Chapter 19
cDNA library Screening
JOHANN PETER HOSSLE
lntroduction
cDNA libraries are collections of recombinant DNA sequences, derived
from and representing entire mRNA populations present at a given stage
of development or differentiation in certain tissues or cell types. cDNA libraries can be self-made and tailored according to individual experimental
needs. Alternatively, they can be purchased from a commercial distributor,
if the library representing the desired mRNA source is available. In any case,
the collection of cDNA sequences is cloned into a vector, a plasmid or phage,
allowing the manipulation of the cloned sequences and propagation of the
whole library in suitable hast bacteria.
The major purpose of cDNA libraries is to serve as a source for isolation
and cloning of individual recombinant DNA sequences of interest. Individual recombinants within libraries are identified by a number of possible
screening methods. The most obvious method is to screen for homology
with a known nucleic acid sequence, thereby using labeled complementary
nucleic acid sequences as probes. Expression libraries, which reflect the protein biosynthesis potential of the original mRNA source, allow for functional and antibody screening.
Vector and host selection
cDNA libraries can be constructed using either plasmid or bacteriophage
vectors. Plasmid libraries offer the advantage of ease of manipulation but
are somewhat limiting if large numbers of recombinant clones need to be
screened. They are suitable for screening up to about 10,000 to 50,000 reJohann Peter Hossle, University Children's Hospital, Division of Immunology/Hematology, Steinwiesstraße 75, Zürich, 8032, Switzerland (phone +41-1-266-73-41;fax +41-1266-71-71; e-mail hosslejp@kispi.unizh.ch)
cDNA library Screening
JOHANN PETER HOSSLE
lntroduction
cDNA libraries are collections of recombinant DNA sequences, derived
from and representing entire mRNA populations present at a given stage
of development or differentiation in certain tissues or cell types. cDNA libraries can be self-made and tailored according to individual experimental
needs. Alternatively, they can be purchased from a commercial distributor,
if the library representing the desired mRNA source is available. In any case,
the collection of cDNA sequences is cloned into a vector, a plasmid or phage,
allowing the manipulation of the cloned sequences and propagation of the
whole library in suitable hast bacteria.
The major purpose of cDNA libraries is to serve as a source for isolation
and cloning of individual recombinant DNA sequences of interest. Individual recombinants within libraries are identified by a number of possible
screening methods. The most obvious method is to screen for homology
with a known nucleic acid sequence, thereby using labeled complementary
nucleic acid sequences as probes. Expression libraries, which reflect the protein biosynthesis potential of the original mRNA source, allow for functional and antibody screening.
Vector and host selection
cDNA libraries can be constructed using either plasmid or bacteriophage
vectors. Plasmid libraries offer the advantage of ease of manipulation but
are somewhat limiting if large numbers of recombinant clones need to be
screened. They are suitable for screening up to about 10,000 to 50,000 reJohann Peter Hossle, University Children's Hospital, Division of Immunology/Hematology, Steinwiesstraße 75, Zürich, 8032, Switzerland (phone +41-1-266-73-41;fax +41-1266-71-71; e-mail hosslejp@kispi.unizh.ch)