Process for producing two or more copies of nucleic acids in a library, and process for detecting or quantifiying more than one nucleic acid in a library

• US 7,807,352 B2
• Filed: 07/29/2004
• Issued: 10/05/2010
• 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: Stavrianopoulos, Jannis G., Donegan, James J., Rabbani, Elazar, Coleman, Jack
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Tung; Joyce
• Application Number:
  US10/902,629
• Publication Number:
  US 20050202455A1
• Time in Patent Office: 2,259 Days
• Field of Search: 435/6, 435/91.2, 435/91.51, 536/23.1, 536/23.4, 536/22.1
• US Class Current: 435/6.11
• 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 producing two or more copies of nucleic acids of interest in a library comprising the steps of:

a) providing;

(i) an array of solid surfaces comprising a plurality of discrete areas;

wherein at least two of said discrete areas each comprises fixed or immobilized thereto;

(1) a first set of nucleic acid primers; and

(2) a second set of nucleic acid primers;

wherein the nucleotide sequences in said first set of nucleic acid primers are different from the nucleotide sequences in said second set of nucleic acid primers;

wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ from each other by at least one base; and

wherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are the same or identical;

(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest;

(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acids of interest;

b) contacting a fixed or immobilized primer of said first set with a complementary sequence in said nucleic acid of interest;

c) separating said fixed or immobilized extended first primer from said nucleic acid of interest template;

d) extending said fixed or immobilized primer in the first set using said nucleic acid of interest as a template to generate a fixed or immobilized extended first primer;

e) contacting a primer in said second set with a complementary sequence in said extended fixed or immobilized first primer;

f) extending said fixed or immobilized primer in the second set using said fixed or immobilized extended first primer as a template to generate a fixed or immobilized extended second primer;

g) separating said fixed or immobilized extended second primer from said extended first primer template;

h) contacting a fixed or immobilized primer in the first set with a complementary sequence in said fixed or immobilized extended second primer;

i) extending said fixed or immobilized primer in the first set using said fixed or immobilized extended second primer as a template to generate a fixed or immobilized extended first primer;

j) separating said fixed or immobilized extended first primer from said extended second primer template; and

k) repeating steps e) through j) above one or more times.

2. The process of claim 1, wherein said nucleic acid primers comprise DNA, RNA, DNA analogs or RNA analogs, and combinations thereof.

3. The process of claim 2, wherein said analogs comprise PNA.

4. The process of claim 2 or 3, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

5. The process of claim 1, wherein said solid support is porous or non-porous.

6. The process of claim 5, wherein said porous solid support comprises polyacrylamide or agarose.

7. The process of claim 5, wherein said non-porous solid support comprises glass or plastic.

8. The process of claim 1, wherein said solid support is transparent, translucent, opaque or reflective.

9. The process of claim 1, wherein nucleic acid primers are directly fixed or immobilized to said solid support.

10. The process of claim 1, wherein said nucleic acid primers are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

11. The process of claim 1, wherein said library of analytes is derived from a biological source comprising organs, tissues or cells, and combinations thereof.

12. The process of claim 1, wherein said analytes comprise genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, or snRNA, and a combination of any of the foregoing.

13. The process of claim 1, wherein said polymerizing means comprise 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, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript or Omniscript, and a combination of any of the foregoing.

33. The process of claim 1, wherein said providing step a), the primers are covalently fixed or immobilized.

35. The process of claim 1, wherein separating in any or all of steps c), g) and i) is carried out by means of heat denaturation.

14. 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 solid surfaces comprising a plurality of discrete areas;

wherein at least two of said discrete areas each comprises fixed or immobilized thereto;

(1) a first set of nucleic acid primers; and

(2) a second set of nucleic acid primers;

wherein the nucleotide sequences in said first set of nucleic acid primers are different from the nucleotide sequences in said second set of nucleic acid primers;

wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ from each other by at least one base; and

wherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical;

(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest;

(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acids of interest; and

(iv) non-radioactive signal generating means capable of being attached to or incorporated into nucleic acids;

b) contacting a fixed or immobilized primer of said first set with a complementary sequence in said nucleic acid of interest;

c) extending said fixed or immobilized primer in the first set using said nucleic acid of interest as a template to generate a fixed or immobilized extended first primer;

d) separating said fixed or immobilized extended first primer from said nucleic acid of interest template;

e) contacting a primer in said second set with a complementary sequence in said extended fixed or immobilized first primer;

f) extending said fixed or immobilized primer in the second set using said fixed or immobilized extended first primer as a template to generate an a fixed or immobilized extended second primer;

g) separating said fixed or immobilized extended second primer from said extended first primer template;

h) contacting a fixed or immobilized primer in the first set with a complementary sequence in said fixed or immobilized extended second primer;

i) extending said fixed or immobilized primer in the first set using said fixed or immobilized extended second primer as a template to generate a fixed or immobilized extended first primer;

j) separating said fixed or immobilized extended first primer from said extended second primer template;

k) repeating steps d) through i) above one or more times; and

l) detecting or quantifying by means of said non-radioactive signal generating means attached to or incorporated into any of said extended primers in steps c), f), i), and k).

15. The process of claim 14, wherein said nucleic acid primers comprise DNA, RNA, DNA analogs or RNA analogs, and combinations thereof.

16. The process of claim 15, wherein said analogs comprise PNA.

17. The process of claim 15 or 16, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

18. The process of claim 14, wherein said solid support is porous or non-porous.

19. The process of claim 18, wherein said porous solid support comprises polyacrylamide or agarose.

20. The process of claim 18, wherein said non-porous solid support comprises glass or plastic.

21. The process of claim 14, wherein said solid support is transparent, translucent, opaque or reflective.

22. The process of claim 14, wherein nucleic acid primers are directly fixed or immobilized to said solid support.

23. The process of claim 14, wherein said nucleic acid primers are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

24. The process of claim 14, wherein said library of analytes is derived from a biological source comprising organs, tissues and or cells, and combinations thereof.

25. The process of claim 14, wherein said analytes comprise genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, or snRNA, and a combination of any of the foregoing.

26. The process of claim 14, wherein said polymerizing means comprise E. coli DNA Pol I, Kienow 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, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript or Omniscript, and a combination of any of the foregoing.

27. The process of claim 14, wherein said non-radioactive signal generating means comprise labeled nucleotides, intercalating dyes, or universal detection elements, and a combination of any of the foregoing.

28. The process of claim 14, wherein said extended primers further comprise one or more signaling entities attached or incorporated thereto.

29. The process of claim 28, wherein said signaling entities generate a signal directly or indirectly.

30. The process of claim 29, wherein said direct signal generation comprises a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, or an energy transfer compound, and a combination of any of the foregoing.

31. The process of claim 29, wherein said indirect signal generation comprises an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, or an enzyme, and a combination of any of the foregoing.

32. The process of claim 31, wherein said enzyme catalyzes a reaction comprises a fluorogenic reaction, a chromogenic reaction or a chemiluminescent reaction.

34. The process of claim 14, wherein said providing step a), the primers are covalently fixed or immobilized.

36. The process of claim 14, wherein separating in any or all of steps d), g) and i) is carried out by means of heat denaturation.