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[9] pEMBL: A new family of single-stranded plasmids for sequencing DNA

1987; Academic Press; Linguagem: Inglês

10.1016/0076-6879(87)55011-x

1557-7988

Luciana Dente, Riccardo Cortese,

RNA and protein synthesis mechanisms

This chapter describes a series of plasmids, the “pEMBL family,” that have the property of being encapsidated as single-stranded deoxyribonucleic acid (DNA) upon superinfection with phage F1. The plasmids can be used for DNA sequencing and for all the other uses that require single-stranded DNA, but, in the absence of F1 helper phage, they replicate as any other doubles-stranded plasmid. This is very useful for the preparation of a large quantity of plasmid and for the stability of longer inserts. The class of M13 derivative vectors, largely used for DNA sequencing with the dideoxy method, has, in fact, the limitation that large inserts tend to be unstable. The chapter discusses the principle of the method and characteristics of pEMBL vectors along with the construction of pEMBL plasmids. The chapter discusses the property of a short segment of F1 genome containing all the cis-acting elements for DNA replication and morphogenesis. To have a series of polylinkers containing a large number of unique sites for restriction endonucleases, in different reciprocal orientation is very useful, because it allows the insertion of DNA fragments in a preselected orientation or in a preselected frame inside the sequence encoding the a-fragment of th/3-galactosidase. The DNA, which is prepared through the procedures described in the chapter, is suitable for DNA sequencing experiments with the dideoxy method.