Processes for genetic manipulations using promoters

• US 5,891,636 A
• Filed: 09/03/1997
• Issued: 04/06/1999
• Est. Priority Date: 09/22/1989
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Eberwine, James H., Von Zastrow, Mark E., Barchas, Jack D., Van Gelder, Russell N.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US08/922,965
• Time in Patent Office: 580 Days
• Field of Search: 435/6, 435/91.21, 435/91.51, 536/23.1, 536/24.1, 536/24.3, 536/24.33, 536/24.5
• 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 gene expression library derived from a cell or tissue sample, comprising two or more specific nucleic acid messages of various abundances, whose levels of representation relative to other messages within a population reflect the physiologic state of the sample, thereby permitting diagnosis of a disease or condition, wherein said library is prepared by the following steps:

(a) adding a primer complex to a population of messenger RNAs (mRNAs) from said cell or cell population, said primer complex comprising;

(i) a primer sequence complementary to a plurality of said population of mRNAs of said cell or cell population, and

(ii) a promoter sequence in antisense orientation, wherein said primer complex hybridizes to said plurality population of mRNAs;

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

(i) extending said primer complex to form a first cDNA strand, and

(ii) synthesizing a second cDNA strand complementary to said first cDNA strand without using an exogenous primer, wherein said second cDNA strand comprises said promoter sequence in sense orientation;

(c) linearly transcribing multiple copies of double-stranded cDNA into antisense RNAs (aRNAs) initiated from the promoter region of the primer complex; and

(d) quantitating specific aRNAs corresponding to specific mRNAs,

wherein a plurality of said aRNAs represents an expression spectrum in said cell or cell population.

2. The gene expression library of claim 1 wherein the relative levels of pathogen-specific sequences allow a determination of infection.

3. The gene expression library of claim 1 wherein two or more specific nucleic acid messages are detected by hybridization.

4. The gene expression of claim 3 wherein hybridization is by northern or Southern blot.

5. An expression library of a cell or cell population, prepared by the following steps:

(a) adding a primer complex to a population of messenger RNAs (mRNAs) from said cell or cell population, said primer complex comprising;

(i) a primer sequence complementary to a plurality of said population of mRNAs of said cell or cell population, and

(ii) a promoter sequence in antisense orientation, wherein said primer complex hybridizes to said plurality of said population of mRNAs;

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

(i) extending said primer complex to form a first cDNA strand, and

(ii) synthesizing a second cDNA strand complementary to said first cDNA strand without using an exogenous primer, wherein said second cDNA strand comprises said promoter sequence in sense orientation;

(c) linearly transcribing multiple copies of double-stranded cDNA into antisense RNAs (aRNAs) initiated from the promoter region of the primer complex; and

(d) quantitating specific aRNAs corresponding to specific mRNAs,

wherein a plurality of said aRNAs represents an expression library in said cell or cell population.

6. The expression library of claim 5 wherein the sample is brain, spleen, bone, heart, vascular lung, kidney, liver, pituitary, endocrine glands, lymph node, dispersed primary cells, or tumor cells.

7. The expression library of claim 5 wherein the sample is blood and the expression of the hemoglobin is reduced relative to other messages and is diagnostic of thalassemia.

8. The expression library of claim 5 wherein said population comprises two or more RNAs.

9. The expression library claim 5, wherein said primer sequence consists essentially of polythymidylate (poly(dT)).

10. The expression library of claim 5, wherein said promoter is a bacteriophage promoter.

11. The expression library of claim 10, wherein said promoter is a T3, T7, or SP6 promoter.

12. The expression library of claim 11, further comprising the expression spectrum of a different cell or cell population or, alternatively, within the same cell or cell population in a different physiologic state wherein a difference in spectrum is indicative of an abnormal physiologic state.

13. The expression library of claim 5, wherein said aRNAs are quantitated by hybridization to nucleic acid sequences complementary to said aRNAs.

14. The expression library of claim 13, wherein said nucleic acid sequences are detected by the presence of a label or by Southern hybridization.

