Ng Patrick, Wei Chia-Lin, Ruan Yijun
Genome Institute of Singapore, Singapore.
Curr Protoc Mol Biol. 2007 Jul;Chapter 21:Unit 21.12. doi: 10.1002/0471142727.mb2112s79.
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. A recent innovation (Multiplex Sequencing of Paired-End ditags; MS-PET) enables PETs to be sequenced using high-throughput 454 sequencing, greatly increasing the amount of data that can be collected in each run.
双末端标记（PET）程序能够从任何连续DNA片段的两端获取序列信息。这是通过一系列酶促操作实现的，即在构建质粒文库过程中，在每个DNA插入片段的两侧直接引入MmeI位点。随后的MmeI消化和自身连接会产生共价连接的双末端标记（PET），可以将其提取出来，然后连接起来进行高效测序。通过将PET序列定位到组装好的基因组上，可以精确确定PET所源自的原始DNA片段的位置。本单元详细介绍了PET技术的两种应用。在GIS-PET中，对转化为全长cDNA的mRNA进行双末端标记可实现全转录组分析，包括新基因鉴定、基因预测验证和基因表达研究。在ChIP-PET中，对染色质免疫沉淀富集的基因组DNA片段进行双末端标记可实现转录因子结合位点的全基因组定位。最近的一项创新（双末端标记多重测序；MS-PET）使PET能够使用高通量454测序进行测序，大大增加了每次运行可收集的数据量。
