Reseach On New Methods Of Dnasei Hypersensitive Sites

DNase I hypersensitive sites(DHSs) are found to be the markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions during the research of last 28 years. DHSs always represent the active cis-regulatory part of DNA. Finding the noncoding functional elements in genome is meaningful to understanding the complicated gene expression. One of the important approaches is seeking the regions on chromatin that are sensitive to the DNaseI. Through researching the DHSs and locating the expression regulatory regions, we can study the regulatory model of creatures.Here, we have developed a new method which reduces the initial cells number based on the DNaseI-seq. Our new method reduced the initial amout of cells, so can be used more flexible. Since we used agarose to protect DNA from degrade and non-digestion break so that we can improve the sencitivity of DNaseI and reduce the signal noisy and false-positive data. To reduce the amount of samples, we used the phi29 DNA polymerase that has unique displacement activity and strong synthesis ability to amplify our DNA sample after DNaseI digestion. First, cells have to be softly digested by detergent to make the DNaseI difused easily through the membrance. Then optimized congcentration of enzyme and digestion time has to be operated, the digested DNA product should be 20-50 kbp that can be amplified by Phi29 efficiently. The same volume of 1.2% low melt-point agarose has to be added to the product after digestion, the DNA will be protected from degrading after the gel is solidified. Solidified gel plugs have to be soaked in lysis buffer to release the naked DNA and after the lysis buffer is washed out thoroughly, the ends of DNA have to be blunted and adapter ligated. The ends of DNA are authenticclly to be the DHSs. At last, the gel plugs have to be melted and add water to make the agarose concentration less than 0.2% so the agarose will not be solidified. We quantified the gDNA and adapters with QPCR to make sure the adapter was ligated efficiently. After being amplified by Phi29, the DNA can be put into library and sequenced by Complete Genomics sequencer. DHSs can be finded by mapping the data to genome and adapter sequence.