Method for serial analysis of gene expression

• US 5,695,937 A
• Filed: 09/12/1995
• Issued: 12/09/1997
• Est. Priority Date: 09/12/1995
• Status: Expired due to Term

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Velculescu, Victor E., Kinzler, Kenneth W., Vogelstein, Bert, Zhang, Lin
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): Weiss, Bonnie D.
• Application Number:
  US08/527,154
• Time in Patent Office: 819 Days
• Field of Search: 435/172.3, 435/6, 435/91.1, 536/24.2, 536/24.3, 536/22.1, 935/1, 935/5, 935/6, 935/8, 935/9, 935/16, 935/19
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2539/103 Serial analysis of gene expression [SAGE]

Abstract

Serial analysis of gene expression, SAGE, a method for the rapid quantitative and qualitative analysis of transcripts is provided. Short defined sequence tags corresponding to expressed genes are isolated and analyzed. Sequencing of over 1,000 defined tags in a short period of time (e.g., hours) reveals a gene expression pattern characteristic of the function of a cell or tissue. Moreover, SAGE is useful as a gene discovery tool for the identification and isolation of novel sequence tags corresponding to novel transcripts and genes.

Claims

1. An isolated oligonucleotide composition comprising at least one ditag, wherein the ditag comprises two covalently joined defined nucleotide sequence tags in opposite orientation, wherein each tag corresponds to at least one expressed gene.

2. The composition of claim 1, wherein the oligonucleotide consists of about 1 to 200 ditags.

3. The composition of claim 2, wherein the oligonucleotide consists of about 8 to 20 ditags.

4. A method for the detection of gene expression comprising:

producing complementary deoxyribonucleic acid (cDNA) oligonucleotides;

isolating a first defined nucleotide sequence tag from a first cDNA oligonucleotide and a second defined nucleotide sequence tag from a second cDNA oligonucleotide;

linking the first tag to a first oligonucleotide linker thereby forming a first linked nucleic acid, wherein the first oligonucleotide linker comprises a first enzyme recognition site that allows DNA cleavage at a site in the first defined nucleotide sequence distant from the first recognition site;

linking the second tag to a second oligonucleotide linker thereby forming a second linked nucleic acid, wherein the second oligonucleotide linker comprises a second enzyme recognition site that allows DNA cleavage at a site in the second defined nucleotide sequence distant from the second recognition site;

cleaving the first and the second linked nucleic acids with at least one enzyme that recognizes each of the recognition sites;

ligating the first and second tags to form a ditag; and

determining the nucleotide sequence of at least one tag of the ditag to detect gene expression.

5. The method of claim 4, wherein the first oligonucleotide linker comprises a first amplification primer hybridization sequence, and the second oligonucleotide linker comprises a second amplification primer hybridization sequence:

and

further comprising amplifying the ditag oligonuclcotide.

6. The method of claim 4, further comprising producing concatemcrs of the ditag.

7. The method of claim 6, wherein the concatemer consists of about 2 to 200 ditags.

8. The method of claim 7, wherein the concatemer consists of about 8 to 20 ditags.

9. The method of claim 4, wherein the first and second oligonucleotide linkers comprise the same nucleotide sequence.

10. The method of claim 4, wherein the first and second oligonucleotide linkers comprise different nucleotide sequences.

11. The method of claim 10, wherein the first and second oligonucleotide linkers have a sequence:

5'"'"'-TTTTACCAGCTTATTCAATTCGGTCCTCTCGCACAGGGACATG-3'"'"' (SEQ ID NO;

     1)

'"'"' -ATGGTCGAATAAGTTAAGCCAGGAGAGCGTGTCCCT-5'"'"' (SEQ ID NO;

2)

or

5'"'"'-TTTTTGTAGACATTCTAGTATCTCGTCAAGTCGGAAGGGACATG-3'"'"' (SEQ ID NO;

3)

3'"'"'- AACATCTGTAAGATCATAGAGCAGTTCAGCCTTCCCT-5'"'"', (SEQ ID NO;

4)

wherein A is dideoxy A.

12. The method of claim 4, wherein at least one of the enzyme recognition sites is a type IIS endonuclease recognition site.

13. The method of claim 12, wherein the type IIS endonuclease is selected from the group consisting of BsmFI and FokI.

14. The method of claim 4, wherein the ditag is about 12 to 60 base pairs.

15. The method of claim 14, wherein the ditag is about 18 to 22 base pairs.

16. The method of claim 5, wherein the amplifying is by polymerase chain reaction (PCR).

17. The method of claim 16, wherein primers for PCR are selected from the group consisting of

5'"'"'-CCAGCTTATTCAATTCGGTCC-3'"'"' (SEO ID NO:

5) and

5'"'"'-GTAGACATTCTAGTATCTCGT-3'"'"' (SEQ ID NO;

6).

