Process for detecting or quantifying nucleic acids in a library
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 comprising the steps of:
a) providing:
(i) a first 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;
(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a) a second array comprising a first set of primers, wherein said first set of primers are complementary, in part or in whole, to sequences of said nucleic acids of interest, and b) a second set of primers, wherein said first set of primers are fixed or immobilized to the second array, and wherein said second set of primers comprises at least two segments, the first segment at the 3′ end comprising random sequences, and the second segment comprising at least one RNA promoter; and
(iv) means for synthesizing labeled RNA copies from said RNA promoter;
b) contacting said library of nucleic acid analytes with said first set of primers on the second array to form more than one first bound entity;
c) extending said bound first set of primers on the second array by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;
c′) separating or removing said template sequences from said first copies;
d) contacting said first copies on the second array 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 first copies to form more than one complex comprising first copies and second copies formed by extension of said second set of primers;
f) synthesizing labeled RNA copies from the RNA promoter in said second copies;
g) hybridizing said RNA copies formed in step f) to said first array of nucleic acids provided in step a) (i); and
h) detecting or quantifying any of said copies hybridized to said first array in step g).
2. The process of claim 1 , wherein said second array comprises a set of beads.
3. The process of claim 2 , wherein said beads are magnetic.
4. The process of claim 1 , wherein said first nucleic acid array comprises 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 claim 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 first nucleic acid array is fixed or immobilized to a solid support.
8. The process of claim 7 , wherein said solid support is porous or non-porous.
9. The process of claim 8 , wherein said first array is a porous solid support comprising a material selected from the group consisting of polyacrylamide and agarose.
10. The process of claim 8 , wherein said first array is a non-porous solid support comprising 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 12 , 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, cells and any combination thereof.
15. The process of claim 1 , wherein said library of nucleic acids analytes are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.
16. The process of claim 1 , wherein said first set of primers comprise one or more sequences which are complementary to an inherent universal detection target (UDT), wherein the inherent UDT is selected from the group consisting of a signal sequence for poly A addition, a splicing element, a multicopy repeat, and a combination of any of the foregoing.
17. The process of claim 1 , wherein said RNA polymerase promoter is a phage promoter.
18. The process of claim 17 , wherein said phage promoter is selected from the group consisting of a T3 promoter, a T7 promoter and an SP6 promoter.
19. The process of claim 1 , wherein said label generates a signal directly.
20. The process of claim 19 , wherein said direct signal generation is 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.
21. The process of claim 1 , wherein said label generates a signal indirectly.
22. The process of claim 21 , wherein said indirect signal is generated from an entity 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.
23. The process of claim 22 , wherein said indirect signal is generated from an enzyme that catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.
24. The process of claim 1 , wherein said polymerizing means of step a)(iii) comprises an enzyme 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, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.
25. The process of claim 1 , further comprising the step of repeating step b) after step c′.
26. The process of claim 1 , further comprising the step of using unlabeled nucleotides in step (f), isolating unlabeled RNA copies made in step (f), contacting said unlabeled RNA copies with said first set of primers on another second array and carrying out steps (e) through (h).
27. The process of claim 1 , wherein step g) is carried out repeatedly.