SELECTIVE AMPLIFICATION USING BLOCKING OLIGONUCLEOTIDES

• US 20180216174A1
• Filed: 01/19/2018
• Published: 08/02/2018
• Est. Priority Date: 02/01/2017
• Status: Active Grant

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Cellular Research, Inc. (Becton, Dickinson & Co)
• Inventors: Shum, Eleen, Fu, Glenn K.
• Granted Patent: US 11,319,583 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• 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 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Disclosed herein include methods and compositions for selectively amplifying and/or extending nucleic acid target molecules in a sample. The methods and compositions can, for example, reduce the amplification and/or extension of undesirable nucleic acid species in the sample, and/or allow selective removal of undesirable nucleic acid species in the sample.

Claims

1. A method of selective amplification, comprising:

providing a sample comprising a plurality of nucleic acid target molecules and one or more undesirable nucleic acid species;

providing a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a molecular label sequence and a binding region;

contacting the plurality of oligonucleotide probes with the plurality of nucleic acid target molecules for hybridization;

extending oligonucleotide probes that are hybridized to the plurality of nucleic acid target molecules to generate a plurality of extension products;

providing a blocking oligonucleotide that specifically binds to at least one of the one or more undesirable nucleic acid species; and

amplifying the plurality of extension products to generate a plurality of amplicons,

whereby the amplification or the extension of the undesirable nucleic acid species is reduced by the blocking oligonucleotide.

2. The method of claim 1, wherein the blocking oligonucleotide comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNA/DNA chimera, a PNA/DNA chimera, or any combination thereof.

3. The method of claim 1, comprising providing blocking oligonucleotides that specifically binds to two or more, at least 10, at least 100, or any combination thereof, undesirable nucleic acid species in the sample.

4. The method of claim 1, wherein the blocking oligonucleotide has a T_m of at least 60°

C., 65°

C., or 70°

C.

5. The method of claim 1, wherein the blocking oligonucleotide is unable to function as a primer for a reverse transcriptase or a polymerase.

6. The method of claim 1, wherein the amplification or the extension of the undesirable nucleic acid species is reduced by at least 50%, 80%, 90%, 95%, or 99%.

7. The method of claim 1, wherein the one or more undesirable nucleic acid species amounts to about 50%, 60%, 70%, or 80%, of the nucleic acid content of the sample.

8. The method of claim 1, wherein the undesirable nucleic acid species comprise rRNA, mtRNA, genomic DNA, intronic sequence, high abundance sequence, or any combination thereof.

9. The method of claim 1, wherein the blocking oligonucleotides specifically bind to within 100 nt of the 3′

end of the one or more undesirable nucleic acid species, within 100 nt of the 5′

end of the one or more undesirable nucleic acid species, within 100 nt of the middle of the one or more undesirable nucleic acid species, or any combination thereof.

10. The method of claim 1, wherein the amplifying comprises PCR amplification of the plurality of extension products.

11. The method of claim 1, wherein the plurality of oligonucleotide probes comprises at least 100, 1000, or 10000, different molecular label sequences.

12. The method of claim 1, wherein the plurality of amplicons comprises a cDNA library.

13. The method of claim 1, wherein the sample comprises a single cell, a plurality of cells, a tissue sample, or any combination thereof.

14. The method of claim 1, further comprising sequencing the plurality of amplicons.

15. The method of claim 1, wherein the undesirable nucleic acid species represents less than 50%, 20%, 10%, or 5%, of the plurality of amplicons.

16. The method of claim 1, wherein the plurality of oligonucleotide probes are immobilized on a particle.

17. The method of claim 1, wherein the plurality of nucleic acid target molecules comprises mRNA target molecules.

18. The method of claim 1, wherein the binding region comprises poly-dT sequence.

19. A method of selective extension, comprising:

providing a sample comprising a plurality of nucleic acid target molecules and one or more undesirable nucleic acid species;

providing a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a molecular label sequence and a binding region;

contacting the plurality of oligonucleotide probes with the plurality of nucleic acid target molecules for hybridization;

providing a blocking oligonucleotide that specifically binds to at least one of the one or more undesirable nucleic acid species; and

extending oligonucleotide probes that are hybridized to the plurality of nucleic acid target molecules to generate a plurality of extension products;

whereby the extension of the undesirable nucleic acid species is reduced by the blocking oligonucleotide.

20. The method of claim 19, wherein the blocking oligonucleotide comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNA/DNA chimera, a PNA/DNA chimera, or any combination thereof.

21. The method of claim 19, further comprising removing the hybridized complex formed between the blocking oligonucleotide and the undesirable nucleic acid species.

22. The method of claim 21, wherein the removing comprises immobilizing the hybridized complex formed between the blocking oligonucleotide and the undesirable nucleic acid species on a solid support.

23. The method of claim 21, wherein the blocking oligonucleotide comprises an affinity moiety.

24. The method of claim 24, wherein the solid support comprises a binding partner of the affinity moiety.

25. The method of claim 24, wherein the affinity moiety comprises biotin, streptavidin, heparin, an aptamer, a click-chemistry moiety, digoxigenin, primary amine(s), carboxyl(s), hydroxyl(s), aldehyde(s), ketone(s), or any combination thereof.

26. A kit for selective amplification of a plurality of nucleic acid target molecules in a sample comprising:

a plurality of oligonucleotide probes, wherein each of the plurality of oligonucleotide probes comprises a molecular label sequence and a binding region; and

a plurality of blocking oligonucleotides that specifically binds to a plurality of undesirable nucleic acid species in the sample, wherein each blocking oligonucleotide probe is unable to function as a primer for a reverse transcriptase or a polymerase.

27. The kit of claim 26, wherein the blocking oligonucleotide comprises a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA/PNA chimera, an LNA/DNA chimera, a PNA/DNA chimera, or any combination thereof.

28. The kit of claim 26, wherein the blocking oligonucleotide is 10 nt to 50 nt long, 20 nt to 30 nt long, or about 25 nt long.

29. The kit of claim 26, wherein each of the plurality of oligonucleotide probes comprises a cell label sequence, a sample label sequence, a location label sequence, a binding site for a universal primer, or any combination thereof.

30. The kit of claim 26, further comprising an enzyme, wherein the enzyme comprises a reverse transcriptase, a polymerase, a ligase, a nuclease, or any combination thereof.