Our novel technology in 2 steps
The SuRE method is in essence a strategy to analyze millions of DNA elements for their activity as promoters or enhancers in a single experiment. By analyzing ~300 million DNA elements of ~300bp length, an entire human genome can be analyzed exhaustively at an approximate 30 fold coverage.
The only required input is DNA and once a SuRE library is established it can be analyzed and compared to other DNA sources in the controlled environment of any transfectable cell type. Thus, without the requirement of invasive biopsies, any genome of interest can be analyzed in relevant cell types and under relevant (e.g. stimulatory) conditions.
1
Library Construction
We first construct a plasmid library containing millions of DNA elements. A human genome is randomly sheared, size-selected and cloned into a plasmid library that contains no promoter of its own. These plasmids contain a unique 20bp sequence referred to as a barcode which allows us to uniquely identify and follow each cloned DNA element. This library is characterized (i.e. barcodes are associated to their respective DNA element) by paired-end sequencing.
barcoded plasmid
Fragmented human genome
0.2 - 2 kb
barcoded plasmid
Plasmid library containing > 300 million DNA elements
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2
Library Expression Profiling
In step 2, the plasmid library is transfected into a cell type of interest. DNA elements containing promoters or enhancers will transcribe the barcode which can now be detected in the RNA by high-throughput sequencing. By comparing these barcode frequencies with the frequencies in the plasmid library, a quantitative map of promoter and enhancer activity can be constructed for the human genome.
Transfection plasmid library into ~1*10 cells
8
Quantify expressed barcodes
Quantify all barcodes
('input')
Ratio
Promoter and enhancer activity for entire genome
