Serial analysis of transcript expression using long tags

• US 20030008290A1
• Filed: 07/27/2001
• Published: 01/09/2003
• Est. Priority Date: 07/28/2000
• Status: Active Grant

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Sparks, Andrew, Velculescu, Victor E., Kinzler, Kenneth W., Vogelstein, Bert
• Granted Patent: US 6,498,013 B1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

Serial analysis of gene expression, SAGE, a method for the rapid quantitative and qualitative analysis of transcripts, has been improved to provide more genetic information about each analyzed transcript. In SAGE, 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. In a method for detecting expressed transcripts in which a first defined nucleotide sequence tag is isolated from a first cDNA oligonucleotide and a second defined nucleotide sequence tag is isolated from a second cDNA oligonucleotide, and the first defined nucleotide sequence tag is linked to a first oligonucleotide linker thereby forming a first linked nucleic acid, wherein the first oligonucleotide linker comprises a recognition site for a restriction endonuclease that allows DNA cleavage at a site in the first defined nucleotide sequence tag distant from the first recognition site;

and the second defined nucleotide sequence tag is linked to a second oligonucleotide linker thereby forming a second linked nucleic acid, wherein the second oligonucleotide linker comprises a second recognition site for the restriction endonuclease that allows DNA cleavage at a site in the first defined nucleotide sequence tag distant from the second recognition site;

wherein the first and the second linked nucleic acids are cleaved with said restriction endonuclease;

wherein the first and second tags are ligated to form ditags; and

the nucleotide sequence of at least one tag of the ditag is determined to detect gene expression, the improvement comprising;

using MmeI as the restriction endonuclease to form 3′

overhanging ends on said first and second tags.

2. A method for the detection of transcript 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 recognition site for MmeI restriction endonuclease;

linking the second tag to a second oligonucleotide linker thereby forming a second linked nucleic acid, wherein the second oligonucleotide linker comprises a second recognition site for MmeI restriction endonuclease;

cleaving the first and the second linked nucleic acids with MmeI restriction endonculease to form 3′

overhanging ends;

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 transcript expression.

3. The method of claim 2 wherein the first oligonucleotide linker comprises a first amplification primer hybridization sequence, and the second oligonucleotide linker comprises a second amplification primer hybridization sequence;

said method further comprising the step of amplifying the ditag oligonucleotide using primers which hybridize to the first and second amplification primer hybridization sequences.

4. The method of claim 2 further comprising producing concatemers of the ditags prior to the step of determining.

5. The method of claim 4 wherein the concatemer consists of about 2 to 200 ditags.

6. The method of claim 2 wherein said 3′

overhanging ends are not removed to form blunt ends prior to said step of ligating.

7. Te method of claim 2 wherein the first and second oligonucleotide linkers comprise the same nucleotide sequence.

8. Te method of claim 2 wherein the first and second oligonucleotide linkers comprise different nucleotide sequences.

9. The method of claim 4 wherein the concatemer consists of about 8 to 20 ditags.

10. The method of claim 2 wherein the ditag is about 38 to 60 base pairs.

11. The method of claim 10 wherein the ditag is about 38 to 42 base pairs.

12. The method of claim 3 wherein the step of amplifying is performed by polymerase chain reaction (PCR).

13. The method of claim 2 further comprising the step of comparing the nucleotide sequence determined to a database comprising mammalian genomic sequences whereby matching sequences are identified.

15. The method of claim 14 further comprising amplifying the at least one ditag.

16. The method of claim 14 wherein the 3′

overhanging ends are not removed to form blunt ends prior to said step of ligating.

17. The method of claim 14 wherein the first restriction endonuclease has a four base pair recognition site.

18. The method of claim 17 wherein the first restriction endonuclease is NlallI.

19. The method of claim 14 wherein the cDNA comprises a means for capture.

20. The method of claim 19 wherein the means for capture is a binding element.

21. The method of claim 20 wherein the binding element is biotin.

22. The method of claim 14 wherein the oligonucleotide linkers comprise a homogeneous population having a single nucleotide sequence.

23. The method of claim 14 wherein the oligonucleotide linkers comprise a first and second linker each having a distinct nucleotide sequence.

24. The method of claim 14 wherein said 3′

overhanging ends are removed to form blunt ends prior to said step of ligating.

25. The method of claim 14 wherein the ditag is about 38 to 60 base pairs.

26. The method of claim 14 further comprising ligating the ditags to produce a concatemer.

27. The method of claim 26 wherein the concatemer consists of about 2 to 200 ditags.

28. The method of claim 27 wherein the concatemer consists of about 8 to 20 ditags.

29. The method of claim 15 wherein the amplifying is by polymerase chain reaction (PCR).

30. The method of claim 14 wherein the oligonucleotide linkers comprise an amplification primer hybridization sequence.

14. A method for detection of transcript 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 pool of tags;

ligating the pool of tags with oligonucleotide linkers having a recognition site for a second restriction endonuclease which is MmeI which forms 3′

overhanging ends;

cleaving the tags with MmeI restriction endonuclease to form 3′

overhanging ends;

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

determining the nucleotide sequence of at least one ditag, wherein the nucleotide sequence of the ditag corresponds to sequence from at least one expressed transcripts.