Method for gene identification signature (GIS) analysis

a gene identification and signature technology, applied in the field of gene and transcript expression, can solve the problems of prohibitively expensive to tag every transcript in the transcriptome, the sage method currently cannot generate any longer tags to improve specificity, and the inability to complete sequencing analysis of all different transcriptomes

Inactive Publication Date: 2005-11-17
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0052] Such ditags can directly guide the process of recovering the full-length nucleic acid molecule corresponding to the newly identified genes.

Problems solved by technology

However, due to the complexity and immense volume of transcripts expressed in the various developmental stages of an organism's life cycle, complete sequencing analysis of all different transcriptomes still remains unrealistic.
Though ESTs are effective in identifying genes, it is prohibitively expensive to tag every transcript in a transcriptome.
Limited by the availability of type II restriction enzymes that can cut longer than 2 lbp, the SAGE method currently cannot generate any longer tags to improve specificity.
Further, SAGE and MPSS methods only produce a single signature per transcript within the gene.
In view of the “internal” nature of the tag in a transcript, these methods provide only limited positional and structural information.
Therefore, despite their usefulness in enhancing sequencing efficiency, the utility of methods such as SAGE or MPSS is severely undermined by their lack of specificity and consequent inconclusiveness.

Method used

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  • Method for gene identification signature (GIS) analysis
  • Method for gene identification signature (GIS) analysis
  • Method for gene identification signature (GIS) analysis

Examples

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example 1

The Method

[0263] The experimental procedure of GIS ditag analysis has been carried out according to the following modules of cDNA library construction and analysis: [0264] (1) The full-length cDNA library which introduces the MmeI sites flanking both ends of each cDNA insert; [0265] (2) The GIS ditag library in which each clone contains a 5′ 18 bp signature and a 3′ 18 bp signature of a transcriptional unit; [0266] (3) The GIS library for clones of concatenated GIS ditags; [0267] (4) GIS sequencing analysis.

1. GIS Full-Length cDNA Library with Addition of MmeI Sites for each cDNA Inserts

[0268] The outline of procedure of this section was as follows: starting from high quality mRNA, the first cDNA was synthesized with a GsuI-oligo dT primer (SEQ ID NO:1).

[0269] The first strand cDNA / RNA hybrids was subjected to a full-length enrichment procedure by the biotinylation-based cap-trapper approach. Any cap-trapper approach known in the art can be used, for example Caminci et al., 19...

example 2

2. GIS Ditag Library

[0331] The cDNA clones made from steps 1-1 to 1-8 contained a MmeI site (TCCGAC) at the 5′ side and another MmeI site (TCCAAC) in reverse orientation at the 3′ end. Note that these two MmeI recognition sites are two isoforms that can be recognized by MmeI (TCCRAC 20 / 18, where R=(A / G)). The sequence difference here will be useful later for directional indication. MmeI restriction enzyme will cleave these clones 20 bp into the cDNA fragments from their 5′ and 3′ ends. Consequently, despite the variable sizes of the digested cDNA, the vector plus the 20 bp cDNA signature tags on each end of all clones will be of a constant size that can be easily recognized upon agarose gel electrophoresis, and can be easily purified from the unwanted cDNA fragments.

[0332] The gel-purified vector plus tags can then be self-ligated to give a “tagged plasmid” containing the 5′ and 3′ GIS signature tags.

2-1. Plasmid Preparation

[0333] The GIS full-length cDNA library was amplified...

example 3

Experimental Summary

[0403] Our experimental strategy (FIG. 13) was to directly clone p53 ChIP-enriched DNA into a plasmid vector for GIS analysis. This preserves the information content of the experiment in an infinitely renewable format. GIS ditag sequences representing the ChIP DNA fragments can then be mapped to the genome to define the genome regions corresponding to the original ChIP-enriched material. Relative tag counts will allow the distinguishing regions of interest from the (inevitable) nonspecific background.

[0404] For this study, we used the colorectal cancer cell line HCT116 (ATCC CCL-247), which contains wildtype p53. The cells were treated with genotoxic 5-Fluorouracil (5-FU) to activate p53 and induce target gene expression. At different time points before and after 5-FU treatment, the cells were treated with 1% formaldehyde for 10 min at room temperature. Formaldehyde was inactivated by addition of 125 mM glycine. After lysis and sonication, chromatin extracts c...

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Abstract

A method of identifying at least a nucleic acid molecule fragment to which a protein of interest binds, comprising: (i) preparing at least one nucleic acid molecule fragment to which a protein binds; (ii) isolating the 5′ terminus and the 3′ terminus of the nucleic acid fragment(s) and linking the 5′ terminus and 3′ terminus to create the at least one ditag; (iii) sequencing the ditag; and (iv) mapping the ditag sequence(s) to the genome.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] The present application is a continuation-in-part of U.S. Ser. No. 10 / 664,234 filed Sep. 17, 2003, the whole content of which is herein incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates generally to the field of gene and transcript expression and specifically to a method for the serial analysis of a large number of transcripts by identification of a Gene Identification Signature (GIS) corresponding to defined regions within a transcript. Further, the present invention relates to a method of identification of protein binding sites, in particular transcription factor binding sites. BACKGROUND OF THE INVENTION [0003] One of the most important goals of the human genome project is to provide complete lists of genes for the genomes of human and model organisms. Complete genome annotation of genes relies on comprehensive transcriptome analysis by experimental and computational approaches. Ab initio predictio...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09C12N15/10C12Q1/68
CPCC12N15/1065C12N15/1093C12N15/1096C12Q1/6809C12Q1/6855C12Q2525/191C12Q2525/131C12Q2521/313C07H21/02
Inventor NG, PATRICKWEI, CHIALINRUAN, YIJUN
Owner AGENCY FOR SCI TECH & RES
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