UNIT 21.12 Paired-End diTagging for Transcriptome and Genome Analysis
Published Online: 1 AUG 2006
Copyright © 2006 by John Wiley & Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 AUG 2006
- Published Print: JUL 2006
This is not the most recent version of the article. View current version (1 JUL 2007)
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.
- DNA sequencing;