Preferential isolation of DNA fragments associated with CpG islands.
Preferential isolation of DNA fragments associated with CpG islands.
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优先分离与 CpG 岛相关的 DNA 片段。
DOI：
10.1073/pnas.92.10.4229
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发表时间：
1995
影响因子：
11.1
通讯作者：
Sekiya,T
中科院分区：
文献类型：
--
作者：
Shiraishi,M;Lerman,LS;Sekiya,T
我们描述了一个程序优先分离的DNA片段与G+ C-丰富的部分。这些片段存在于CpG岛内或附近的已知基因中。由于约56%的人类基因与CpG岛相关，这些片段的分离允许检测和探测更大的DNA片段内的许多基因，例如尚未测序的Cosmoprotein或酵母人工染色体。将用四种限制性内切酶消化的克隆DNA片段进行变性梯度凝胶电泳。片段中富含长G+ C的部分在片段达到梯度中的阻滞水平后抑制链解离;在大多数其他片段消失后，这些片段保留在凝胶中。保留片段的核苷酸序列显示，这些片段中约有一半似乎来自CpG岛。北方分析表明存在与大多数保留片段互补的RNA。一个启发式的方法之间的关系的碱基序列和动力学的链解离的部分熔融的分子似乎占保留和nonretention。CpG岛片段将在变性梯度中保留的片段中富集的预期得到基于应用于已知序列的解链理论的速率估计的支持。该方法被命名为SPM，用于分离部分解链的分子，有望提供一种从未测序的DNA中方便有效地分离基因的方法。
We describe a procedure for preferential isolation of DNA fragments with G+C-rich portions. Such fragments occur in known genes within or adjacent to CpG islands. Since about 56% of human genes are associated with CpG islands, isolation of these fragments permits detection and probing of many genes within much larger segments of DNA, such as cosmids or yeast artificial chromosomes, which have not been sequenced. Cloned DNA fragments digested with four restriction endonucleases were subjected to denaturing gradient gel electrophoresis. Long G+C-rich sections in fragments inhibit strand dissociation after the fragments reach retardation level in the gradient; such fragments are retained in the gel after most others disappear. Nucleotide sequences of the retained fragments show that about half of these fragments appear to be derived from CpG islands. Northern analysis indicated the presence of RNA complementary to most of the retained fragments. A heuristic approach to the relation between base sequence and the kinetics of strand dissociation of partly melted molecules appears to account for retention and nonretention. The expectation that CpG island fragments will be enriched among fragments retained in a denaturing gradient is supported by rate estimates based on melting theory applied to known sequences. This method, designated SPM for segregation of partly melted molecules, is expected to provide a means for convenient and efficient isolation of genes from unsequenced DNA.