15. A method of determining the presence of thalassemia disease, comprising the steps of:

(a) adding primer complexes to a population of mRNAs from a mammalian cell or cell population, each primer complex comprising;

(i) a primer sequence complementary to a plurality of said mRNAs of said cell or cell population, and

(ii) a promoter sequence in antisense orientation, wherein said primer complexes hybridize to said plurality of said mRNAs;

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

(i) extending said primer complex to form a first cDNA strand, and

(ii) synthesizing a second cDNA strand complementary to said first cDNA strand without using an exogenous primer, wherein said second cDNA strand comprises said promoter sequence in sense orientation;

(c) linearly transcribing multiple copies of double-stranded cDNA into antisense RNAs (aRNAs) initiated from the promoter region of the primer complexes; and

(d) quantitating specific aRNAs corresponding to specific mRNAs;

wherein quantitation of said aRNAs generates an expression spectrum reflecting the population of mRNAs in said cell or cell population, allowing for the identification of abnormal hemoglobin gene expression, thus permitting determination of a thalassemia disease.

16. The method of claim 15, wherein said mammalian cell or cell population is from blood.

17. The method of claim 15, wherein said mammalian cell or cell population is a human cell or cell population.

18. The method of claim 15, comprising the additional step of comparing said expression spectrum with a normal expression spectrum of a healthy cell or cell population, wherein abnormal levels of hemoglobin gene expression is indicative of a thalassemia disease.

19. The method of claim 15, wherein said promoter is a bacteriophage promoter.

20. The method of claim 19, wherein said promoter is a T3, T7, or SP6 promoter.

21. The method of claim 15, wherein said aRNAs are quantitated by hybridization to a nucleic acid sequence complementary to said aRNAs.

22. The method of claim 21, wherein said aRNAs are detected by the presence of a label.

23. The method of claim 21, wherein said aRNAs are detected by Southern hybridization.

24. A kit for detecting a thalassemia disease comprising a plurality of primer complex species and instructions for use of said primer complex species in a suitable container means, wherein said primer complex species comprise at least one primer sequence complementary to a plurality of hemoglobin genes and a promoter sequence in antisense orientation.

25. The kit according to claim 24, further comprising:

(a) a reverse transcriptase;

(b) an RNA polymerase;

(c) dATP, dCTP, dGTP, and dTTP nucleotides;

or

(d) rATP, rCTP, rGTP, and UTP nucleotides.

26. A method of detecting the expression of mutant hemoglobin genes in a mammalian cell or cell population, comprising the steps:

(a) adding primer complexes to a population of mRNAs from said mammalian cell or cell population, each primer complex comprising

(i) a primer sequence complementary to a plurality of said mRNAs of said cell or cell population, and

(ii) a promoter sequence in antisense orientation,

wherein said primer complexes hybridize to said plurality of said mRNAs;

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

(i) extending said primer complex to form a first cDNA strand, and

(ii) synthesizing a second cDNA strand complementary to said first cDNA strand without using an exogenous primer, wherein said second cDNA strand comprises said promoter sequence in sense orientation;

(c) linearly transcribing multiple copies of double-stranded cDNA into antisense RNAs (aRNAs) initiated from the promoter region of said primer complexes; and

(d) quantitating specific aRNAs corresponding to specific mRNAs;

wherein the quantitation of said aRNAs indicates expression of mutant hemoglobin genes in said cell or cell population, as compared to expression of hemoglobin genes in a normal mammalian cell or cell population.

27. A method of identifying a pathogen-specific nucleic acid sequence in a cell or tissue of a mammal, comprising the steps:

(a) adding a primer complex to a population of nucleic acid sequences from said cell or tissue, said primer complex comprising

(i) a primer sequence complementary to a plurality of pathogen-specific nucleic acid sequences, and

(ii) a promoter sequence in antisense orientation,

wherein said primer complex hybridizes to said plurality of pathogen-specific nucleic acid sequences;

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

(i) extending said primer complex to form a first cDNA strand, and

(ii) synthesizing a second cDNA strand complementary to said first cDNA strand without using an exogenous primer, wherein said second cDNA strand comprises said promoter sequence in sense orientation; and

(c) linearly transcribing multiple copies of double-stranded cDNA into antisense RNAs (aRNAs) initiated from the promoter region of said primer complex;

wherein the presence of said aRNAs is indicative of at least one pathogen-specific nucleic acid sequence in said cell or tissue.

28. The method of claim 27, wherein said mammal is a human.

29. The method of claim 27, wherein said cell or tissue is obtained from blood, brain, spleen, bone, heart, vascular tissue, lung, kidney, liver, pituitary, endocrine glands, lymph node, dispersed primary cells and tumor cells.

