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Genomics
Promises: for huge improvements in human & animal healthcare and production Contemporary Study: Genomes are read out as linear sequences In the cell- complex interactions and mechanisms to transduce DNA information into biological function Conventional DNA sequencing -explore genetic elements and structure Practical Challenges: high sequencing costs and low throughputs limited in-depth analysis of genomic elements
Emulsion PCR
Bridge PCR
Enyzmatic
extension
with
ESTs are sequences from each end of the cDNA inserts Unigene cluster is an group of overlapping ESTs, likely from one gene
This method covalently links the 5 tag and 3 tag of a DNA fragment into a ditag structure for sequencing analysis, thus combining the benefits of the cost-effective SAGE and the linkage information from paired-end sequencing
(Hong, 1981)
Nebulization:
Tags of 106 bp size unbalanced with tags under 15 bp
Cloning Method
Benefit: Preserves original flcDNA/ ChIP DNA fragments as library clones Limitation: - Construction process long (2-4 weeks) - Technically challenging
Cloning-free Method
Applications of PET
Limitation: no information of internal exons Unique Feature: Ability to detect unconventional fusion genes Early version of RNA-PET was cloning based method i.e. GIS-PET analysis
RNA-PET for Transcriptome Studies Early version of RNA-PET was cloning based method i.e. GIS (Gene Identification signature)-PET analysis It has revealed its ability to high-throughput identification of fusion genes Now, Cloning-free methods Alternatively, perform shotgun paired-end sequencing of cDNA templates
Sequencing based approach to to characterize ChIP-enriched DNA fragments ChIP-PET provides linked 5 and 3 sequences for ChIP-enriched DNA molecules, which are mapped to the reference genome such that the complete ChIP DNA fragment can be inferred from the genome sequence in between the 5 and 3 tags, and the enriched TFBS can be determined
ChIA-PET : identifying chromatin interactions Introduce a linker sequence in the junction of two DNA fragments during nuclear proximity ligation to build connectivity of DNA fragments that are tethered together by protein factors All linker connected ligation products can be extracted for the taglinkertag constructs that can be analyzed by ultra-high-throughput PET sequencing
When mapped to reference genome, the ChIAPET sequences are read out to detect the relationship of two DNA fragments in chromatin interactions captured by chromatin proximity ligation ChIA-PET has the potential to be an unbiased genome-wide approach for de novo detection of chromatin interactions
DNA-PET: Genome Structure Analysis Ideal method for sequencing and assembling genomes as well as studying genome structural variations First demonstrated in resequencing an evolved E. coli genome using the polony sequencing-by ligation method Applied to map cancer genome rearrangements DNA-PET can become a vital part in the concept of personal genomics for personal medicine
Future of PET Technology Limitations: - require more starting material - involve more molecular manipulations - certain DNA portions not recovered Versatile readily adapt to new sequencing technology Couple methods for asking biological questions with NGS Future is bright.
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