Processes for detecting or quantifying nucleic acids using an array of fixed or immobilized nucleic acids
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.
1. A process for detecting or quantifying more than one nucleic acid of interest in a library, said process comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids 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; and
(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers and a second set of primers; wherein said first set of primers consists of sequences that are complementary to inherent UDTs, said inherent UDTs consisting of 3′ poly A segments, consensus sequences, or both, wherein the consensus sequence is selected from a sequence for poly A addition, splicing elements, multicopy repeats, or a combination of any of the foregoing, and wherein said second set of primers comprises at least two segments, the first segment at the 3′ end comprising random sequences comprising at least a hexameric sequence, and the second segment comprising at least one production center comprising an RNA promoter;
(iv) means for synthesizing nucleic acid copies by transcription;
b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity;
c) extending said bound first set of primers by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;
d) contacting said extended first copies with said second set of primers to form more than one second bound entity;
e) extending said bound second set of primers by means of template sequences provided by said extended first copies to form more than one complex comprising extended first copies and extended second set of primers;
f) transcribing said complex from an RNA promoter in said second set of primers in said complexes;
g) hybridizing said nucleic acid copies formed in step f) to said array of nucleic acids provided in step a) (i); and
h) detecting or quantifying any of said hybridized copies obtained in step g).
2. The process of claim 1 , wherein said nucleic acid array comprises members selected from the group consisting of 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 nucleic acid array is fixed or immobilized to a solid support.
6. The process of claim 5 , wherein said solid support is porous or non-porous.
7. The process of claim 6 , wherein said porous solid support comprises polyacrylamide or agarose.
8. The process of claim 6 , wherein said non-porous solid support comprises glass or plastic.
9. The process of claim 5 , wherein said solid support is transparent, translucent, opaque or reflective.
10. The process of claim 5 , wherein said nucleic acids are indirectly fixed or immobilized to said solid support.
11. The process of claim 10 , wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.
12. The process of claim 1 , wherein said library of nucleic acid analytes is derived from a biological source comprising organs, tissues or cells, and combinations thereof.
13. The process of claim 1 , wherein said library of nucleic acids analytes are derived from genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, or snRNA, or a combination of any of the foregoing.
14. The process of claim 1 , wherein said RNA promoters comprise phage promoters.
15. The process of claim 14 , wherein said phage promoters comprises T3, T7 or SP6, and combinations thereof.
16. The process of claim 1 , wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.
17. The process of claim 16 , wherein said signaling entities generate a signal directly or indirectly.
18. The process of claim 17 , 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.
19. The process of claim 17 , 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.
20. The process of claim 19 , wherein said enzyme catalyzes a reaction comprising a fluorogenic reaction, a chromogenic reaction or a chemiluminescent reaction.
21. 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).
22. 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).
23. The process of claim 1 , wherein step g) is carried out repeatedly.
24. The process of claim 1 , wherein said polymerizing means comprises 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 or Sensiscript and Omniscript, and combinations thereof.
25. The process of claim 1 , wherein said nucleic acids are directly fixed or immobilized to said solid support.
26. The process of claim 1 , wherein said at least one production center in steps a) (iii) or f) or both comprise primer binding sites, and wherein said synthesizing means in step a) (iv) or said synthesizing step f) is by strand displacement or secondary structure amplification, or both.
27. The process of claim 1 , wherein said at least one production center in steps a) (iii) or f) or both comprise RNA promoters, and wherein said synthesizing means in step a) (iv) or said synthesizing step f) is by transcription.