测定重复序列的一种有效方法——定向删除法

An Efficient Method to Sequence Repetitive Sequences——The Nested Deletion Strategy

基因组中存在大量的重复序列,它们具有调节基因表达和染色体的生理代谢等重要的生物功能。重复序列由于其结构的特殊性,不但鸟枪法基因组测序不能有效地测定它们的序列,而且采用引物步移法等一般的定向测序方法也不能对其准确测定。本研究利用核酸外切酶Ⅲ定向删除技术,结合菌液电泳等快速筛选方法,对黄蜂蜘蛛三个高度重复序列丝蛋白基因MaSp2、CySp1和CySp2进行了准确的测定。研究结果表明核酸外切酶Ⅲ定向删除法是一种测定高度重复序列的有效方法,利用普通的琼脂糖电泳技术,该方法可以单向地有效测定重复序列的长度可达10kb,同时我们还发现合适内切酶的选择,定向删除亚克隆的筛选与排序以及序列的准确拼接是该方法有效而准确测定高度重复序列的关键。

Many repetitive sequences exist in the genome. Being able to regulate gene expression and physiological metabolism of chromosomes, they are thought to be of great biological importance. Owing to their special structures, repetitive sequences cannot be accurately sequenced by the shot gun method or primer walking, which are commonly used strategies in directed sequencing. In a study reported in the present paper, we sequenced three highly repetitive sequences, MaSp2, CySpl and CySp2, which are all silk protein genes from wasp spider, Argiope bruennichi, by generating a series of nested deletions using exonuclease Ⅲin combination with a bacterial liquid electrophoresis strategy to select suitable subclones. Our results indicated that the nested deletion strategy is an efficient method to sequence highly repetitive sequences as large as lOkb using the technique of com- mon agarose gel electrophoresis. Some critical parameters of sequencing repetitive sequences are discussed, such as suitable restriction enzymes, selection and ranking of deletion subclones and accu- rately overlapping of the subclone＇s sequences.