We describe a method for the construction of large DNA fragment libraries in plasmid vectors, in which complementary, single-stranded extensions are llgated onto both vector and insert DNA using un-phosphorylated adaptor oligonucleotides. Special consideration has been taken of the requirements of expression screening as follows: (1) cDNA synthesis using random oligonucleotide primers is described which maximises the probability of obtaining open reading frame fragments from large mRNA molecules, (2) the adaptors use codons found in high abundance E.coli proteins to minimise problems of premature termination when using strong promoters, and (3) the sequence encoded by the adaptors, when cloned into the bacterial expression vector pEX1, promotes a surface location for the foreign antigenic determinant where it is accessible to antibodies used for screening.
ASJC Scopus subject areas
- Statistics, Probability and Uncertainty
- Applied Mathematics
- Health, Toxicology and Mutagenesis