通过焦磷酸测序对双链DNA上的单核苷酸多态性进行直接分析。

Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing.

作者信息

Nordström T, Ronaghi M, Forsberg L, de Faire U, Morgenstern R, Nyrén P

机构信息

Department of Biotechnology, The Royal Institute of Technology, SE-100 44 Stockholm, Sweden.

出版信息

Biotechnol Appl Biochem. 2000 Apr;31(2):107-12. doi: 10.1042/ba19990104.

DOI:10.1042/ba19990104

PMID:10744955

Abstract

Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.

摘要

焦磷酸测序是一种新的DNA测序方法，在许多不同类型的DNA分析中得到了广泛应用。该方法利用单管检测中的四种偶联酶，通过发光检测系统实时监测DNA合成。在此，我们展示了焦磷酸测序用于直接分析双链PCR产物上的单核苷酸多态性。分析了来自多个个体的人类谷胱甘肽过氧化物酶基因（GPX1）的焦磷酸测序数据，确定并证实了三种不同的等位基因变体。还讨论了进一步简化测序和模板制备步骤的可能性。