Dual polarity analysis of nucleic acids

• US 9,777,312 B2
• Filed: 05/31/2006
• Issued: 10/03/2017
• Est. Priority Date: 06/30/2001
• Status: Expired due to Term

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Rabbani, Elazar, Donegan, James J.
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US11/444,151
• Publication Number:
  US 20070281863A1
• Time in Patent Office: 4,143 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2531/143 Promoter based amplification, e.g. NASBA, 3SR, TAS
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded

Abstract

This invention provides methods for characterizing the amounts of nucleic acids, including plus/minus determinations, the use of different constructs, the use of a library and a reference library. Expression may also be compared in two or more samples using the methods of this invention. Also provided are heterophasic arrays comprising labeled positive copies of nucleic acids hybridized to the array and labeled negative copies of nucleic acids hybridized to the array, in which the labeled positive copies are separately quantifiable from the labeled negative copies.

Claims

1. A method of determining the amounts of RNA transcripts in a sample comprising the steps of:

(i) generating a library of double-stranded DNA constructs from said RNA transcripts by a process comprising the steps of;

(a) synthesizing first strand DNA [−

] copies of said RNA transcripts; and

(b) converting said first strand DNA copies into double-stranded DNA copies by synthesizing second strand DNA [+] copies using said first strand DNA copies as templates,

wherein each double-stranded DNA construct generated comprises a first strand DNA [−

] copy of an RNA transcript of the RNA transcripts and a second strand DNA [+] copy of the RNA transcript;

(ii) preparing

(a) labeled [+] nucleic acid copies using said first strand DNA [−

] copies of the double-stranded DNA constructs as template strands; and

(b) labeled [−

] nucleic acid copies using said second strand DNA [+] copies of the double-stranded DNA constructs as template strands,

wherein said labeled [+] nucleic acid copies and said labeled [−

] nucleic acid copies are complementary to each other, and

wherein said complementary nucleic acid copies are made using the same double stranded DNA constructs;

(iii) after step (ii), hybridizing said labeled [+] nucleic acid copies and said labeled [−

] nucleic acid copies to a nucleic acid array or arrays; and

(iv) measuring the amounts of hybridization of said labeled [+] nucleic acid copies and said labeled [−

] nucleic acid copies to said array or arrays,

wherein the amounts of hybridization of said labeled [+] nucleic acid copies and the amounts of hybridization of said labeled [−

] nucleic acid copies are independently quantified, thereby determining the amounts of said transcripts,

wherein,

(A) said labeled [+] nucleic acid copies and said labeled [−

] nucleic acid copies are generated by transcription from a collection of linear nucleic acid constructs derived from said library, wherein a sequence for a first RNA promoter is located at one end of a linear nucleic acid construct and a sequence for a second RNA promoter is located at the other end of said linear nucleic acid construct, wherein said first RNA promoter and said second RNA promoter are different, and wherein said labeled [+] nucleic acid copies are generated by transcribing from said first promoter and said labeled [−

] nucleic acid copies are generated by transcribing from said second promoter, or

(B) said labeled [+] nucleic acid copies and said labeled [−

] nucleic acid copies are generated by transcription from a collection of linear nucleic acid constructs derived from said library, wherein a first portion of said linear nucleic acid constructs comprises a first RNA promoter directing transcription of said [+] nucleic acid copies and wherein a second portion of said linear nucleic acid constructs comprises a second RNA promoter directing transcription of said [−

] nucleic acid copies, or

(C) said labeled [+] nucleic acid copies and said labeled [−

] nucleic acid copies are synthesized from a collection of linear nucleic acid constructs derived from said library, wherein asymmetric PCR is used to amplify one strand of said linear nucleic acid constructs to provide said labeled [+] nucleic acid copies in one reaction and asymmetric PCR is used to amplify the other strand of said linear nucleic acid constructs to provide said labeled [−

] nucleic acid copies in a second reaction.

2. The method of claim 1, wherein said labeled [+] nucleic acid copies and said labeled [−

] nucleic acid copies are generated by transcription from a collection of linear nucleic acid constructs derived from said library, wherein a sequence for a first RNA promoter is located at one end of a linear nucleic acid construct and a sequence for a second RNA promoter is located at the other end of said linear nucleic acid construct, wherein said first RNA promoter and said second RNA promoter are different, and wherein said labeled [+] nucleic acid copies are generated by transcribing from said first promoter and said labeled [−

] nucleic acid copies are generated by transcribing from said second promoter.

3. The method of claim 1, wherein said labeled [+] nucleic acid copies and said labeled [−

] nucleic acid copies are generated by transcription from a collection of linear nucleic acid constructs derived from said library, wherein a first portion of said linear nucleic acid constructs comprises a first RNA promoter directing transcription of said [+] nucleic acid copies and wherein a second portion of said linear nucleic acid constructs comprises a second RNA promoter directing transcription of said [−

] nucleic acid copies.

4. The method of claim 1, wherein said labeled [+] nucleic acid copies and said labeled [−

] nucleic acid copies are synthesized from a collection of linear nucleic acid constructs derived from said library, wherein asymmetric PCR is used to amplify one strand of said linear nucleic acid constructs to provide said labeled [+] nucleic acid copies in one reaction and asymmetric PCR is used to amplify the other strand of said linear nucleic acid constructs to provide said labeled [−

] nucleic acid copies in a second reaction.

