Unit

UNIT 21.12 Paired-End diTagging for Transcriptome and Genome Analysis

  1. Patrick Ng,
  2. Chia-Lin Wei,
  3. Yijun Ruan

Published Online: 1 AUG 2006

DOI: 10.1002/0471142727.mb2112s75

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Ng, P., Wei, C.-L. and Ruan, Y. 2006. Paired-End diTagging for Transcriptome and Genome Analysis. Current Protocols in Molecular Biology. 21:21.12.

Author Information

  1. Cloning and Sequencing Group, Genome Institute of Singapore, Singapore

Publication History

  1. Published Online: 1 AUG 2006
  2. Published Print: JUL 2006

This is not the most recent version of the article. View current version (1 JUL 2007)

Abstract

The Paired-End diTagging (PET) procedure enables one to obtain sequence information from both termini of any contiguous DNA fragment. This is achieved by a series of enzymatic manipulations that introduce MmeI sites directly flanking each DNA insert during the construction of a plasmid library. Subsequent MmeI digestion and self-ligation results in the production of covalently-linked paired-end ditags (PETs) that can be extracted and then concatenated for efficient sequencing. By mapping the PET sequences to assembled genomes, the original DNA fragments from which the PETs were derived can be precisely localized. This unit details two applications of PET technology. In GIS-PET, ditagging of mRNA converted to full-length cDNA enables whole-transcriptome analysis, including novel gene identification, gene prediction validation, and gene expression studies. In ChIP-PET, ditagging of chromatin immunoprecipitation–enriched genomic DNA fragments enables the global mapping of transcription factor binding sites.

Keywords:

  • PET;
  • ditag;
  • paired-end;
  • mate-pair;
  • SAGE;
  • DNA sequencing;
  • GIS-PET;
  • ChIP-PET