Processes for genetic manipulations using promoters

• US 5,716,785 A
• Filed: 04/19/1996
• Issued: 02/10/1998
• Est. Priority Date: 09/22/1989
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Eberwine, James H., Von Zastrow, Mark E., Barchas, Jack D., Van Gelder, Russell N.
• Primary Examiner(s): Elliott, George C.
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US08/636,748
• Time in Patent Office: 662 Days
• Field of Search: 435/6, 435/91.2, 435/240.2, 435/91.1, 536/23.1, 536/24.1, 536/24.31, 536/25.3
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Abstract

This invention relates to the use of promoters for ribonucleic acid amplification and other genetic manipulations. Processes are provided wherein complementary deoxyribonucleic acid (cDNA) is synthesized from a ribonucleic acid (RNA) sequence using a complementary primer linked to an RNA polymerase promoter region complement and then anti-sense RNA (aRNA) is transcribed from the cDNA by introducing an RNA polymerase capable of binding to the promoter region. Additional processes using the resulting aRNA are also described.

Claims

1. A process for producing a subtractive hybridization probe comprising:

(a) synthesizing a first double-stranded cDNA collection by treating a first mRNA population with a primer complex, wherein the primer is complementary to the RNA sequence and is operably linked to a first promoter region for transcription of the cDNA strand complementary to the primer;

(b) transcribing the first cDNA into anti-sense RNA by introducing a first RNA polymerase that binds to the first promoter region;

(c) hybridizing the anti-sense RNA to a second mRNA population, whereby an unhybridized subpopulation of the second RNA population is separated;

(d) generating a second double-stranded cDNA collection from the unhybridized subpopulation using a second primer complex comprising a second promoter region in an orientation for transcribing anti-sense RNA complementary to the unhybridized subpopulation; and

(e) transcribing the second cDNA into a ribonucleotide probe by introducing a second RNA polymerase that binds to the second promoter region and transcribing RNA which is complimentary to the mRNA in the unhybridized subpopulation of the second RNA population.

2. A method for making a cDNA library from a collection of mRNA molecules comprising:

(a) hybridizing one or more primer complexes to a plurality of the mRNA'"'"'s wherein each complex comprises an oligonucleotide primer linked to a promoter sequence;

(b) producing a collection of double-stranded cDNAs by extending the primers of a plurality of any hybridization duplexes formed between the mRNAs and the complexes wherein each double stranded cDNA comprises a first cDNA strand which is complementary to one mRNA molecule and operably linked to the promoter, and a second strand which is complementary to the first cDNA strand operably linked to the promoter;

(c) transcribing multiple copies of anti-sense RNA off of the second strand; and

(d) preparing a cDNA library from the anti-sense RNA copies.

3. A process for amplifying at least one target nucleic acid sequence comprising:

(a) synthesizing a double-stranded nucleic acid by;

i) hybridizing a primer complex to the target nucleic acid sequence and extending the primer complex to form a first DNA strand complementary to the target sequence, wherein said primer complex comprises a promoter and a primer region complementary to the target nucleic acid sequence, and

ii) synthesizing a second DNA strand complementary to the first DNA strand without using an exogenous primer complementary to the first DNA strand; and

,

(b) transcribing copies of RNA initiated from the promoter region of the primer complex, wherein said copies of RNA are complementary to the second DNA strand.

4. A process according to claim 3, wherein the primer region consists essentially of polythymidylate (poly(dT)).

5. A process according to claim 3, wherein the promoter is a naturally occurring prokaryotic promoter.

6. A process according to claim 3 wherein the second DNA strand is synthesized by formation of a hairpin loop formed spontaneously at the 3'"'"' end of the first DNA strand.

7. A process according to claim 3 wherein said target nucleic acid sequence is a pathogen-specific sequence.

8. A process of claim 3 comprising:

(a) hybridizing a primer complex to the target sequence, wherein said primer complex comprises a promoter and a polythymidylate (poly(dT)) region complementary to the target sequence, and extending the primer to form a first DNA strand complementary to the target sequence;

(b) adding a homopolymeric tail to the 3'"'"' terminus of the first DNA strand and synthesizing a second cDNA strand complementary to the first cDNA strand, with the proviso that said synthesis does not comprise extension from an exogenous primer complementary to the first cDNA strand; and

,

(c) transcribing copies of RNA initiated from the promoter region of the primer complex, wherein said copies of RNA are complementary to the second cDNA strand.

