MICROARRAY SYSTEM WITH IMPROVED SEQUENCE SPECIFICITY

• US 20090143243A1
• Filed: 08/12/2008
• Published: 06/04/2009
• Est. Priority Date: 08/12/2007
• Status: Active Grant

• Current Assignee: Integrated DNA Technologies (Danaher Corporation)
• Original Assignee: Integrated DNA Technologies (Danaher Corporation)
• Inventors: Behlke, Mark Aaron, Gunning, Kerry B.
• Granted Patent: US 8,067,164 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: Y10T 436/108331 Preservative, buffer, anticoagulant or diluent

Abstract

The invention provides a novel array method for nucleic acid sequence detection with improved specificity which allows for detection of genetic variation, from simple SNPs (where the variation occurs at a fixed position and is of limited allelic number) to more complex sequence variation patterns (such as with multigene families or multiple genetic strains of an organism where the sequence variation between the individual members is neither fixed nor consistent). The array is comprised of short, synthetic oligonucleotide probes attached to a solid surface which are hybridized to single-stranded targets. Single stranded targets can be produced using a method that employs primers modified on the 5′ end to prohibit degradation by a 5′-exonuclease that is introduced to degrade the unprotected strand. The invention further provides for printing buffers/solutions for the immobilization of oligonucleotide probes to an array surface. The invention also provides hybridization and wash buffers and conditions to maximize hybridization specificity and signal intensity, and reduce hybridization times.

Claims

1. A method of preparing a detectably labeled single-stranded polynucleotide target from a double stranded DNA comprising the detectably labeled polynucleotide target hybridized to a complementary polynucleotide comprising:

contacting the double stranded DNA with a 5′

to 3′

exonuclease under suitable conditions to degrade at least a portion of the complementary polynucleotide to form a detectably labeled single-stranded polynucleotide target, the polynucleotide target comprising at least one of;

(1) a cyanine dye moiety positioned between the second and third nucleotides from the 5′

end of the polynucleotide target;

or

(2) a cyanine dye moiety positioned between first and second nucleotides from the 5′

end of the polynucleotide target and having a modified linkage between the second and third nucleotides from the 5′

end of the polynucleotide target;

or

(3) a cyanine dye moiety attached at the 5′

end of the polynucleotide target, with the complementary strand modified with;

(a) a 5′

-phosphate group;

or

(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′

-residue of said polynucleotide being an A or a T base;

or

(4) a dye moiety attached at the 5′

end of the polynucleotide target and the first 5′

-residue of said polynucleotide being a G or C base, with the complementary strand modified with;

(a) a 5′

-phosphate group;

or

(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′

-residue of said polynucleotide being an A or a T base.

2. The method of claim 1, the polynucleotide target comprising a cyanine dye moiety positioned between first and second nucleotides from the 5′

end of the polynucleotide target and having a modified linkage between the second and third nucleotides from the 5′

end of the polynucleotide target, wherein the modified linkage comprises a nuclease resistant bond.

3. The method of claim 2, wherein the nuclease resistant bond is a phosphorothioate bond.

4. The method of claim 1, wherein the polynucleotide target comprises a cyanine dye moiety positioned between the second and third nucleotides from the 5′

end of the polynucleotide target and the 5′

-3′

exonuclease is T7 exonuclease.

5. The method of claim 1, wherein the polynucleotide target comprises a cyanine dye moiety positioned between first and second nucleotides from the 5′

end of the polynucleotide target and has a modified linkage between the second and third nucleotides from the 5′

end of the polynucleotide target, and wherein the 5′

-3′

exonuclease is T7 exonuclease.

6. The method of claim 1, wherein the polynucleotide target comprises a cyanine dye moiety attached at the 5′

end of the polynucleotide target, wherein the complementary strand is modified with a 5′

-phosphate group or a primary amine with an aliphatic linker arm connected to the complementary polynucleotide via a phosphate linkage and the first 5′

-residue of the complementary polynucleotide is an A or a T base, and wherein the 5′

-3′

exonuclease is lambda exonuclease.

7. The method of claim 1, wherein the polynucleotide target comprises a dye moiety attached at the 5′

end of the polynucleotide target and the first 5′

-residue of the target polynucleotide is a G or C base, wherein the complementary strand is modified with a 5′

-phosphate group or a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage, the first 5′

-residue of said polynucleotide being an A or a T base, and wherein the 5′

-3′

exonuclease is lambda exonuclease.

8. The method of claim 1 wherein the modified nucleic acids are produced using single-stranded oligonucleotide primers comprising the modifications listed using the polymerase chain reactions to enzymatically produce the double-stranded DNA product.

30. A method of detecting the presence or absence of hybridization of a detectably labeled single-stranded polynucleotide target to one or more oligonucleotide probes attached to a support comprising:

contacting the support with the target under conditions that allow hybridization of the target to the probe, wherein the target was generated by the method of claim 1;

removing unhybridized target from the support by washing the support; and

detecting the presence or absence of labeled target on the washed support.

31. The method of claim 30, wherein the support is contacted with the target under relatively high stringency conditions.

32. The method of claim 30, wherein the conditions include TMAC in a concentration in the range of from about 2M to about 3M.

33. The method of claim 30, wherein the conditions include Sarkosyl in a concentration in the range of from about 0.1% to about 1%.

34. The method of any of claim 30, wherein the probes are the same length.

35. The method of any of claim 30, wherein the target comprises at least one phosphorothioate group on the 5′

end of the target.

36. The method of any of claim 30, wherein the probes are from 9 to 25 bases in length.

37. The method of claim 36, wherein the probes are from 15 to 19 bases in length.

38. The method of claim 36, wherein the probes are 17 bases in length.

39. The method of any of claim 30, wherein the probes are deposited onto the support using a spotting buffer comprises a monobasic sodium phosphate, an ethylene oxide based non-ionic detergent, and ethylene glycol.

