METHODS OF NONSPECIFIC TARGET CAPTURE OF NUCLEIC ACIDS

• US 20080286775A1
• Filed: 08/01/2007
• Published: 11/20/2008
• Est. Priority Date: 08/01/2006
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: BECKER, Michael M., MAJLESSI, Mehrdad R.
• Granted Patent: US 9,051,601 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 2310/18 acting by a non-sequence specific mechanism other than C12N2310/16 or C12N2310/17
• CPC Class Codes: C12N 2310/31 of the backbone
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2330/30 chemically synthesised
• 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 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

Methods for capturing a target nucleic acid from a sample by using a capture probe that binds nonspecifically to the target nucleic acid and binds specifically to an immobilized probe via a specific binding pair that has one member on the capture probe and one member on the immobilized probe are disclosed. Compositions that include a capture probe that binds nonspecifically to a target nucleic acid and specifically to an immobilized probe via binding of members of a specific binding pair in a solution phase of a reaction mixture are disclosed.

Claims

1. A method for isolating a target nucleic acid from a sample, comprising:

mixing a sample containing a target nucleic acid with a nonspecific capture probe made up of an oligonucleotide sequence that hybridizes nonspecifically with the target nucleic acid and a means for linking the target nucleic acid to a support, wherein the oligonucleotide sequence is selected from the group consisting of;

a poly-U sequence,

a random poly-(k) sequence comprising G and T nucleotides or G and U nucleotides, two random oligonucleotide sequences separated by a non-random sequence consisting of 10 or fewer nucleotides,

two random oligonucleotide sequences separated by a non-random sequence consisting of 10 or fewer nucleotide base analogs,

two random oligonucleotide sequences separated by a non-nucleotide spacer compound, a plurality of random oligonucleotide sequences separated by non-random sequences consisting of 10 or fewer nucleotides or nucleotide base analogs, a plurality of random oligonucleotide sequences separated by non-nucleotide spacer compounds, and

a non-random poly-GU sequence made up of at least two GU units, incubating a reaction mixture containing the support, the target nucleic acid, and the nonspecific capture probe in hybridization conditions that allow nonspecific hybridization of the capture probe and the target nucleic acid to form a hybridization complex linked to the support, and

separating the support from a solution phase of the reaction mixture to separate the hybridization complex linked to the support from other sample components, thereby isolating the target nucleic acid from other sample components.

2. The method of claim 1 wherein the means for linking the target nucleic acid to the support is direct attachment of the capture probe to the support.

3. The method of claim 1 wherein the means for linking the target nucleic acid to the support is a specific binding pair made up of a first specific binding partner (SBP) attached to the capture probe that binds specifically to a second specific binding partner (SBP′

) that is linked to the support.

4. The method of claim 3 wherein the SBP′

is part of an immobilized probe that is linked to the support.

5. The method of claim 3 wherein the mixing step includes mixing the sample with the SBP attached to the capture probe and the SBP′

linked to the support, and the incubating step includes binding the SBP and the SBP′

to link the hybridization complex to the support.

6. The method of claim 3 wherein the SBP and the SBP′

are substantially complementary nucleic acid sequences.

7. The method of claim 3 wherein the SBP and the SBP′

are non-nucleic acid moieties.

8. The method of claim 7 wherein the non-nucleic acid moieties are selected from the group consisting of:

(a) a receptor and ligand pair, (b) an enzyme and substrate pair, (c) an enzyme and cofactor pair, (d) an enzyme and coenzyme pair, (e) an antibody and antigen pair, (f) an antibody fragment and antigen pair, (g) a sugar and lectin pair, (h) a ligand and chelating agent pair, (i) biotin and avidin, (j) biotin and streptavidin, and (k) nickel and histidine.

9. The method of claim 1 further comprising a washing step after the separating step to remove other sample components from the hybridization complex linked to the support, wherein the washing step mixes the hybridization complex linked to the support with a washing solution and then separates the hybridization complex linked to the support from the washing solution.

10. The method of claim 1 wherein the incubating step is performed at about 25°

C. for about 5 minutes to about 90 minutes.

11. The method of claim 1 further comprising a step after the separating step of detecting the presence of the target nucleic acid isolated from other sample components, amplifying in vitro a sequence contained in the target nucleic acid isolated from other sample components, or determining a sequence contained the target nucleic acid isolated from other sample components.