9. A process according to claim 3, wherein the promoter is a bacteriophage promoter.

10. A process according to claim 9, wherein the promoter is a T3, T7, or SP6 promoter.

11. A process according to claim 3, wherein the target nucleic acid sequence is eukaryotic mRNA.

12. A process according to claim 11 wherein the eukaryotic mRNA comprises a genetic polymorphism.

13. A process according to claim 11, wherein the mRNA is located in situ in a tissue sample or in a cultured cell and synthesis of the first DNA strand is performed in situ.

14. A process for detecting expression of at least one gene in a preselected cell population comprising:

(a) synthesizing double-stranded complementary deoxyribonucleic acid (cDNA) by;

(i) hybridizing a primer complex comprising a promoter and a polythymidylate (poly(dT)) region complementary to mRNA present in the cell population,

(ii) extending the primer complex to form a first cDNA strand, and,

(iii) synthesizing a second cDNA strand complementary to the first cDNA strand without using an exogenous primer complementary to the first cDNA strand;

(b) transcribing the double-stranded cDNA into anti-sense RNA (aRNA); and

,

(c) determining the presence of aRNA corresponding to the gene or genes, wherein the presence of aRNA complementary to a gene corresponds to expression of a gene.

15. A process according to claim 14, wherein the preselected cell population is a tissue sample or a cell culture and the step of synthesizing first strand cDNA is performed in situ.

16. A method for subtractive hybridization comprising the steps:

(a) binding a primer to sense RNA molecules in a first population, wherein the primer is operably linked to a promoter sequence in an anti-sense orientation;

(b) synthesizing a first complementary DNA (cDNA) strand by elongation from the primer;

(c) synthesizing a second cDNA strand without using an exogenous primer complementary to the first strand, whereupon a functional promoter is generated;

(d) initiating RNA synthesis from the promoter by adding RNA polymerase, whereby anti-sense RNA (aRNA) is produced;

(e) introducing the aRNA in molar excess to a second population of sense RNA molecules, whereby complementary RNA sequences from the two populations hybridize; and

(f) isolating remaining single-stranded sense RNA.

17. A method according to claim 16, wherein the aRNA is labeled during synthesis.

18. A method for detecting the expression in one or more cells of at least one gene, said method comprising the steps:

(a) hybridizing a primer complex to RNA molecules in a population of RNA molecules from said cell, wherein the primer complex comprises a promoter sequence and a primer sequence complementary to at least one RNA in the population;

(b) synthesizing a first complementary DNA (cDNA) strand by elongation from said primer;

(c) synthesizing a second complementary cDNA strand without using an exogenous primer complementary to the first cDNA strand, wherein a functional promoter is generated;

(d) initiating RNA synthesis from the promoter by adding RNA polymerase, thereby producing amplified antisense RNA (aRNA); and

(e) detecting the presence of aRNA complementary to the mRNA transcribed from the gene and relating the presence of said aRNA to the expression of the gene in the cell.

19. The method of claim 18 wherein the primer sequence consists essentially of polythymidylate (poly(dT)).

20. A method according to claim 18, wherein the cells are obtained from human tissue.

21. The method according to claim 18, wherein the aRNA is analyzed by hybridization to a DNA having a sequence from the gene.

22. A method according to claim 21, wherein the DNA sequence is immobilized on a Southern blot.

23. A method according to claim 21 wherein expression from a single gene is detected.

24. A method for amplifying a brain cell mRNA comprising:

(a) introducing into a brain cell a reaction mixture comprising;

(I) a primer complex operably bound to a promoter sequence in the anti-sense orientation,

ii) reverse transcriptase, and

iii) dATP, dCTP, dGTP, and dTTP, thereby generating a first strand cDNA;

(b) harvesting the brain cell;

(c) precipitating nucleic acids from the brain cell and recovering the first strand cDNA;

(d) synthesizing second-strand cDNA without using an exogenous primer complementary to the first cDNA strand;

(e) initiating transcription by adding RNA polymerase, rATP, rCTP, rGTP and UTP;

(f) whereby anti-sense RNA is produced and a brain cell mRNA is amplified.