5. The method of claim 2 or 3, wherein said first RNA promoter and said second RNA promoter are the same promoter.

6. The method of claim 5, wherein said first promoter and said second promoter comprise a T7 RNA promoter, a T3 promoter, or an SP6 promoter.

7. The method of claim 5, wherein said first RNA promoter comprises a T7 RNA promoter sequence, a T3 promoter sequence or an SP6 promoter sequence, and said second RNA promoter comprises a T7 RNA promoter sequence, a T3 promoter sequence or an SP6 promoter sequence.

8. The method of claim 1, wherein said array or arrays is a biphasic array or a heterophasic array.

9. The method of claim 1, wherein said label comprises a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound, or a combination of any of the foregoing.

10. The method of claim 1, wherein said RNA transcripts comprise hnRNA, mRNA, or both.

27. The method of claim 8, wherein said array or arrays is a heterophasic array.

28. The method of claim 8, wherein said array or arrays is a biphasic array.

11. A method of analyzing nucleic acids in a sample comprising the steps of

a) providing RNA to be analyzed, said RNA having been divided into a first portion and a second portion;

b) adding a first sequence to the 3′

ends of the first portion of said RNA;

c) binding a set of first primers to said first sequence added to said first portion, wherein said first primers comprise a first RNA promoter sequence;

d) extending said first primers using said first portion of RNA as templates and generating first cDNA copies of said first portion;

e) removing said first portion RNA templates;

f) adding a second sequence to the 3′

ends of said first cDNA copies of said first portion generated in step d);

g) binding a set of second primers to said second sequence added to said first cDNA copies of said first portion;

h) extending said set of second primers using said first cDNA copies of said first portion as templates, to form double-stranded copies of said first portion;

i) adding a third sequence to the 3′

ends of said second portion of said RNA;

j) binding a set of third primers to said third sequence added to said second portion;

k) extending said third primers using said second portion of RNA as templates and generating first cDNA copies of said second portion;

l) removing said second portion RNA templates;

m) adding a fourth sequence to the 3′

ends of said first cDNA copies of said second portion generated in step k);

n) binding a set of fourth primers to said fourth sequence added to said first cDNA copies of said second portion, wherein said fourth primers comprise a second RNA promoter sequence;

o) extending said set of fourth primers using said first cDNA copies of said second portion as templates to form double-stranded copies of said second portion;

p) carrying out a transcription reaction with said double-stranded copies of said first portion made in step h) to generate labeled [−

] copies of said RNA;

q) carrying out a transcription reaction with said double-stranded copies of said second portion made in step o) to generate labeled [+] copies of said RNA, wherein said [+] copies and said [−

] copies are complementary to each other; and

r) hybridizing said labeled [+] copies and said labeled [−

] copies to an array or arrays and separately quantifying the amount of hybridization of said labeled [+] copies and said labeled [−

] copies.

12. The method of claim 11, further comprising a step wherein said RNA is fragmented prior to said addition steps.

13. The method of claim 11 or 12, further comprising a step wherein 3′

ends that are blocked for extension are unblocked.

14. The method of claim 13, wherein said unblocking step comprises treatment with a kinase, or a phosphatase.

15. The method of claim 12, wherein said fragmentation step is carried out by physical, chemical or enzymatic means.

16. The method of claim 15, wherein said enzymatic means comprise treatment with a nuclease or an RNase.

17. The method of claim 11, wherein one or more of said addition steps comprises a ligation step or a polymerization step.

18. The method of claim 11, wherein said first RNA promoter and said second RNA promoter are the same promoter.

19. The method of claim 18, wherein said RNA promoter comprises a T7 RNA promoter sequence, a T3 promoter sequence or an SP6 promoter sequence.

21. The method of claim 11, wherein the label of said [+] copies and the label of said [−

] copies comprise different labels.

22. The method of claim 11, wherein said labeled [+] copies and said labeled [−

] copies are hybridized to the same array or arrays.

23. The method of claim 11, wherein said array or arrays comprise a nucleic acid target at one site and a complementary nucleic acid target at a different site.

20. A method of analyzing nucleic acids in a sample comprising the steps of:

a) providing RNA to be analyzed;

b) adding a first sequence to the 3′

ends of said RNA;

c) binding a set of first primers to said first added sequence wherein said first primers comprise a first RNA promoter sequence;

d) extending said first primers using said RNA as templates and generating first cDNA copies of said RNA;

e) removing said RNA templates;

f) adding a second sequence to the 3′

ends of said first cDNA copies;

g) binding a set of second primers to said second added sequence wherein said second primers comprise a second RNA promoter sequence;

h) extending said second set of primers using said first cDNA copies as templates to form double-stranded copies of said RNA;

i) carrying out a transcription reaction with said double-stranded copies of said RNA to generate labeled [−

] copies of said RNA;

j) carrying out a transcription reaction with said double-stranded copies of said RNA to generate labeled [+] copies of said RNA; and

k) hybridizing said labeled [+] copies and said labeled [−

] copies to an array and separately quantifying the amount of hybridization of said labeled [+] copies and said labeled [−

] copies,

wherein said array is a biphasic array or a heterophasic array.

24. The method of claim 20, wherein the label of said [+] copies and the label of said [−

] copies comprise different labels.

25. The method of claim 24, wherein the array is a biphasic array.

26. The method of claim 20, wherein said array is a heterophasic array comprising a nucleic acid target at one site and a nucleic acid complementary to the nucleic acid target at a different site.