Method for gene expression analysis

• US 5,876,932 A
• Filed: 05/17/1996
• Issued: 03/02/1999
• Est. Priority Date: 05/19/1995
• Status: Expired due to Fees

• Current Assignee: Sygnis Bioscience GmbH & Co. KG
• Original Assignee: Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften e.V. (Max-Planck-Gesellschaft zur Frderung der Wissenschaften e)
• Inventors: Fischer, Achim
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Yucel, Irem
• Application Number:
  US08/649,511
• Time in Patent Office: 1,019 Days
• Field of Search: 536/23.1, 435/91.1, 435/91.2, 435/91.3, 435/6
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2539/103 Serial analysis of gene expression [SAGE]

Abstract

The present invention concerns a method for the general analysis of gene expression as well as a method for the differential analysis of the expression of members of a gene family.

Claims

1. A method for the differential analysis of the expression of members of a gene family said method comprising:

(a) isolating mRNA molecules from at least one tissue sample to be analyzed,

(b) synthesizing cDNA first strand molecules from the mRNA molecules using a cDNA primer,

(c) preparing amplification products by selectively amplifying the cDNA first stand molecules of members of a gene family of interest using oligonucleotide primers comprising at least one gene family specific primer wherein said gene family specific primer is an oligonucleotide which hybridizes under PCR conditions with a conserved domain within the gene family of interest and a reverse primer which is an oligonucleotide directed against a sequence that is complementary to the sequence of the cDNA primer a reverse primer and labeling the amplification products,

(d) transcript-specific shortening of the amplification products obtained in (c) by cleaving the amplification products with at least one restriction endonuclease,

(e) separating the cleaved amplification products obtained in (d) according to their length and

(f) analyzing the separated amplification products.

2. The method as claimed in claim 1, wherein the synthesis of the cDNA first strand molecules in step (b) is carried out by reverse transcription using an oligo-dT nucleotide as a cDNA primer.

3. The method as claimed in claim 2, wherein the oligo-dT primer has an extension at its 5'"'"' end or at its 3'"'"' end or at both its 5'"'"' and 3'"'"' ends.

4. The method as claimed in claim 1, wherein the cDNA primer is a random primer or a mixture of random primers.

5. The method as claimed in claim 1, wherein the cDNA primer is a gene family-specific primer or a mixture of gene family-specific primers.

6. The method as claimed in claim 1, wherein the cDNA primer is an oligo-dT primer with a 5'"'"' extension and the reverse primer in step (c) has a sequence which is directed against a sequence that is complementary to the sequence of the 5'"'"' extension.

7. The method as claimed in claim 1, wherein the restriction endonuclease cleaves the amplification products with a high statistical probability.

8. The method of claim 7, wherein the restriction endonuclease has a four-base recognition sequence.

9. The method as claimed in claim 1, wherein the label is incorporated by using a primer which carries a marker group.

10. The method as claimed in claim 1, wherein the label is incorporated by using labeled nucleotides in the amplification.

11. The method as claimed in claim 1, wherein label is incorporated into the amplification products by primer extension wherein said amplification products are templates.

12. The method as claimed in claim 1, wherein separating the amplification products is by gel electrophoresis.

13. The method as claimed in claim 1, wherein the analysis of the separated amplification products is by direct sequencing of said amplification products.

14. The method as claimed in claim 1, wherein the first strand cDNA molecules are provided with a specific nucleotide sequence at their 3'"'"' ends and an oligonucleotide directed against the specific nucleotide sequence is used as a reverse primer for the amplification in step (c).

15. The method as claimed in claim 14, wherein the specific nucleotide sequence comprises a homopolymer or an arbitrary sequence.

16. The method as claimed in claim 1, wherein the first strand cDNA molecules are converted into oligomeric or circular molecules or both oligomeric and circular molecules by self ligation and an oligonucleotide which is at least partially complementary to the cDNA primer is used as a reverse primer for the amplification in step (c).

17. The method of claim 16, wherein said oligonucleotide has an extension at its 3'"'"' end.

18. The method as claimed in claim 1, further comprising before step (c) and after step (b) converting the first strand of cDNA molecules into oligomeric or circular molecules or oligomeric and circular molecules by means of self-ligation and wherein two oligonucleotides directed against a sequence that is complementary to the sequence of the cDNA primer are used as primers for the amplification in step (c) wherein said oligonucleotides are in opposite orientation relative to each other.

