Method for gene identification signature (GIS) analysis

• US 8,222,005 B2
• Filed: 09/17/2003
• Issued: 07/17/2012
• Est. Priority Date: 09/17/2003
• Status: Expired due to Fees

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Agency For Science Technology and Research
• Inventors: Ruan, Yijun, Ng, Patrick, Wei, Chialin
• Primary Examiner(s): JOHANNSEN, DIANA B
• Application Number:
  US10/664,234
• Publication Number:
  US 20050059022A1
• Time in Patent Office: 3,226 Days
• Field of Search: None
• US Class Current: 435/91.52
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

An isolated oligonucleotide comprising at least one ditag, wherein the ditag comprises two joined first and second sequence tags, wherein the first tag comprises the 5′-terminus sequence and the second tag comprises the 3′-terminus sequence of a nucleic acid molecule or a fragment thereof. The ditag analysis is useful for gene discovery and genome mapping.

Claims

1. A method for preparing at least one ditag comprising the steps of:

(i) producing at least one full-length cDNA transcript, said transcript having a 5′

terminus including the starting point of the full-length cDNA transcript and a 3′

terminus including the ending point of the full-length cDNA transcript;

(ii) cleaving the full-length cDNA transcript at its 5′

terminus to extract a 5′

tag 8-20 base pairs (bp) in length, said 5′

tag having a 5′

end and a 3′

end and including the starting point of the full-length cDNA transcript, and cleaving the full-length cDNA transcript at its 3′

terminus to extract a 3′

tag 8-20 bp in length, said 3′

tag having a 5′

end and a 3′

end and including the ending point of the full-length cDNA transcript; and

(iii) generating at least one ditag by ligating the 3′

end of the 5′

tag to the 5′

end of the 3′

tag.

2. A method of mapping a gene on a genome, comprising:

preparing at least one ditag according to the method of claim 1, and

mapping the 5′ and

3′

tags of the at least one ditag on a genome; and

defining the entire structural region of the corresponding gene on the genome, wherein the region being defined includes exons and introns of the gene.

3. A method of gene discovery comprising:

preparing at least one ditag according to the method of claim 1,

determining the sequence of the at least one ditag and

comparing the sequence of the at least one ditag with a genome sequence and a gene database;

wherein detecting matching of the sequence of the 5′ and

3′

tags of the at least one ditag with the genome sequence but detecting no match with one or more gene databases indicates a new gene.

4. The method of claim 3, further comprising recovering a full-length cDNA corresponding to the at least one ditag by PCR or directly from target RNA samples by RT-PCR.

5. A method for preparing at least one ditag comprising the steps of:

(i) providing at least one full-length cDNA transcript having a 5′

terminus including the starting point of the full-length cDNA transcript and a 3′

terminus including the ending point of the full-length cDNA transcript, and flanked by two adapters;

(ii) cleaving the full-length cDNA transcript at its 5′

terminus to extract a 5′

tag 8-20 base pairs (bp) in length, said 5′

tag having a 5′

end and a 3′

end and including the starting point of the full length cDNA transcript and an adapter, and cleaving the full-length cDNA transcript at its 3′

terminus to extract a 3′

tag 8-20 by in length, said 3′

tag having a 5′

end and a 3′

end and including the ending point of the full length cDNA transcript and an adapter; and

(iii) generating at least one ditag flanked by two adapters by ligating the 3′

end of the 5′

tag to the 5′

end of the 3′

tag.

6. The method of claim 5, further comprising the step of creating a concatemer of ditags.

7. The method of claim 5, further comprising including the at least one ditag flanked by the adapters in a vector.

8. The method of claim 7, wherein the vector further comprises two adapters flanking the at least one ditag, wherein each adapter includes at least one restriction site.

9. The method of claim 8, wherein each adapter comprises at least a first restriction site which is an asymmetric restriction site and a second restriction site.

10. The method of claim 9, wherein the backbone of the vector does not comprise the asymmetric restriction site or the second restriction site.

11. The method of claim 10, wherein the asymmetric restriction site is a type II restriction site.

12. The method of claim 7, wherein each adapter comprises at least:

a first restriction site which is a type II restriction site and at least a second restriction site, and wherein the backbone of the vector does not comprise the type II restriction site or the second restriction site.

13. The method of claim 12, wherein the type II restriction site is a MmeI site.

14. The method of claim 7, wherein the vector comprises SEQ ID No:

18.

15. The method of claim 5, further comprising the step of determining the nucleotide sequence of the at least one ditag.

16. The method of claim 5, further comprising:

determining the sequence of the at least one ditag; and

mapping the 5′ and

3′

tags of the at least one ditag nucleotide sequence to a database comprising genomic sequences.

17. The method of claim 5, wherein each adapter comprises at least one restriction site and step (ii)

includes adding at least one restriction enzyme capable of recognizing the at least one restriction site to the full-length cDNA transcript flanked by the two adapters from step (i).

18. The method of claim 17, wherein the at least one restriction site is an asymmetric recognition site.

19. The method of claim 18, wherein the asymmetric recognition site is a restriction endonuclease asymmetric cleavage site sequence recognizable by a type II restriction enzyme selected from the group consisting of AarI, AceIII, AloI, BaeI, Bbr7I, BbvI, BbvII, BccI, Bce83I, BceAI, BcefI, BcgI, BciVI, BfiI, BinI, BplI, BsaXI, BscAI, BseMII, BseRI, BsgI, BsmI, BsmAI, BsmFI, Bsp24I, BspCNI, BspMI, BsrI, BsrDI, BstF5I, BtgZI, BtsI, CjeI, CjePI, EciI, Eco31I, Eco57I, Eco57MI, Esp3I, FalI, FauI, FokI, GsuI, HaeIV, HgaI, Hin4I, HphI, HpyAV, Ksp632I, MboII, MlyI, MmeI, MnlI, PleI, PpiI, PsrI, RleAI, SapI, SfaNI, SspD5I, Sth132I, StsI, TaqII, TspDTI, TspGWI, TspRI and Tth111II.

20. The method of claim 18, wherein the asymmetric recognition site is a homing endonuclease asymmetric recognition site sequence-recognizable by a homing endonuclease selected from the group consisting of:

I-CeuI, PI-SceI, PI-PspI and I-SceI.

21. The method of claim 5 wherein each adapter comprises at least one restriction site which is a MmeI recognition site;

and step (ii) comprises

cleaving the full-length cDNA transcript flanked by the two adapters from step (i) with MmeI.

22. The method of claim 5, wherein the at least one ditag is 34-38 nucleotides in length.