12. A nonspecific capture probe comprising a first specific binding partner (SBP) that binds specifically to a second specific binding partner (SBP′

) linked to a support and an oligonucleotide sequence that hybridizes nonspecifically to a target nucleic acid, wherein the oligonucleotide sequence is selected from the group consisting of;

a poly-U sequence,

a random poly-(k) sequence comprising G and T nucleotides or G and U nucleotides,

two random oligonucleotide sequences separated by a non-random sequence consisting of 10 or fewer nucleotides,

two random oligonucleotide sequences separated by a non-random sequence consisting of 10 or fewer nucleotide base analogs,

a plurality of random oligonucleotide sequences separated by non-random sequences consisting of 10 or fewer nucleotides or nucleotide base analogs,

two random oligonucleotide sequences separated by a non-nucleotide spacer compound,

a plurality of random oligonucleotide sequences separated by non-nucleotide spacer compounds, and

a non-random poly-GU sequence made up of at least two GU units.

13. The capture probe of claim 12 wherein the oligonucleotide sequence of about 5 to 100 nucleotides in length.

14. The capture probe of claim 12 wherein the oligonucleotide sequence of about 12 to about 25 nucleotides in length.

15. The capture probe of claim 12 wherein the oligonucleotide sequence contains:

(a) standard RNA bases and linkages, (b) standard DNA bases and linkages, (c) RNA bases with 2′

modified linkages, (d) DNA bases in which at least part of the sequence is in a locked nucleic acid (LNA) conformation, (e) one or more nucleotide base analogs, (f) one or more abasic residues, (g) one or more non-nucleic acid spacer compounds, or (h) a combination of elements selected from groups (a) to (g) above.

16. The capture probe of claim 15 wherein the base analogs exhibit alternative base pairing properties compared to standard DNA or RNA base pairing.

17. The capture probe of claim 16 wherein the base analogs are inosine or 5-nitroindole.

18. The capture probe of claim 15 wherein the oligonucleotide sequence contains a mixture of nucleotides in LNA conformation and standard DNA conformation.

19. The capture probe of claim 15 wherein poly(k) bases are the RNA bases with 2′

modified linkages.

20. The capture probe of claim 12 wherein the SBP that binds specifically to the SBP′

is a member of a specific binding pair selected from the group consisting of;

(a) a pair of complementary nucleic acid sequences, (b) a receptor and ligand pair, (c) an enzyme and substrate pair, (d) an enzyme and cofactor pair, (e) an enzyme and coenzyme pair, (t) an antibody and antigen pair, (g) an antibody fragment and antigen pair, (h) a sugar and lectin pair, (i) a ligand and chelating agent pair, (j) biotin and avidin, (k) biotin and streptavidin, and (l) nickel and histidine.

21. The capture probe of claim 12 wherein the oligonucleotide sequence is linked to the SBP at a 5′

terminal position of the oligonucleotide, a 3′

terminal position of the oligonucleotide, or at an internal position of the oligonucleotide via a linker compound.

22. The capture probe of claim 12 wherein the oligonucleotide sequence comprises at least one random poly-(k)₆ sequence.

23. The capture probe of claim 12 wherein the oligonucleotide sequence comprises a random poly-(k)₁₂, poly-(k)₁₈, or poly-(k)₂₅ sequence.

24. The capture probe of claim 12 wherein the oligonucleotide sequence is selected from the group consisting of:

(n)₆-U₃-(n)₆,

(n)₆-Ni₅-(n)₆ in which “

Ni”

stands for 5-nitroindole,

(k)₆-Ni₅-(k)₆ in which “

Ni”

stands for 5-nitroindole,

(k)₆-C9-C9-(k)₆ in which “

C9”

stands for a 9 carbon non-nucleotide spacer,

(k)₆-C9-(k)₆-C9-(k)₆ in which “

C9”

stands for a 9 carbon non-nucleotide spacer,

(k)₁₂,

(k)₆-U₃-(k)₆,

(k)₆-U₆-(k)₆,

(k)₆-T₃-(k)₆,

(k)₁₈,

(k)₂₄,

d(G or T)₁₈,

L(k)₆-dT₃-L(k)₆,

L(k)₄-d(k)₂-dT₃-L(k)₄-d(k)₂,

U₁₈,

L(k)₄-d(k)₃-L(k)₄-d(k)₃-L(k)₄,

L(k)₃-d(k)₃-L(k)₃-d(k)₃-L(k)₃-d(k)₃,

(GU)₉,

L(k)₆-dT₃-L(k)₆,

L(k)₄-d(k)₂-dT₃-L(k)₄-d(k)₂,

L(k)₂-d(k)₄-L(k)₂-d(k)₄-L(k)₂-d(k)₄,

d(k)₆-dT₃-L(k)₆, and

L(k)₄-d(k)₃-L(k)₄-d(k)₃-L(k)₄-d(k)₃-L(k)₄.

25. The capture probe of claim 24 wherein the oligonucleotide sequence is joined to a homopolymeric nucleic acid sequence that is the SBP.