DIRECT GENOME SEQUENCING--MYCOPLASMA CAPRICOLUM

我们将对800个碱基的绵羊支原体进行测序 染色体。这将为自由生活提供最小的模型基因组 能够或在特定的介质中生长的有机体。因为支原体只有 大约500个基因，人们可以希望对其进行全面的了解 基因组测序的结果是生物学。山羊分枝杆菌是一种病原体。 对于山羊，它与一种人类病原体肺炎支原体有关； 对这种生物体的了解也将有助于阐明它的机制 传染性。 这种生物将通过一种直接的技术进行测序，这种技术不涉及 克隆和作图：基于寡核苷酸的多重基因组行走 揭示染色体DNA序列的程序。聚合酶(聚合酶) 链式反应)方法将被用来解决困难区域和任何 顺序模糊。第一轮鸟枪式克隆和测序将是 需要建立大约800个潜在的步行起始点 策略。 将开发一个关于这种生物的信息数据库，将 包含基因组序列，所有转录可能性，都是开放的 阅读框，并通过以下方式识别大多数阅读框 序列同源。该数据库将包含相关的信息 其他生物体中的基因，并最终将包括关于 支原体的所有基因。此数据库将分发到 光盘。 这个以技术人员为基础的小组专门从事直接测序 微生物应达到1兆基数/完成年的速度 第二年的双链序列。排序方法将是 开发和简化，以使每年两到三兆基数的速度 将在第三年实现。在完成支原体序列后， 这些相同的直接方法将被应用于对大染色体进行测序 来自酵母或其他简单的真核生物。

We shall sequence Mycoplasma capricolum which has an 800 kilobase chromosome. This will provide the smallest model genome for a free-living organism capable or growing in a defined medium. Since mycoplasma has only about 500 genes, one can hope to develop a complete understanding of its biology as a result of the genome sequencing. M. capricolum is a pathogen for goats, and its related to a human pathogen, M. pneumoniae; the understanding of the organism will also shed light on its mechanism of infectivity. This organism will be sequenced by a direct technique that does not involve cloning and mapping: multiplex genomic walking, an oligonucleotide-based procedure that reveals the sequence of chromosomal DNA. PCR (polymerase chain reaction) methods will be used to resolve difficult regions and any sequence ambiguities. One round of shotgun cloning and sequencing will be needed to establish about 800 potential initiation points for the walking strategy. A database of information about this organism will be developed that will contain the genomic sequence, all transcription possibilities, all open reading frames, and the identification of most of those reading frames by sequence homologies. The database will contain information about related genes in other organisms and, eventually, will include information about all of the genes of mycoplasma. This database will be distributed on compact discs. This technician-based group specializing in the direct sequencing of microorganisms should achieve a rate of one megabase/year of finished double-stranded sequence by the second year. Sequencing methods will be developed and simplified so that a rate of two to three megabases/year will be achieved by the third year. After completing the mycoplasma sequence, these same direct methods will be applied to sequence a large chromosome from yeast or an other simple eukaryote.