40. The method of claim 39, wherein the ethylene oxide based non-ionic detergent is Nonidet P-40.

41. The method of any of claim 30, wherein the probes comprise a hydrazide modification through which they are attached to the support.

42. The method of any of claim 30, wherein the support comprises epoxide moieties.

9. A method of generating a double stranded DNA comprising a detectably labeled polynucleotide target hybridized to a complementary polynucleotide, the detectably labeled the polynucleotide target comprising at least one of the following modifications:

(1) a cyanine dye moiety positioned between the second and third nucleotides from the 5′

end of the polynucleotide target;

or

(2) a cyanine dye moiety positioned between first and second nucleotides from the 5′

end of the polynucleotide target and having a modified linkage between the second and third nucleotides from the 5′

end of the polynucleotide target;

or

(3) a cyanine dye moiety attached at the 5′

end of the polynucleotide target, with the complementary strand modified with;

(a) a 5′

-phosphate group;

or

(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′

-residue of said polynucleotide being an A or a T base;

or

(4) a dye moiety attached at the 5′

end of the polynucleotide target and the first 5′

-residue of said polynucleotide being a G or C base, with the complementary strand modified with;

(a) a 5′

-phosphate group;

or

(b) a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage and the first 5′

-residue of said polynucleotide being an A or a T base;

the method comprising;

amplifying the polynucleotide target and the complementary polynucleotide from a sample comprising the polynucleotide target and complementary polynucleotides using oligonucleotide primers comprising the modification or modifications.

10. The method of claim 9, the polynucleotide target comprising a cyanine dye moiety positioned between first and second nucleotides from the 5′

end of the polynucleotide target and having a modified linkage between the second and third nucleotides from the 5′

end of the polynucleotide target, wherein the modified linkage comprises a nuclease resistant bond.

11. The method of claim 10, wherein the nuclease resistant bond is a phosphorothioate bond.

12. The method of claim 9, wherein the polynucleotide target comprises a cyanine dye moiety positioned between the second and third nucleotides from the 5′

end of the polynucleotide target.

13. The method of claim 9, wherein the polynucleotide target comprises a cyanine dye moiety positioned between first and second nucleotides from the 5′

end of the polynucleotide target and has a modified linkage between the second and third nucleotides from the 5′

end of the polynucleotide target.

14. The method of claim 9, wherein the polynucleotide target comprises a cyanine dye moiety attached at the 5′

end of the polynucleotide target, wherein the complementary strand is modified with a 5′

-phosphate group or a primary amine with an aliphatic linker arm connected to the complementary polynucleotide via a phosphate linkage and the first 5′

-residue of the complementary polynucleotide is an A or a T base.

15. The method of claim 9, wherein the polynucleotide target comprises a dye moiety attached at the 5′

end of the polynucleotide target and the first 5′

-residue of the polynucleotide target is a G or C base, wherein the complementary strand is modified with a 5′

-phosphate group or a primary amine with an aliphatic linker arm connected to the polynucleotide via a phosphate linkage, the first 5′

-residue of said polynucleotide being an A or a T base.

16. An array comprising a surface comprising epoxide moieties and a plurality of oligonucleotides each comprising a 5′

hydrazide linker attached to the surface of the array through a bond formed between the linker and an epoxide moiety.

17. A method of forming a microarray on a surface comprising epoxide moieties, comprising:

depositing a plurality of samples onto the surface in discrete domains, each sample comprising a spotting buffer and an oligonucleotide comprising a 5′

hydrazide linker, under conditions that allow formation of a bond between the hydrazide linker and the epoxide moiety.

18. The method of claim 17 wherein the spotting buffer having a pH of less than about 8.5 and comprising monobasic sodium phosphate, an ethylene oxide based nonionic detergent, and ethylene glycol.

19. The method of claim 18, wherein the nonionic detergent is Nonidet P-40.

20. The method of any of claim 18, wherein the pH is in the range of from about 4.0 to about 8.0.

21. The method of 20, wherein the pH is in the range of from about 4.5 to about 5.5.

22. A buffer having a pH in the range of from about 4.0 to about 8.0 and comprising sodium phosphate (monobasic) in a concentration in the range of from about 1 mM to about 1M, an ethylene oxide based nonionic detergent in a concentration in the range of from about 0.001% to about 1% (v/v), and ethylene glycol in a concentration in the range of from about 10% to about 90% (v/v).

23. The buffer of claim 22, wherein nonionic detergent is Nonidet P-40.

24. The buffer of claim 22, wherein the pH is in the range of from about 4.5 to about 5.5.

25. The buffer of claim 22, wherein the buffer has a pH of about 4.5 and comprises 300 mM sodium phosphate (monobasic), 0.01% NP-40, and 45% ethylene glycol.

26. A method of detecting the presence of a specific nucleic acid sequence within a pool of detectably labeled target polynucleotides by hybridization to one or more oligonucleotide probes attached to a support comprising:

contacting the support with the polynucleotide under high stringency conditions in a buffer comprising a tertiary alkyl ammonium salt and formamide for a period of about 6 hours or less;

removing unhybridized target polynucleotides from the support by washing the support under conditions similar to those used during the hybridization for a period about 30 minutes or less; and

detecting the presence or absence of labeled target polynucleotides on the washed support.

27. The method of claim 26, wherein the high stringency conditions include TMAC in a concentration of from about 2M to about 3M.

28. The method of claim 27, wherein the concentration of TMAC is about 2.5M.

29. The method of claim 26, wherein the conditions further comprise the presence of Sarkosyl in a concentration in the range of from about 0.1% to about 1%.