Method to generate or determine nucleic acid tags corresponding to the terminal ends of DNA molecules using sequences analysis of gene expression (terminal SAGE)

• US 8,158,355 B2
• Filed: 06/03/2005
• Issued: 04/17/2012
• Est. Priority Date: 12/04/2002
• Status: Expired due to Fees

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research
• Inventors: Ruan, Yijun, Wei, Chialin
• Primary Examiner(s): Woolwine, Samuel
• Assistant Examiner(s): Pande, Suchira
• Application Number:
  US11/145,005
• Publication Number:
  US 20060084083A1
• Time in Patent Office: 2,510 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2539/103 Serial analysis of gene expression [SAGE]

Abstract

A method of providing an indication of an instance of expression of a gene is described herein, the method which may comprise the steps of: (a) providing a complementary deoxyribonucleic acid (cDNA) having a terminus comprising a terminal transcribed sequence of a gene; (b) linking the cDNA to an linker sequence thereby forming a linked nucleic acid, in which the linker sequence comprises a first recognition site for a first nucleic acid cleavage enzyme, preferably a restriction endonuclease, that allows nucleic acid cleavage at a site distant from the first recognition site; and (c) cleaving the linked nucleic acid with the first nucleic acid cleavage enzyme to provide a linked tag, in which the linked tag comprises a nucleotide sequence tag representative of a terminal transcribed sequence of the gene; and (d) detecting the presence or identity of the linked tag or the nucleotide sequence tag to provide an indication of an instance of gene expression.

Claims

1. A method of obtaining a nucleotide sequence tag from a terminus of a nucleic acid, the method comprising the steps of:

(a) providing a first nucleic acid sequence comprising a full-length first strand cDNA comprising a 5′

untranslated region, a coding sequence, a 3′

untranslated region and a poly A tail;

(b) without prior cleavage of said first nucleic acid sequence, linking the 5′

end of said full length first strand cDNA to a linker sequence to form a linked nucleic acid, in which the linker sequence comprises;

(i) a first recognition site for a first nucleic acid cleavage enzyme that allows cleavage of the first nucleic acid sequence at a site distant from the first recognition site, and

(ii) a second recognition site for a second nucleic acid cleavage enzyme that allows nucleic acid cleavage at a site distant from the second recognition site, said cleavage site located at a position within the first recognition site;

in which the linked nucleic acid has the structure;

5′

-second recognition site-first recognition site-first nucleic acid-3′

(c) cleaving the linked nucleic acid with the first nucleic acid cleavage enzyme to provide;

(i) a linked tag comprising the linker sequence linked to a nucleotide sequence tag representative of the first nucleic acid sequence, the nucleotide sequence tag comprising the 5′

terminus of the full length first strand cDNA; and

(ii) a second nucleic acid sequence comprising a remainder portion of the first nucleic acid.

2. The method according to claim 1, in which the second nucleic acid cleavage enzyme comprises a restriction endonuclease having a recognition site of 6 bases or greater.

3. The method according to claim 2, wherein the restriction endonuclease is MmeI.

4. The method according to claim 1, wherein the first nucleic acid cleavage enzyme is the restriction endonuclease BseRI.

5. A method of sequentially generating a plurality of nucleic acid sequences each comprising a nucleotide sequence tag from a nucleic acid, the method comprising performing the steps of the method of claim 1, followed by repeating at least once the steps of:

A) linking a linker sequence as described in step (b)(i) and (b)(ii) of claim 1 to said second nucleic acid sequence comprising a remainder portion of the first nucleic acid; and

B) cleaving the linked second nucleic acid with the first nucleic acid cleavage enzyme to provide a linked tag comprising the linker sequence linked to a nucleotide sequence tag comprising the 5′

terminus of said second nucleic acid, and a cleaved remainder portion of said second nucleic acid.

6. A method of providing an indication of an instance of expression of a gene, the method comprising the method according to claim 1, and further comprising the step of detecting the presence of the linked tag to provide an indication of an instance of gene expression.

7. A method of detecting gene expression, the method comprising the steps of:

(a) providing a first linked tag and a second linked tag, each independently produced by the method according to claim 1;

(b) linking the first linked tag and the second linked tag such that the nucleotide sequence tag portion of one linked tag is linked to the nucleotide sequence tag of the other linked tag to form a ditag, the ditag comprising terminal transcribed sequences from first and second genes; and

(c) detecting the presence or identity of the ditag, or at least one nucleotide sequence tag comprised therein, to detect gene expression.

8. The method according to claim 7, in which each of the first and second linker sequences comprised in the first and second linked tags comprises an amplification primer sequence, and in which the method further comprises a step of amplifying the ditag.

9. The method according to claim 8, wherein the ditag is amplified by polymerase chain reaction (PCR).

10. The method according to claim 7, further comprising the steps of:

(d) cleaving the ditag with second nucleic acid cleavage enzyme;

(e) linking a plurality of the resultant trimmed ditags to form a concatamer; and

(f) obtaining the nucleic acid sequence of at least a portion of the concatamer.