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 20060084083A1
• Filed: 06/03/2005
• Published: 04/20/2006
• Est. Priority Date: 12/04/2002
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research (Government of Singapore)
• Original Assignee: Agency For Science Technology and Research (Government of Singapore)
• Inventors: Ruan, Yijun, Wei, Chialin
• Granted Patent: US 8,158,355 B2
• 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 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;

(b) linking the first nucleic acid sequence to an 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 or about 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 and comprising a terminal portion thereof; and

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

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 a method according to any preceding claim;

(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 or

a method of providing an indication of an instance of expression of a gene, the method comprising 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 or

a method of detecting expression of a gene, the method comprising;

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

(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 or

A method for detecting expression of a gene, the method comprising the steps of;

(a) providing a first complementary deoxyribonucleic acid (cDNA) having a terminus comprising a terminal transcribed sequence of a first gene;

(b) providing a second complementary deoxyribonucleic acid (cDNA) having a terminus comprising a terminal transcribed sequence of a second gene;

(c) linking the first cDNA so produced to a first linker sequence thereby forming a first linked nucleic acid, in which the first linker sequence comprises a first recognition site for a first nucleic acid cleavage enzyme, preferably a first restriction endonuclease, that allows nucleic acid cleavage at a site distant from the first recognition site;

(d) linking the second cDNA so produced to a second linker sequence thereby forming a second linked nucleic acid, in which the second linker sequence comprises a second recognition site for a second nucleic acid cleavage enzyme, preferably a second restriction endonuclease, that allows nucleic acid cleavage at a site distant from the second recognition site;

(e) cleaving the first linked nucleic acid with the first nucleic acid cleavage enzyme to provide a first linked tag, in which the first linked tag comprises a first nucleotide sequence tag representative of a terminal transcribed sequence of the first cDNA.

(f) cleaving the second linked nucleic acid with the second nucleic acid cleavage enzyme to provide a second linked tag, in which the second linked tag comprises a second nucleotide sequence tag representative of a terminal transcribed sequence of the second cDNA.

(g) ligating the first and second tags to form a ditag; and

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

2. A method of obtaining a nucleotide sequence tag from a terminus of a nucleic acid according to claim 1, in which the first nucleic acid comprises a complementary deoxyribonucleic acid (cDNA) having a terminus comprising a 5′

terminal transcribed sequence of a gene, and in which the linker sequence is linked to said terminus or

in which the first nucleic acid comprises a complementary deoxyribonucleic acid (cDNA) having a terminus comprising a 3′

terminal transcribed sequence of a gene, and in which the linker sequence is linked to said terminus or

in which the second nucleic acid cleavage enzyme comprises a restriction endonuclease, preferably a Type IIS restriction endonuclease, whose recognition site is 6 bases or greater, preferably MmeI or

in which the first nucleic acid cleavage enzyme comprises a restriction endonuclease, preferably a Type IIS restriction endonuclease, preferably BseRI or

a method of sequentially generating a plurality of nucleic acid sequences each comprising a nucleotide sequence tag from a nucleic acid, the method comprising repeating steps (a) to (c) of claim 1 at least once, in which the first nucleic acid sequence of step (a) is provided by the second nucleic acid sequence of step (c)(ii) or

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

3. A method of detecting gene expression according to claim 1, 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, preferably by means of the polymerase chain reaction (PCR) or

further comprising the steps of:

(d) cleaving the ditag with the or each 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.

4. A method of providing an indication of an instance of expression of a gene according to claim 1, in which the 5′

end of the cDNA comprises a sequence corresponding to the 5′

terminal transcribed sequence of the gene, and in which the linker sequence is linked to the 5′

end of the cDNA or

in which the nucleotide sequence tag comprises a 5′

terminal transcribed sequence of the gene, preferably at least the first 16 bases of the transcribed portion of the gene, more preferably the first 20 bases of the transcribed portion of the gene or

in which the 3′

end of the cDNA comprises a sequence corresponding to the 3′

terminal transcribed sequence of the gene, and in which the linker sequence is linked to the 3′

end of the cDNA or

in which the nucleotide sequence tag comprises a 3′

terminal transcribed sequence of the gene, preferably at least the last 16 bases of the transcribed portion of the gene, more preferably the last 20 bases of the transcribed portion of the gene or

in which step (a) comprises;

(i) deriving a cDNA from an mRNA by reverse transcription with an primer comprising a sequence 5′

-NV(T)¹³CCGGCCGG-3′

(SEQ ID NO;

     4), in which N=A, C, G or T and V=A, C or G;

(ii) producing a double stranded cDNA therefrom and digesting the double stranded

cDNA with FseI to produce a cleaved cDNA comprising

$3^{,}<br><br>\frac{\mathrm{GGCCGG}}{\mathrm{CC}}$

overhang;

