Method for serial analysis of gene expression

• US 6,746,845 B2
• Filed: 03/14/2002
• Issued: 06/08/2004
• Est. Priority Date: 09/12/1995
• Status: Expired due to Fees

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Velculescu, Victor E., Kinzler, Kenneth W., Zhang, Lin, Vogelstein, Bert
• Primary Examiner(s): SITTON, JEHANNE SOUAYA
• Application Number:
  US10/096,596
• Publication Number:
  US 20030049653A1
• Time in Patent Office: 817 Days
• Field of Search: 435/6, 435/91.1, 435/91.5, 435/196, 536/23.1, 536/24.2
• US Class Current: 435/6.11
• 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. A method for making a tag which identifies a complementary deoxyribonucleic acid (cDNA) oligonucleotide, comprising:

providing a complementary deoxyribonucleic acid (cDNA) oligonucleotide comprising a 5′ and

a 3′ and

;

cleaving said cDNA oligonucleotide with a restriction enzyme at a first restriction endonuclease site to provide cDNA fragments;

isolating the 5′

or 3′

end of said cDNA oligonucleotide;

linking an oligonucleotide linker to an isolated cDNA fragment comprising the 5′

or 3′

end of said cDNA oligonucleotide, wherein the oligonucleotide linker comprises a recognition site for a second restriction endonuclease enzyme which cleaves at a site distant from the recognition site; and

cleaving the isolated cDNA fragment with the second restriction endonuclease enzyme to provide a tag which identifies a cDNA oligonucleotide.

2. The method of claim 1 wherein the second restriction endonuclease is a type IIS endonuclease.

3. The method of claim 2 wherein the type IIS endonuclease is selected from the group consisting of BsmFI and FokI.

4. A method for making a defined sequence tag which identifies a complementary deoxyribonucleic acid (cDNA) oligonucleotide, comprising the steps of:

cleaving a cDNA sample with a first restriction endonuclease, wherein the endonuclease cleaves the cDNA at a defined position relative to the 5′

or 3′

terminus of the cDNA thereby producing defined sequence tags;

isolating the defined sequence tags;

ligating an oligonucleotide linker to the defined sequence tags, wherein the oligonucleotide linker comprises a recognition site for a second restriction endonuclease which cleaves at a site outside of its recognition sequence;

cleaving the defined sequence tags with the second restriction endonuclease at the site outside of its recognition sequence to provide defined sequence tags having a defined length.

5. The method of claim 4, wherein the first restriction enzyme has a four base pair recognition site.

6. The method of claim 5 wherein the first restriction endonuclease is NlaIII.

7. The method of claim 4 wherein the cDNA comprises a means for capture.

8. The method of claim 7 wherein the means for capture is a binding element.

9. The method of claim 8 wherein the binding element is biotin.

10. The method of claim 4 wherein the second restriction endonuclease is a type IIS endonuclease.

11. The method of claim 10 wherein the type IIS endonuclease is selected from the group consisting of BsmFI and FokI.