25. A process for determining the level of expression of at least one mRNAs in a preselected cell or cell population relative to the level of expression of the other mRNAs in the same cell or cell population comprising:

(a) adding a primer complex to a mixture comprising a population of mRNAs from said preselected cell or cell population, said primer complex comprising

i) a primer sequence complementary to a plurality of said mRNAs, and

ii) a promoter sequence in antisense orientation;

whereupon the primer complex hybridizes to said plurality of mRNAs;

(b) synthesizing double-stranded complementary deoxyribonucleic acid (cDNA) by

i) extending the hybridized primer complexes to produce first strand cDNAs,

ii) synthesizing second strand cDNAs without using an exogenous primer, wherein the second strand comprises the promoter sequence in sense orientation;

(c) transcribing multiple copies of antisense RNA (aRNA) initiated from the promoter region of the primer complexes to produce a population of aRNAs; and

,

(d) analyzing the population of aRNAs to determine the level of representation of a specific aRNA sequence relative to other aRNA sequences within the population of aRNAs, wherein the amount of a specific aRNA in a population of aRNAs corresponds to the amount of the corresponding mRNA in the population of mRNAs.

26. A process of claim 27 wherein the aRNA is labeled.

27. A process of claim 25 wherein the primer sequence consists essentially of polythymidylate (poly(dT)).

28. A process of claim 27 wherein step (d) comprises hybridizing the aRNA to immobilized nucleic acid sequences.

29. A process of claim 27 wherein step (d) comprises hybridizing the aRNA to immobilized nucleic acid sequences.

30. A process of claim 29 wherein the immobilized nucleic acid sequences are polydeoxyribonucleotides immobilized on nitrocellulose.

31. A process of claim 29 wherein the cell or cell population is obtained from the group consisting of blood, brain, spleen, bone, heart, vascular tissue, lung, kidney, liver, pituitary, endocrine glands, lymph node, dispersed primary cells and tumor cells.

32. A process of claim 25 wherein an abnormal level of expression of an RNA is detected.

33. A process of claim 32 wherein the RNA is a hemoglobin mRNA and the presence of an abnormal level of said hemoglobin mRNA is correlated to a thalassemia.

34. A process for comparing mRNA expression in different tissues or different physiologic states of the same tissue, said process comprising:

(a) adding a primer complex to a mixture comprising a population of mRNAs from a first cell population, said primer complex comprising

i) a primer sequence complementary to a plurality of said mRNAs, and

ii) a promoter sequence in antisense orientation;

whereupon the primer complex hybridizes to said plurality of mRNAs;

(b) synthesizing double-stranded complementary deoxyribonucleic acid (cDNA) by

i) extending the hybridized primer complexes to produce first strand cDNAs,

ii) synthesizing second strand cDNAs without using an exogenous primer, wherein the second strand comprises the promoter sequence in sense orientation;

(c) transcribing multiple copies of amplified RNA initiated from the promoter region of the primer complexes to produce a population of amplified RNAs;

(d) carrying out steps (a)-(c) by adding the primer complex of step (a) to a mixture comprising a population of mRNAs from a second preselected cell population, wherein said first and second cell populations are from different tissues or different physiologic states of the same tissue; and

,

(e) comparing the species of amplified RNA produced from the first cell population to the to same species of amplified RNA produced from the second cell population, wherein the amount of the amplified RNA in each population of amplified RNAs corresponds to the amount of the corresponding mRNA in the corresponding cell population.

35. The process of claim 34 wherein the primer sequence consists essentially of polythymidylate (poly(dT)).

36. A process of claim 34, wherein the first and second cell populations differ in a physiologic state selected from the group consisting of a developmental state, an arousal state, a behavioral state, or a state induced by drug treatment.

37. A process of claim 34, wherein the first and second cell populations are from different tissues.

38. A process of claim 37, wherein the tissues are a tumor tissue and a nontumor tissue of the same tissue type.

39. A component kit for the production of amplified RNA comprising:

a container comprising a primer complex component and instructions for use of said primer complex wherein said primer complex component comprises a polythymidylate (poly(dT)) region and a promoter.

40. A component kit according to claim 39, further comprising

(a) a container comprising a reverse transcriptase;

(b) a container comprising an RNA polymerase;

(c) a container comprising dATP, dCTP, dGTP, and dTTP nucleotides;

or

(d) a container comprising rATP, rCTP, rGTP, and UTP nucleotides.

41. A component kit according to claim 39, wherein the polythymidylate (poly(dT)) region is between about 5 and about 50 bases.

42. A component kit for the production of amplified RNA comprising:

a container comprising a plurality of primer complex species, wherein said primer complex species comprise random primers linked to a promoter, and instructions for use of said primer complex species.