(iii) linking the cleaved cDNA to a linker comprising

$\frac{\mathrm{pTCGGA}}{\mathrm{GGCCAGCCT}};<br><br>$

and

(iv) cleaving the resulting molecule with MmeI to produce a cDNA lacking a polyA/T tail or

in which the cDNA comprises the 5′

terminal transcribed sequence of the gene, or the 3′

terminal transcribed sequence of the gene, or both or

in which the cDNA is full length cDNA, preferably comprising substantially all the coding sequence of the gene or

a method of obtaining sequential sequence information from a gene, the method comprising steps (a) to (c) of a method according to claim 5, or any claim dependent thereon, the method further comprising;

(d) providing a second nucleic acid from step (c) comprising 3′

remaining sequences of the cDNA;

(e) linking the second nucleic acid to an linker sequence, thereby forming a linked nucleic acid, in which the linker sequence comprises a recognition site for a nucleic acid cleavage enzyme, preferably a restriction endonuclease, that allows nucleic acid cleavage at a site distant from the recognition site;

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

(i) a linked tag comprising the linker sequence linked to a nucleotide sequence tag comprising a 5′

portion of the second nucleic acid; and

(ii) a fourth nucleic acid sequence comprising a 3′

remainder portion of third nucleic acid;

(g) repeating steps (d) to (f at least once, in which the second nucleic acid sequence of step (d) is provided by the fourth nucleic acid sequence of step (f)(ii); and

(h) detecting the presence, identity or sequence of at least one linked tag or a nucleotide sequence tag comprised therein or

in which the linker sequence further comprises a second recognition site for a second nucleic acid cleavage enzyme, preferably a second restriction endonuclease, that allows nucleic acid cleavage at a site distant from the second recognition site, and in which the second recognition site is located 5′

of the first recognition site in the linker sequence or

in which the linker sequence further comprises a second recognition site for a second nucleic acid cleavage enzyme, preferably a second restriction endonuclease, that allows nucleic acid cleavage at a site distant from the second recognition site, and in which the second recognition site is located 5′

of the first recognition site in the linker sequence and in which the location of the second recognition site with respect to the first recognition site is such that, when the linked tag is exposed to the second nucleic acid cleavage enzyme, cleavage of the nucleic acid occurs at a position within or about the first recognition site or

in which the first recognition site or the second recognition site, or both, comprises a Type IIS restriction enzyme recognition site or

in which the first restriction enzyme comprises MmeI, or in which the first recognition site comprises a MmeI recognition site 5′

-TCC RAC-3′

, preferably 5′

-TCC GAC-3′

or

in which the first recognition site or the second recognition site, or both, comprises a Type IIS restriction enzyme recognition site and in which the second restriction enzyme comprises a restriction enzyme capable of recognising a site which is 6 bases or longer, preferably BseR1, or in which the second recognition site comprises a BseRI recognition site 5′

-GAGGAG-3′

or

in which the first restriction enzyme comprises MmeI, or in which the first recognition site comprises a MmeI recognition site 5′

-TCC RAC-3′

, preferably 5′

-TCC GAC-3′ and

in which the second restriction enzyme comprises a restriction enzyme capable of recognising a site which is 6 bases or longer, preferably BseR1, or in which the second recognition site comprises a BseR1 recognition site 5′

-GAGGAG-3′

or

a method according to claim 5 in which the linker sequence comprises the sequence 5′

-GAGGAGNNNNNNTC_▴

CG^▾

AC-3′

(SEQ ID NO;

     1), preferably 5′

-GAGGAGCGTCTCTCCGAC-3′

(SEQ ID NO;

     2) or

further comprising the step of determining the sequence of a or each linked tag, nucleotide sequence tag, ditag, trimmed ditag or concatamer.

5. A method of detecting expression of a gene according to claim 1, in which each of the first linker sequence of the first linked tag and the second linker sequence of the second linked tag comprises an amplification primer hybridisation sequence or

in which the method further comprises a step of amplifying the ditag, preferably by means of the polymerase chain reaction (PCR) or

in which the method further comprises the step of cleaving the ditag with the or each second nucleic acid cleavage enzyme to provide a trimmed ditag or

in which the trimmed ditag comprises between 12 to 120 base pairs, preferably between 18 to 46 base pairs, preferably 40 base pairs or

in which a plurality of ditags or trimmed ditags are linked to form a concatamer or

in which the concatamer comprises between 2 to 200 ditags or trimmed ditags, preferably between 10 to 40 ditags or trimmed ditags, preferably between 8 to 20 ditags or trimmed ditags or

in which the method further comprising the step of determining the sequence of a or each linked tag, nucleotide sequence tag, ditag, trimmed ditag or concatamer or

in which the method further comprising the step of determining the sequence of a or each linked tag, nucleotide sequence tag, ditag, trimmed ditag or concatamer and further comprising the step of determining the identity of the expressed gene by the comparing the sequence to a nucleotide sequence comprised in a database of nucleotide sequences or

in which the method further comprising the step of determining the sequence of a or each linked tag, nucleotide sequence tag, ditag, trimmed ditag or concatamer and in which the sequence is compared to a database of known genes, such that if the database does not comprise the sequence, the sequence comprises a new gene.

