A POLYMERASE CHAIN REACTION METHOD FOR LARGE DNA REGIONS

这项研究的目标是开发一种“巨型聚合酶链式反应”程序，其中DNA 在两个DNA序列之间有数十万甚至数百万个碱基 可以用酶促扩增和直接从 扩增反应。如果这个程序能够被开发出来，它可能是 与序列标记位点(STSS)相结合，大大简化了主要方面 人类基因组计划的一部分。因此，制造超级聚合酶链式反应是可能的。 来自任何两个短串联重复序列的引物，每个短串联重复序列可能有100万个碱基对 其他的，并用这些来放大中间的DNA。如果是这样的话 不需要克隆基因组，因为STSS两侧的任何区域都可能是 随意放大以供研究。百万碱基对的聚合酶链式反应扩增 DNA区域的成功，所需的STS数量 基因组将减少多达10倍。这将是可能的 从酵母人工染色体或粘粒中扩增一种插入片段 目的之所在。超级聚合酶链式反应将极大地促进感兴趣的克隆 疾病基因，因为所需要的只是两个侧翼 在可扩增距离内克隆基因的标记。因此，超级聚合酶链式反应 将极大地帮助人类基因组计划的许多方面。

The goal of this research is to develop a "mega-PCR" procedure in which DNA between two DNA sequences hundreds of thousands or even millions of base pairs apart can be amplified enzymatically and directly cloned from the amplification reaction. If this procedure can be developed, it could be coupled with sequence tagged sites (STSs) to greatly simplify major aspects of the human genome project. Thus, it would be possible to make mega-PCR primers from any two STSs within, perhaps, one million base pairs of each other and use these to amplify the intervening DNA. It would be unnecessary to clone the genome, since any region flanked by STSs could be amplified at will for study. If PCR amplification of million base pair regions of DNA is successful, the number of STSs required to cover the genome would be reduced by up to a factor of 10. It would be possible to amplify inserts from yeast artificial chromosomes or cosmids for a variety of purposes. Mega-PCR would greatly facilitate cloning of interesting disease genes because all that would be required would be two flanking markers within the amplifiable distance to clone the gene. Thus, mega-PCR would significantly aid many aspects of the human genome initiative.