An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends

Abstract

For the preparation of gene libraries, DNA from λEMBL3 phage was digested with SalI and EcoRI, and the cohesive ends partially filled-in by addition of dTTP, dCTP and Klenow fragment of DNA polymerase I (Pollk). Genomic DNA was cleaved partially with Sau3A and subsequently incubated with dATP, dGTP and Pollk. The phage and genomic DNAs were then mixed and ligated. The recombinant DNAs were packaged in vitro. The efficiency of packaging was 10 5–10 6 of infectious phage λ particles per μg of the genomic DNA (as compared to approx. 10 7 per μg for the wild-type λ DNA). This procedure is very rapid and requires only μg quantities of genomic DNA for preparing an entire gene library. The other important advantage is that multiple independent insertions of genomic DNA cannot occur in a single recombinant phage and self-ligation of phage DNA is blocked. It is also applicable for other SalI-containing vectors.