19 cDNA Library Screening
289
combinants. For screening of larger numbers of recombinants (>100,000),
phage libraries are more suitable, because of library size and efficiency of
screening. Introduction of recombinant DNA into host bacteria for propagation is more efficient by phage infection than by conventional plasmid
transfection procedures. This permits construction of libraries containing
larger numbers of recombinants. Furthermore, screening of phage libraries
can be carried out at very high plaque densities thereby allowing for screening of much larger numbers of recombinants at the same time. Advantages
of both systems have recently been combined in so-called excision vector
systems in which initial screening is phage-based but later cDNA inserts are
excised in vivo and can be further propagated and screened as phagemids
(see below).
Expression libraries can be plasmid or bacteriophage based. They offer
versatility over conventional plaque hybridization screening, since antibody recognition and phenotypic expression can also serve as screening
tools. However, isolation of a particular sequence from an expression library requires screening of at least 6 times the number of recombinants
needed in standard hybridization screening. This is due to possible orientation (factor two) of the cDNA insertrelative to its integration site as well as
its frame relative to the initiation codon from which translation starts (factor three). One potential problern of cDNA expression screening is the
synthesis of a protein product that might be toxic to the host bacteria.
This is one of the reasons why phage expression libraries are often favored
over plasmid expression libraries. Phage vectors such as A.gt11 can easily be
grown to high numbers under lysogenic conditions, before protein expression is induced for detection.
The choice of host bacteria is primarily determined by the vector used.
The most common vectors used are bacteriophage A.gtlO for plaque hybridization screening and A.gt11 (Jendrisak et al. 1987} and pUC plasmid (Helfman and Hughes 1987} for cDNA expression screening. Among the most
widely used excision vectors is the A.ZAP expression vector ( Short and Sorge
1992 ). If the biological activity of a peptide that is expressed from an isolated
clone is to be verified later in an eukaryotic cell, prokaryotic to eukaryotic
shuttle vector systems can be used (Kato et al. 1994).
Library size and status
The size of a cDNA library and the percentage ofindependent recombinants
it contains are of critical importance, for determing the probability that a
specific RNA sequence is represented in the library. To find a specific se-
289
combinants. For screening of larger numbers of recombinants (>100,000),
phage libraries are more suitable, because of library size and efficiency of
screening. Introduction of recombinant DNA into host bacteria for propagation is more efficient by phage infection than by conventional plasmid
transfection procedures. This permits construction of libraries containing
larger numbers of recombinants. Furthermore, screening of phage libraries
can be carried out at very high plaque densities thereby allowing for screening of much larger numbers of recombinants at the same time. Advantages
of both systems have recently been combined in so-called excision vector
systems in which initial screening is phage-based but later cDNA inserts are
excised in vivo and can be further propagated and screened as phagemids
(see below).
Expression libraries can be plasmid or bacteriophage based. They offer
versatility over conventional plaque hybridization screening, since antibody recognition and phenotypic expression can also serve as screening
tools. However, isolation of a particular sequence from an expression library requires screening of at least 6 times the number of recombinants
needed in standard hybridization screening. This is due to possible orientation (factor two) of the cDNA insertrelative to its integration site as well as
its frame relative to the initiation codon from which translation starts (factor three). One potential problern of cDNA expression screening is the
synthesis of a protein product that might be toxic to the host bacteria.
This is one of the reasons why phage expression libraries are often favored
over plasmid expression libraries. Phage vectors such as A.gt11 can easily be
grown to high numbers under lysogenic conditions, before protein expression is induced for detection.
The choice of host bacteria is primarily determined by the vector used.
The most common vectors used are bacteriophage A.gtlO for plaque hybridization screening and A.gt11 (Jendrisak et al. 1987} and pUC plasmid (Helfman and Hughes 1987} for cDNA expression screening. Among the most
widely used excision vectors is the A.ZAP expression vector ( Short and Sorge
1992 ). If the biological activity of a peptide that is expressed from an isolated
clone is to be verified later in an eukaryotic cell, prokaryotic to eukaryotic
shuttle vector systems can be used (Kato et al. 1994).
Library size and status
The size of a cDNA library and the percentage ofindependent recombinants
it contains are of critical importance, for determing the probability that a
specific RNA sequence is represented in the library. To find a specific se-