19. The method of claim 18, wherein the oligonucleotide primers in (c) have 3'"'"' extensions.

20. The method as claimed in claim 1, wherein amplification steps are carried out under long range PCR conditions.

21. A method for isolating new genes comprising steps (a) to (f) of claim 1.

22. The method of claim 21 wherein the new genes are members of a gene family.

24. A method for examining the life cycle of a cell or an organism comprising steps (a) to (f) of claim 1.

23. A method for determining the living stage of a cells or an organism comprising steps (a) to (f) of claim I.

25. A method for the differential analysis of the expression of members of a gene family said method comprising:

(a) isolating mRNA molecules from at least one tissue sample to be analyzed,

(b) synthesizing cDNA first strand molecules from the mRNA molecules using a cDNA primer,

(c) preparing amplification products by selectively amplifying the cDNA first strand molecules of members of a gene family of interest using at least one oligonucleotide gene family-specific primer wherein said gene family-specific primer is an oligonucleotide which hybridizes under PCR conditions with a conserved domain within the gene family of interest and a reverse primer which is an oligonucleotide directed against a sequence that is complementary to the sequence of the cDNA primer, and labeling the amplification products,

(d) transcript-specific shortening of the amplification products obtained in (c)

by cleaving the amplification products with at least one restriction endonuclease,

(e) separating the cleaved amplification products obtained in (d) according to their length and

(f) analyzing the separated amplification products.

26. The method as claimed in claim 25, wherein the linker molecule has a first end which is ligated to said cleaved amplification products and a second end which cannot be ligated to said cleaved amplification products.

27. The method as claimed in claim 25, wherein a nested primer is used in step (f) which is directed against the gene family-specific conserved domain.

28. The method as claimed in claim 25, wherein the amplification or reamplification products are isolated and additionally amplified.

29. The method as claimed in claim 28, wherein the oligonucleotide primer directed against the linker molecule and the gene-family specific primer in (f) are used in the additional amplification.

30. The method as claimed in claim 25, wherein the linker molecules, the cDNA primers or the amplification primers have at least one cleavage site for a restriction endonuclease.

31. The method as claimed in claim 25, wherein amplification steps are carried out under long range PCR conditions.

32. The method of claim 25, wherein the oligonucleotide primers in (d) have 3'"'"' extensions.

33. A method for the differential analysis of gene expression said method comprising:

(a) isolating mRNA molecules from one or several samples to be analyzed,

(aa) attaching an anchor sequence to the 5'"'"' end of the mRNA molecules,

(b) synthesizing cDNA first strand molecules from the mRNA molecules,

(c) amplifying the cDNA first strand molecules and labeling of the amplification products,

(d) transcript-specific shortening of the amplification products by cleaving said amplification products with at least one restriction endonuclease,

(e) separating the amplification products according to their length,

(f) analyzing the separated amplification products.

34. The method as claimed in claim 33, wherein amplification steps are carried out under long range PCR conditions.

35. A method for the detection of transcripts, the method comprising:

(a) isolating mRNA molecules from one or several samples,

(aa) attaching of an anchor sequence to the 5'"'"' end of the mRNA molecules,

(b) synthesizing cDNA first strand molecules from the mRNA molecules,

(c) amplifying the cDNA first strand molecules,

(d) transcript-specific shortening of the amplification products by cleaving said amplification products with at least one restriction endonuclease,

(e) attaching linker molecules to the ends of the cleaved amplification products,

(f) preparing a reamplification product by amplifying and labeling the cleaved amplification products having attached linkers in which a primer directed against the linker molecule attached in step (e) and a primer selected from the group consisting of (i) a primer directed against the anchor sequence attached in step (aa) or (ii) a primer directed against the cDNA primer sequence used in step (b) are used,

(g) separating the reamplification products according to their length and,

(h) detecting and analyzing the separated reamplification products.

36. The method of claim 35, further comprising before step (g) and after step (f),

(ff) an additional amplification and labeling of the cleaved amplification products using nested primers, and

(fff) a subtractive hybridization of two pools of amplification products which are to be compared with one another.

37. The method as claimed in claim 35, wherein amplification steps are carried out under long range PCR conditions.

38. The method of claim 35, wherein the oligonucleotide primers in (d) have 3'"'"' extensions.

45. The method of claim 35, wherein the oligonucleotide primers in (d) have 3'"'"' extensions.

39. A reagent kit for the analysis of the expression of genes comprising:

(a) an enzyme for cDNA first stand synthesis,

(b) at least one enzyme for the synthesis of cDNA second strands and for the amplification of DNA fragments,

(c) at least one double-stranded DNA linker molecule and an agent for attaching the linker molecule to the ends of DNA fragments,

(d) single-stranded nucleic acid primer molecules comprising at least one of (i) a gene family-specific primer, (ii) a set of random primers, (iii) primers according to (i-ii) with a 3'"'"' extension or 5'"'"' extension or a 3'"'"' extension and a 5'"'"' extension, (iv) primers according to (i-iii) which carry marker groups and (v) primers according to (iv) which contain a 3'"'"' extension,

(e) an agent for labeling and for detecting nucleic acids,

(f) at least one oligo-dT nucleotide, and

(g) at least one primer directed against a sequence which is complementary to the sequence of the DNA linker molecule.

