Large-scale phagemid conversion on solid medium

摘要

An important element of genomic analysis programs is the investigation and systematic sequencing of cDNA populations. The method we propose is designed to isolate a large number of different representative cDNAs from phagemid libraries with no perturbation of their relative amounts. Modified lambda phages, called phagemids, have been designed for rapid and efficient subcloning of recombinant fragments, phage display technology, and also DNA delivery. We have used a phagemid (lambda TriplEx2; BD Biosciences Clontech, Palo Alto, CA, USA) based on the Cre/LoxP system (8,9) and propagated it in Escherichia coli XLl-blue . Cre is a recombinase that specifically recognizes a DNA sequence named LoxP. The cloning vector is a lambda phage bearing a plasmid sequence surrounded by two LoxP sites. When lambda TriplEx2 phage is introduced into E. coli BM25.8, a strain that expresses Cre, the phagemid is converted into its plasmid, which can be amplified in the bacteria. This plasmid can then be extracted for sequencing or other procedures. Normally, these types of phagemids are mainly used for subcloning particular sequences after library screening, and the conversion process is done in liquid media. Large-scale conversions are also done in liquid media. Under these conditions, the relative abundance of different sequences in the population may be artifactually altered, because phage replication will depend on the length and/or cytotoxicity of the inserted sequence.