6. A method of providing an indication useful in the diagnosis of a disease in an individual, the method comprising:

(a) providing a cell known to be affected by the disease;

(b) determining if a gene is expressed in the cell of (b) by a method according to any preceding claim;

(c) providing a cell of an individual suspected of suffering from the disease; and

(d) determining whether the same gene is expressed in the cell of (c) by a method according to any preceding claim; and

(e) comparing the expression, or lack thereof, of the gene between the cell of (b) and the cell of (c) or

a method of producing determining the transcriptome of a cell, or obtaining a gene expression profile of a cell, the method comprising providing cDNA from the cell, subjecting said cDNA to a method according to any preceding claim, and determining whether a particular gene, or a particular set of genes, is expressed by the cell or

a method of providing an indication useful in the diagnosis of a disease in an individual, the method comprising comparing the gene expression profile of a cell known to be affected by the disease, with a cell of an individual suspected of suffering from the disease, in which either or both of the gene expression profiles are produced by a method according a method of producing determining the transcriptome of a cell, or obtaining a gene expression profile of a cell, the method comprising providing cDNA from the cell, subjecting said cDNA to a method according to any preceding claim, and determining whether a particular gene, or a particular set of genes, is expressed by the cell or

a method of determining the sequence of a control sequence of a gene, preferably a promoter or enhancer sequence, the method comprising;

(a) obtaining a nucleotide sequence tag representative of the 5′

terminal transcribed sequence of the gene by a method according to claims 1, 2, or 3, or any claim dependent thereon; and

(b) obtaining a sequence of the gene 5′

to the terminal transcribed sequence of (a), in which the sequence comprises a promoter or enhancer consensus sequence or

a method of determining the sequence of a control sequence of a gene, preferably a promoter or enhancer sequence, the method comprising;

(a) obtaining a nucleotide sequence tag representative of the 5′

terminal transcribed sequence of the gene by a method according to claims 1, 2, or 3, or any claim dependent thereon; and

(b) obtaining a sequence of the gene 5′

to the terminal transcribed sequence of (a), in which the sequence comprises a promoter or enhancer consensus sequence, in which the sequence of the gene 5′

to the terminal transcribed sequence of (a) is obtained by means of (a) chromosome walking, (b) SAGE walking, (c) nucleic acid hybridisation of a genomic library;

or (d) querying a database of genomic sequences.

7. A database comprising a plurality of records, each record comprising an indication whether a gene is expressed by a particular cell, which indication is provided by a method according to claim 1.

8. A database comprising a plurality of records, each record comprising an indication whether a gene is expressed by a particular cell, which indication is provided by a method according to claim 6.

9. A computer readable medium comprising a database according to claim 7.

10. A computer readable medium comprising a database according to claim 8.

11. A nucleic acid sequence comprising:

(a) a recognition site for a nucleic acid cleavage enzyme, preferably a restriction endonuclease, that allows nucleic acid cleavage at a site distant from the first recognition site, and (b) a nucleotide sequence tag representative of a terminal transcribed sequence of a gene or

a linker sequence comprising;

(a) a first recognition site for a nucleic acid cleavage enzyme, preferably a restriction endonuclease, that allows nucleic acid cleavage at a site distant from the first recognition site;

(c) a second recognition site for a second nucleic acid cleavage enzyme, preferably a second restriction endonuclease, that allows nucleic acid cleavage at a site distant from the second recognition site;

in which in which the cleavage site for the second nucleic acid cleavage enzyme is located within or about the first recognition site.

12. A nucleic acid sequence according to claim 11, further comprising:

(c) a second recognition site in which the second recognition site is for a second nucleic acid cleavage enzyme, preferably a second restriction endonuclease, that allows nucleic acid cleavage at a site distant from the second recognition site, and in which the cleavage site for the second nucleic acid cleavage enzyme is located within or about the first recognition site or

in which the nucleic acid sequence comprises the sequence 5′

-GAGGAGNNNNNNTC_▴

CG^▾

AC-3(SEQ ID NO;

     1)′

, preferably 5′

-GAGGAGCGTCTCTCCGAC-3′

(SEQ ID NO;

     2).

13. A nucleic acid according to claim 11, or a linker sequence according to claim 11, in which the first recognition site and the second recognition site are spaced such that when the nucleic acid is exposed to the second nucleic acid cleavage enzyme, cleavage of the nucleic acid occurs at a position within the first recognition site.