Peterson Daniel G, Schulze Stefan R, Sciara Erica B, Lee Scott A, Bowers John E, Nagel Alexander, Jiang Ning, Tibbitts Deanne C, Wessler Susan R, Paterson Andrew H
Center for Applied Genetic Technologies and Department of Crop and Soil Sciences, University of Georgia, Athens, Georgia 30602, USA.
Genome Res. 2002 May;12(5):795-807. doi: 10.1101/gr.226102.
Cot-based sequence discovery represents a powerful means by which both low-copy and repetitive sequences can be selectively and efficiently fractionated, cloned, and characterized. Based upon the results of a Cot analysis, hydroxyapatite chromatography was used to fractionate sorghum (Sorghum bicolor) genomic DNA into highly repetitive (HR), moderately repetitive (MR), and single/low-copy (SL) sequence components that were consequently cloned to produce HRCot, MRCot, and SLCot genomic libraries. Filter hybridization (blotting) and sequence analysis both show that the HRCot library is enriched in sequences traditionally found in high-copy number (e.g., retroelements, rDNA, centromeric repeats), the SLCot library is enriched in low-copy sequences (e.g., genes and "nonrepetitive ESTs"), and the MRCot library contains sequences of moderate redundancy. The Cot analysis suggests that the sorghum genome is approximately 700 Mb (in agreement with previous estimates) and that HR, MR, and SL components comprise 15%, 41%, and 24% of sorghum DNA, respectively. Unlike previously described techniques to sequence the low-copy components of genomes, sequencing of Cot components is independent of expression and methylation patterns that vary widely among DNA elements, developmental stages, and taxa. High-throughput sequencing of Cot clones may be a means of "capturing" the sequence complexity of eukaryotic genomes at unprecedented efficiency.
基于 Cot 值的序列发现是一种强大的方法，通过该方法可以对低拷贝和重复序列进行选择性、高效的分级分离、克隆和表征。根据 Cot 分析的结果，利用羟基磷灰石色谱法将高粱（双色高粱）基因组 DNA 分级分离为高度重复（HR）、中度重复（MR）和单拷贝/低拷贝（SL）序列组分，随后将这些组分进行克隆，构建了 HRCot、MRCot 和 SLCot 基因组文库。滤膜杂交（印迹法）和序列分析均表明，HRCot 文库富含传统上在高拷贝数中发现的序列（例如，反转录元件、rDNA、着丝粒重复序列），SLCot 文库富含低拷贝序列（例如，基因和“非重复 EST”），而 MRCot 文库包含中度冗余的序列。Cot 分析表明，高粱基因组约为 700 Mb（与先前的估计一致），HR、MR 和 SL 组分分别占高粱 DNA 的 15%、41%和 24%。与先前描述的对基因组低拷贝组分进行测序的技术不同，Cot 组分的测序与 DNA 元件、发育阶段和分类群之间差异很大的表达和甲基化模式无关。对 Cot 克隆进行高通量测序可能是以前所未有的效率“捕获”真核基因组序列复杂性的一种手段。
