Factors which equalize the representation of genome segments in recombinant libraries

• Published in: Gene Vol. 49; no. 2; pp. 263 - 271
• Main Authors: Wyman, Arlene R., Wertman, Kenneth F., Barker, David, Helms, Cynthia, Petri, William H.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 1986; Amsterdam Elsevier; New York, NY
• Subjects: Animals; Bacterial Proteins - genetics; Bacterial Proteins - physiology; Bacteriophage lambda - genetics; Base Sequence; Biological and medical sciences; Chromatin. Chromosome; cloning; DNA, Recombinant; Drosophila genomic library; Drosophila melanogaster - genetics; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins; Exodeoxyribonuclease V; Exodeoxyribonucleases - genetics; Exodeoxyribonucleases - physiology; Fundamental and applied biological sciences. Psychology; gene libraries; Genetic Vectors; genomes; human genomic libraries; Humans; Molecular and cellular biology; Molecular genetics; Palindromes; phage lambda; phage λ vectors; Polymorphism, Genetic; recB recC sbcB; recD; recombinant DNA; recombinants; Repetitive Sequences, Nucleic Acid; Viral Proteins - genetics; Viral Proteins - physiology; phage λ vectors; Drosophila genomic library; recD; chi; human genomic libraries; kb; Palindromes; recombinant DNA; PIC; bp; recB recC sbcB; Virus; Human; Lambda phage group; Bactériophage lambda; Styloviridae; Drosophila; Bactériophage; Gene library; Molecular cloning; Vector; Palindromic sequence
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(86)90287-8

Genomic segments which contain inverted repetitions longer than 300 bp are frequently lost from recombinant libraries grown on rec + hosts. We have found that 9% of phage A clones that contain 15–20-kb insertions of human or Drosophila DNA are inhibited on rec + hosts and as a result will become underrepresented in amplified genomic libraries. We have therefore examined several factors of both host and vector origin which affect the fidelity of representation of genomic sequences in recombinant DNA libraries constructed in bacteriophage λ vectors. This loss may be diminished if the vector carries either a chi element or a functional gam gene. The most successful approach, however, involves using a host with mutations in recB, recC, and sbcB, or in recD. We have shown that recombinant clones which require such mutant hosts for growth are somewhat more likely to contain DNA derived from loci in the genome which are polymorphic than are clones recovered on conventional hosts.