Process for detecting or quantifying nucleic acids in a library

• US 9,765,387 B2
• Filed: 07/29/2015
• Issued: 09/19/2017
• Est. Priority Date: 06/30/2001
• Status: Expired due to Term

• Current Assignee: Enzo Biochem Incorporated
• Original Assignee: Enzo Biochem Incorporated
• Inventors: Rabbani, Elazar, Stavrianopoulos, Jannis G., Donegan, James J., Coleman, Jack
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/812,487
• Publication Number:
  US 20150376688A1
• Time in Patent Office: 783 Days
• Field of Search: 435 912
• 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 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2545/114 involving a quantitation step
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• 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 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C40B 40/00 : Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

This invention provides novel compositions and processes for analyte detection, quantification and amplification. Nucleic acid arrays and libraries of analytes are usefully incorporated into such compositions and processes. Universal detection elements, signaling entities and the like are employed to detect and if necessary or desirable, to quantify analytes. Amplification of target analytes are also provided by the compositions and processes of this invention.

Claims

1. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:

a) providing;

(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;

(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;

(iii) a first set of primers and a second set of primers, wherein said first set of primers are fixed or immobilized to a solid support, and wherein said first set or said second set of primers comprise at least one production center;

(iv) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes using said first and second sets of primers;

(v) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of said second set of primers;

(vi) ligating means for ligating said set of oligonucleotides or polynucleotides (v); and

(vii) synthesizing means for synthesizing nucleic acid copies from a production center under isothermal or isotactic conditions;

b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity, each comprising a first set primer hybridized to a nucleic acid analyte;

c) using said polymerizing means, extending said bound first set primers of the first bound entities using said nucleic acid analytes of the first bound entities as templates to form first copies of said analytes;

d) using said ligating means, ligating said set of oligonucleotides or polynucleotides a) (v) to the 3′

end of said first copies formed in step c) to form more than one ligation product;

e) contacting said more than one ligation product with said second set of primers to form more than one second bound entity, each comprising a second set primer hybridized to a ligated oligonucleotide or polynucleotide of one of the more than one ligation product;

f) using said polymerizing means, extending said bound second set primers of the second bound entities using said ligation products of the second bound entities as templates to form more than one complex comprising said ligation products and said extended second set primers;

g) using said synthesizing means, synthesizing under isothermal or isostatic conditions from a production center in said first or second set primers incorporated in said complexes one or more nucleic acid copies;

h) hybridizing said nucleic acid copies formed in step g) to said array of nucleic acids provided in step a) (i); and

i) detecting or quantifying any of said hybridized copies obtained in step h).

2. The process of claim 1, wherein said solid support comprises beads.

3. The process of claim 2, wherein said beads are magnetic.

4. The process of claim 1, wherein the fixed or immobilized nucleic acids of said array comprise members selected from the group consisting of DNA, RNA and analogs thereof.

5. The process of claim 4, wherein said analogs comprise PNA.

6. The process of claims 4 or 5, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

7. The process of claim 1, wherein said nucleic acid array is fixed or immobilized to a solid support.

8. The process of claim 7, wherein said solid support non-porous.

9. The process of claim 7, wherein said solid support is porous.

10. The process of claim 8, wherein said non-porous solid support comprises glass or plastic.

11. The process of claim 7, wherein said solid support is transparent, translucent, opaque or reflective.

12. The process of claim 7, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

13. The process of claim 7, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

14. The process of claim 1, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

15. The process of claim 1, wherein said library of nucleic acid analytes is selected from the group consisting of a genomic DNA library, an episomal DNA library, an unspliced RNA library, an mRNA library, an rRNA library, an snRNA library, and a combination of any of the foregoing.

16. The process of claim 1, wherein nucleic acid analytes of the library comprise inherent universal detection targets (UDTs) and said first set of primers comprise one or more sequences which are complementary to said inherent universal detection targets (UDTs).

17. The process of claim 16, wherein said inherent UDTs are selected from the group consisting of 3′

poly A segments, consensus sequences, and a combination of both.

18. The process of claim 17, wherein said inherent UDTs comprise consensus sequences selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

19. The process of claim 1, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, and a combination of both.

20. The process of claim 19, wherein said production center comprises a phage RNA promoter.

21. The process of claim 20, wherein said phage RNA promoter is selected from the group consisting of T3, T7 and SP6 phage RNA promoters.

22. The process of claim 1, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.

23. The process of claim 22, wherein said signaling entities generate a signal directly or indirectly.

24. The process of claim 23, wherein said one or more signaling entities generate a signal directly and are selected from the group consisting of 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 and a combination of any of the foregoing.

25. The process of claim 24, wherein said one or more signaling entities generate a signal indirectly and are selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

26. The process of claim 25, wherein said one or more signaling entities comprise an enzyme that catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

27. The process of claim 1, wherein said polymerizing means comprise a nucleic acid polymerase selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, and HIV-1 reverse transcriptase.

28. The process of claim 1, wherein said ligating means comprise T4 DNA ligase.

29. The process of claim 1, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

30. The process of claim 1, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

31. The process of claim 1, wherein step g) is carried out repeatedly.

32. The process of claim 1, wherein said synthesizing means synthesize nucleic acid copies under isothermal or isostatic conditions by one or more of RNA transcription, strand displacement amplification and secondary structure amplification.

33. The process of claim 9, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.