloma virus.

23. The method of claim 12, wherein the active or latent infection is selected from the group consisting of toxoplasmosis, Hansen disease, listeriosis, influenza, toxoplasmosis, hepatitis B, Coxsackie virus, Epstein-Barr virus, varicella-zoster virus, human parvovirus B19, rubella, cytomegalovirus and Group B Streptococci (GBS).

24. The method of claim 12, wherein the individual is pregnant.

25. The method of claim 12, wherein junctions for ligation for the second set of oligonucleotides probes are different than junctions for ligation for the first set of oligonucleotides probes.