40. The reagent kit of claim 39 further comprising at least one restriction enzyme.

41. The reagent kit of claim 40 further comprising reagents selected from the group consisting of RNase inhibitors, buffers, streptavidin-coated reaction vessels and magnetic particles.

42. A method for the detection of transcripts, said method comprising:

(a) isolating mRNA molecules from at least one tissue sample to be analyzed,

(b) synthesizing cDNA first strand molecules from the mRNA molecules,

(c) preparing double stranded cDNA from said cDNA first strand molecules,

(d) cleaving said double stranded cDNA with at least one restriction endonuclease,

(e) separating the cleaved double stranded cDNA according to their length,

(f) attaching linker molecules to the ends of the cleaved amplification products obtained in (d),

(g) preparing amplification products by amplifying and labeling the cleaved amplification products attached to said linker obtained in (f) using a linker primer and a primer directed against the cDNA primer or against a gene family-specific domain, and

(h) detecting and analyzing the reamplification products.

43. The method as claimed in claim 42, wherein the double-stranded cDNA is produced by self-ligation of the first strand cDNA and subsequent second strand synthesis.

44. The method as claimed in claim 42, wherein amplification steps are carried out under long range PCR conditions.

46. A method for the detection of transcripts, said method comprising:

(a) isolating mRNA molecules from at least one tissue sample to be analyzed,

(b) synthesizing cDNA first strand molecules from the mRNA molecules using a cDNA primer,

(c) preparing amplification products by amplifying and labeling the cDNA first strand molecules using oligonucleotides comprising random primers, and labeling the amplification products,

(d) cleaving said amplification products with at least one restriction endonuclease,

(e) separating the amplification products according to their length, and

(f) analyzing the separated amplification products.

47. The method as claimed in claim 46, wherein said random primers have a length of 15-25 nucleotides.

48. The method as claimed in claim 47, wherein the random primers have an adequately high degree of degeneracy in order to statistically bind to a DNA template as frequently as an 8-12 nucleotide long non-degenerate primer.

49. The method as claimed in claim 46, wherein amplification steps are carried out under long range PCR conditions.

50. A method for the detection of transcripts, said method comprising:

(a) isolating MA molecules from at least one tissue sample to be analyzed,

(b) attaching an anchor oligonucleotide to the 5'"'"' end of said mRNA,

(c) synthesizing cDNA first strand molecules from the mRNA molecules using a cDNA primer,

(d) preparing amplification products by amplifying the cDNA first strand molecules using oligonucleotides comprising a primer which is a sequence which is complementary to the cDNA primer or a gene family-primer,

(e) cleaving said amplification products with at least one restriction endonuclease,

(f) attaching a linker molecule to the ends of the cleaved amplification products,

(g) preparing reamplification products by amplifying and labeling the cleaved amplification products ligated to said linker using oligonucleotide primers comprising at least one primer selected from the group consisting of a gene family-specific primer, a primer which is directed against a sequence which is complementary to the sequence of the cDNA primer, a primer directed against the linker molecule and a primer directed against a sequence which is complementary to the sequence of the anchor oligonucleotide,

(h) separating the reamplification products according to their length, and

(i) detecting and analyzing the separated reamplification products.

51. The method as claimed in claim 50, wherein said anchor oligonucleotide is an RNA oligonucleotide and wherein the method further comprises after (a) and before (b) decapping the mRNA and wherein the RNA oligonucleotide is attached to the decapped mRNA molecules 5'"'"' ends by means of a single-strand ligation.

52. The method as claimed in claim 50, wherein said anchor oligonucleotide is an oligonucleotide which specifically hybridizes to the cap structure on said mRNA molecules and wherein said oligonucleotide has a nucleotide sequence which is incorporated into the first strand cDNA molecules during reverse transcription of said mRNA.

53. The method as claimed in claim 50, wherein amplification steps are carried out under long range PCR conditions.

54. The method as claimed in claim 50 further comprising before step (e) a subtractive hybridization step of the amplification products from two tissue samples which are to be compared with one another.

55. The method as claimed in claim 54, wherein the subtractive hybridization is carried out in several subpools which have been generated by using primers with different 3'"'"' extensions but otherwise identical sequence.