18. A method for detection of gene expression comprising:

cleaving a cDNA sample with a first restriction endonuclease, wherein the endonuclease cleaves the cDNA at a defined position in the cDNA thereby producing defined sequence tags;

isolating the defined cDNA tags and forming a first pool of tags;

ligating a first pool of tags with a first oligonucleotide linker having a first enzyme recognition site that allows DNA cleavage at a site distant from the second recognition site;

ligating a second pool of tags with a second oligonucleotide linker having a second enzyme recognition site that allows DNA cleavage at a site distant from the second recognition site;

cleaving the tags with a first and a second tag cleaving restriction endonuclease, wherein the first tag-cleaving restriction endonuclease recognizes a first enzyme recognition site and cleaves at a site distant from the first recognition site and wherein the second tag-cleaving restriction endonuclease recognizes a second enzyme recognition site and cleaves at a site distant from the second recognition site;

ligating the two pools of tags to produce at least one ditag; and

determining the nucleotide sequence of at least one ditag, wherein the ditag(s) correspond to sequence from an expressed gene.

19. The method of claim 18, further comprising amplifying the ditag.

20. The method of claim 18, wherein the first restriction endonuclease has at least one recognition site in the cDNA.

21. The method of claim 20, wherein the first restriction enzyme has a four base pair recognition site.

22. The method of claim 21, wherein the restriction endonuclease is NlallI.

23. The method of claim 18, wherein the cDNA comprises a means for capture.

24. The method of claim 23, wherein the means for capture is a binding element.

25. The method of claim 24, wherein the binding element is biotin.

26. The method of claim 18, wherein the first and second oligonucleotide linkers comprise the same nucleotide sequence.

27. The method of claim 18, wherein the first and second oligonucleotide linkers comprise different nucleotide sequences.

28. The method of claim 27, wherein the first and second oligonucleotide linkers have a sequence:

5'"'"'-TTTTACCAGCTTATTCAATTCGGTCCTCTCGCACAGGGACATG-3'"'"' (SEQ ID NO;

1)

3'"'"'ATGGTCGAATAAGTTAAGCCAGGAGAGCGTGTCCCT-5'"'"' (SEQ ID NO;

2)

or

5'"'"'-TTTTTGTAGACATTCTAGTATCTCGTCAAGTCGGAAGGGACATG-3'"'"' (SEQ ID NO;

3)

3'"'"'-AACATCTGTAAGATCATAGAGCAGTTCAGCCTTCCCT-5'"'"', (SEQ ID NO;

4)

wherein A is dideoxy A.

29. The method of claim 18, wherein at least one of the restriction endonuclease sites is a type IIS endonuclease site.

30. The method of claim 29, wherein the type IIS endonuclease is selected from the group consisting of BsmFI and FokI.

31. The method of claim 18, wherein the ditag is about 12 to 60 base pairs.

32. The method of claim 31, wherein the ditag is about 14 to 22 base pairs.

33. The method of claim 18, further comprising ligating the ditags to produce a concatemer.

34. The method of claim 33, wherein the concatemer consists of about 2 to 200 ditags.

35. The method of claim 34, wherein the concatemer consists of about 8 to 20 ditags.

36. The method of claim 18, wherein the amplifying is by polymerase chain reaction (PCR).

37. The method of claim 36, wherein primers for PCR are selected from the group consisting of

5'"'"'-CCAGCTTATTCAATTCGGTCC-3'"'"' (SEQ ID NO:

5) and

5'"'"'-GTAGACATTCTAGTATCTCGT-3'"'"' (SEQ ID NO;

6).

43. The method of claim 18, wherein the first oligonucleotide linker comprises a fast amplification primer hybridization sequence and the second oligonucleotide linker comprises a second amplification primer hybridization sequence.

38. A kit useful for detection of gene expression wherein the presence of a cDNA ditag is indicative of expression of a gene having a sequence of a tag of the ditag, the kit comprising one or more containers comprising a first container containing a first oligonucleotide linker having a first sequence useful hybridization of an amplification primer;

a second container containing a second oligonucleotide linker having a second oligonucleotide linker having a second sequence useful hybridization of an amplification primer, wherein the linkers further comprise a restriction endonuclease site for cleavage of DNA at a site distant from the restriction endonuclease recognition site; and

a third and fourth container having a nucleic acid primers for hybridization to the first and second unique sequences of the linker.

39. The kit of claim 38, wherein the linkers have a sequence

5'"'"'-TTTTACCAGCTTATTCAATTCGGTCCTCTCGCACAGGGACATG-3'"'"' (SEQ ID NO:

1)

3'"'"'-ATGGTCGAATAAGTTAAGCCAGGAGAGCGTGTCCCT-5'"'"' (SEQ ID NO;

2)

or

'"'"' -TTTTTGTAGACATTCTAGTATCTCGTCAAGTCGGAAGGGACATG-3'"'"' (SEQ ID NO;

3)

3'"'"'-AACATCTGTAAGATCATAGAGCAGTTCAGCCTTCCCT-5'"'"', (SEQ ID NO;

4)

wherein A is dideoxy A.

40. The kit of claim 38, wherein the restriction endonuclease is a type IIS endonuclease.

41. The kit of claim 40, wherein the type IIS endonuclease is BsmFI.

42. The kit of claim 38, wherein the primers for amplification are selected from the group consisting of

5'"'"'-CCAGCTTATTCAATTCGGTCC-3'"'"' (SEO ID NO:

5) and

5'"'"'-GTAGACATTCTAGTATCTCGT-3'"'"' (SEQ ID NO;

6).