[Construction of randomly matrix attachment regions library from the green alga: Dunaliella salina].

Abstract

Matrix attachment region (MAR) is DNA fragment that can bind to the nuclear matrix. In order to isolate the MAR fragment from the halotolerant green alga Dunaliella salina, we created a library of randomly obtained MAR from D. salina. Firstly the intact nuclei were released using 0.5% Triton X-100, then purification was carried out by discontinuous centrifugation using 30% and 70% Percoll gradients. Histones of nuclear matrices were removed using 25mmol/L lithium dioodosalicylate, the DNAs not closely associated with the matrices were removed using restriction enzymes. The remained matrices DNAs were digested by proteinase K, extracted with phenol/chloroform and precipitated with ethanol, and then cut with four kinds of restriction enzymes, the resulting DNAs were subsequently ligated to pUC18-vector and transferred to E. coli JM109 strains, DNA sequencing showed that the DNA fragments had the features of MAR DNA fragments.