56. The method of claim 50, wherein the oligonucleotide primers in (d) have 3'"'"' extensions.

64. The method of claim 50 or 62 wherein said RNA oligonucleotide has a length of 20-30 bases.

57. A method for the detection of transcripts, said method comprising:

(a) isolating mRNA molecules form at least one tissue sample to be analyzed,

(b) synthesizing cDNA first strand molecules from the mRNA molecules using a cDNA primer,

(c) adding said cDNA first strand molecules to an oligonucleotide having a known nucleotide sequence,

(d) preparing amplification products by amplifying the cDNA first strand molecules using at least one oligonucleotide primer directed against the oligonucleotide having a known nucleotide sequence in (b) and another oligonucleotide primer having a sequence that is directed against a sequence which is complementary to the cDNA primer in (c),

(e) cleaving said amplification products with at least one restriction endonuclease,

(f) separating the amplification products according to their length, and

(g) attaching linker molecules to the ends of the cleaved amplification products obtained in (d),

(h) preparing reamplification products by amplifying and labeling the cleaved amplification products attached to said linker obtained in (g) using an oligonucleotide primer selected from the group consisting of a gene family-specific primer, a primer directed against the linker, a primer directed against the sequence added to the cDNA and primer directed against a sequence which is complementary to the cDNA-primer,

(i) detecting and analyzing the separated amplification products.

58. The method as claimed in claim 57, wherein the oligonucleotide having a known nucleotide sequence comprises a homopolymer or an arbitrary sequence.

59. The method as claimed in claim 57, wherein the oligonucleotide having a known nucleotide sequence is modified at its 3'"'"' end in order to prevent further ligation of the oligonucleotides.

60. The method of claim 57, wherein the oligonucleotide primers in (d) have 3'"'"' extensions.

61. The method as claimed in claim 57, wherein amplification steps are carried out under long range PCR conditions.

62. A method for the differential analysis of the expression of members of a gene family said method comprising:

(a) isolating mRNA molecules from at least one tissue sample to be analyzed,

(b) attaching an anchor oligonucleotide to the 5'"'"' end of said mRNA molecules,

(c) synthesizing cDNA first strand molecules from the mRNA molecules using a cDNA primer,

(d) preparing amplification products by selectively amplifying the cDNA first stand molecules of members of a gene family using oligonucleotide primers comprising at least one gene family specific primer, wherein said gene family specific primer is an oligonucleotide which hybridizes under PCR conditions with a conserved domain within the gene family, and an oligonucleotide directed against a sequence which is complementary to the sequence of the anchor oligonucleotide functioning as a reverse primer, and labeling the amplification products,

(e) transcript specific shortening of the amplification products with at least one restriction endonuclease,

(f) separating the reamplification products according to their length, and

(g) analyzing the separated reamplification products.

63. The method as claimed in claim 62, wherein amplification steps are carried out under long range PCR conditions.

65. A method for the detection of transcripts, said method comprising:

(a) isolating mRNA molecules from at least one tissue sample to be analyzed,

(b) synthesizing cDNA first strand molecules from the mRNA molecules using a cDNA primer,

(c) preparing amplification products by amplifying the cDNA first strand molecules using oligonucleotides comprising a primer which is directed against a sequence which is complementary to the cDNA primer or a gene family-primer,

(d) cleaving said amplification products with at least one restriction endonuclease,

(e) attaching a linker molecule to the ends of the cleaved amplification products,

(f) preparing reamplification products by amplifying and labeling the cleaved amplification products ligated to said linker using oligonucleotide primers comprising at least one primer selected from the group consisting of a gene family-specific primer, a primer which is directed against a sequence which is complementary to the sequence of the cDNA primer, a primer directed against the linker molecule and a primer directed against a sequence which is complementary to the sequence of the anchor oligonucleotide.

(g) separating the reamplification products according to their length, and

(h) detecting and analyzing the separated reamplification products.

66. A method for the detection of transcripts, said method comprising:

(a) isolating mRNA molecules from at least one tissue sample to be analyzed,

(b) synthesizing cDNA first strand molecules from the mRNA molecules,

(c) preparing double stranded cDNA from said cDNA first strand molecules,

(d) preparing amplification products by amplifying the double stranded cDNA,

(e) cleaving said double stranded cDNA with at least one restriction endonuclease,

(f) separating the cleaved double stranded cDNA according to their length,

(g) attaching linker molecules to the ends of the cleaved amplification products obtained in (d),

(h) preparing reamplification products by amplifying and labeling the cleaved amplification products attached to said linker obtained in (g) using a linker primer and a primer directed against the cDNA primer or against a gene family-specific domain, and

(i) detecting and analyzing the reamplification products.

67. A reagent kit for the analysis of the expression of genes comprising:

(a) an enzyme for cDNA first strand synthesis,

(b) at least one enzyme for the synthesis of cDNA second strands and for the amplification of DNA fragments,

(c) at least one double-stranded DNA linker molecule and an agent for attaching the linker molecule to the ends of DNA fragments,

(d) an RNA oligonucleotide and attaching agent,

(e) an agent for labeling and for detecting nucleic acids,

(f) a primer directed against a sequence which is complementary to the sequence of the RNA oligonucleotide,

(g) at least one oligo-dT nucleotide, and

(h) at least one primer derived from the sequence of the DNA linker molecule.