30. The method of claim 27, wherein said pathogen-specific nucleic acid sequence is a DNA sequence.

31. The method of claim 27, wherein said pathogen-specific nucleic acid sequence is a RNA sequence.

32. The method of claim 27, wherein said promoter is a bacteriophage promoter.

33. The method of claim 32, wherein said promoter is a T3, T7, or SP6 promoter.

34. The method of claim 27, wherein said aRNAs are quantitated by hybridization to pathogen-specific nucleic acid sequences.

35. The method of claim 34, wherein said nucleic acid sequences are detected by the presence of a label.

36. The method of claim 34, wherein said nucleic acid sequences are detected by Southern hybridization.

37. A method of identifying at least one member of a gene family in a cell or tissue, comprising the steps of:

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

(i) a primer sequence complementary to mRNAs of said gene family, and

(ii) a promoter sequence in antisense orientation,

wherein said primer complex hybridizes to a commonly shared sequence in said mRNAs of said gene family;

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

(i) extending said primer complex to form a first cDNA strand, and

(ii) synthesizing a second cDNA strand complementary to said first cDNA strand without using an exogenous primer, wherein said second cDNA strand comprises said promoter sequence in sense orientation; and

(c) linearly transcribing multiple copies of double-stranded cDNA into antisense RNAs (aRNAs) initiated from the promoter region of said primer complex;

wherein the presence of said aRNAs is indicative of at least one member of a gene family in said cell or tissue.

38. The method of claim 37, wherein said promoter is a bacteriophage promoter.

39. The method of claim 38, wherein said promoter is a T3, T7, or SP6 promoter.

40. The method of claim 37, wherein said aRNAs are quantitated by hybridization to a nucleic acid sequence complementary to said aRNAs.

41. The method of claim 40, wherein said nucleic acid sequence is detected by the presence of a label.

42. The method of claim 40, wherein said nucleic acid sequence is detected by Southern hybridization.

43. A method of identifying alternatively spliced mRNA transcripts of a gene in a cell or tissue, comprising the steps of:

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

(i) a primer sequence complementary to said gene, and

(ii) a promoter sequence in antisense orientation,

wherein said primer complex hybridizes to said MRNA transcripts of said gene;

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

(i) extending said primer complex to form a first cDNA strand, and

(ii) synthesizing a second cDNA strand complementary to said first cDNA strand without using an exogenous primer, wherein said second cDNA strand comprises said promoter sequence in sense orientation; and

(c) linearly transcribing multiple copies of double-stranded cDNA into antisense RNAs (aRNAs) initiated from the promoter region of said primer complex;

wherein the presence of aRNAs of variant lengths reflects alternatively spliced mRNA transcripts of said gene in said cell or tissue.

44. The method of claim 43, wherein said promoter is a bacteriophage promoter.

45. The method of claim 43, wherein said promoter is a T3, T7, or SP6 promoter.

46. The method of claim 43, wherein said aRNAs are quantitated by hybridization to a nucleic acid sequence complementary to said aRNAs.

47. The method of claim 46, wherein said nucleic acid sequence is detected by the presence of a label.

48. The method of claim 46, wherein said nucleic acid sequences are detected by Southern hybridization.

49. A method for identifying genetic polymorphisms of a gene in a cell or cell population, comprising the steps of:

(a) adding a primer complex to a population of mRNAs from a cell or cell population, the primer complex comprising;

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

(ii) a promoter sequence in antisense orientation,

wherein the primer complex hybridizes to said plurality of mRNAs;

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

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

(ii) synthesizing a second cDNA strand complementary to the first cDNA strand without using an exogenous primer, wherein the second cDNA strand comprises said promoter sequence in sense orientation;

(c) linearly transcribing multiple copies of double-stranded cDNA into antisense RNA (aRNA) initiated from the promoter region of the primer complex; and

(d) determining the sequence of the aRNAs;

wherein differences in the aRNA sequences are indicative of genetic polymorphisms in said cell or cell population.

50. The method of claim 49, wherein said promoter is a bacteriophage promoter.

51. The method of claim 49, wherein said promoter is a T3, T7, or SP6 promoter.

52. The method of claim 49, wherein said aRNAs are separated by hybridization to a nucleic acid sequence complementary to said aRNAs prior to sequence determination.

53. The method of claim 52, wherein said nucleic acid sequences are detected by Southern hybridization.

54. The method of claim 52, wherein said nucleic acid sequences are detected by the presence of a label.