18 cDNA Libraries
279
• One can screen cDNA libraries for gene sequences using many more approaches than can be used to screen genomic libraries. Standard hybridization techniques using labeled oligonucleotide, cDNA, RNA or genomic fragments as probes are similar for the two types oflibraries. cDNA
libraries, however, byvirtue ofbeing comprised of expressed sequences,
can also be screened by testing for their gene products. Antihoclies can be
used to identify clones expressing particular antigens. Ligauds can be
used to identify clones expressing particular receptors (or vice versa),
etc. Genes can also be identified on the basis of the functional properties
of the encoded proteins (eg. transporters, channels).
• Inefficient ligation, packaging, transfection or transformation results in
incomplete representation of the transcribed sequences from the tissue
that was the source for the mRNA.
Theoretical considerations for cloning cDNA sequences
Determining the number of clones that must be screened to ensure >95%
coverage ofthe genome is relatively straightforward, as was previously explained. Each genome equivalent is expected to have one copy of a chromosome locus. These calculations, however, are much more difficult for cDNA
libraries since not all genes are expressed in equal abundance in a cell. Some
cells have a large number of copies of mRNA ( -1500/cell) derived from a
handful of genes whereas other mRNA molecules are present at less than 10
copies/cell. Consider a chicken red blood cell (RBC) library: 3 mRNA species
( -1500 copies/cell) comprise over 90% ofthe total mRNA molecules present
in chicken RBCs. One plate ofless than 100 clones has a probability >95% of
having at least one copy of each of the most common genes. Alternatively,
over 1000 fold more plaques would be required to find a message that is
rarely expressed in this cell type. The vast majority of chicken geneswill
never be found no matter how many millions of plaques one screens.
Only approx. 1000 genes, or ~ 1 - 5% of the total, are expressed in this
cell type. Thus, even a perfect cDNA library derived from chicken RBCs
willlack copies of most genes.
In sum, there are four major principles that determine the number of
clones that must be screened.
• The relative abundance of the transcript in the tissue or cell type that is
the source of the RNA is one of the most important factors. N orthern blot
analysis or a quantitative RNA protection assay can help one estimate
expression levels.
279
• One can screen cDNA libraries for gene sequences using many more approaches than can be used to screen genomic libraries. Standard hybridization techniques using labeled oligonucleotide, cDNA, RNA or genomic fragments as probes are similar for the two types oflibraries. cDNA
libraries, however, byvirtue ofbeing comprised of expressed sequences,
can also be screened by testing for their gene products. Antihoclies can be
used to identify clones expressing particular antigens. Ligauds can be
used to identify clones expressing particular receptors (or vice versa),
etc. Genes can also be identified on the basis of the functional properties
of the encoded proteins (eg. transporters, channels).
• Inefficient ligation, packaging, transfection or transformation results in
incomplete representation of the transcribed sequences from the tissue
that was the source for the mRNA.
Theoretical considerations for cloning cDNA sequences
Determining the number of clones that must be screened to ensure >95%
coverage ofthe genome is relatively straightforward, as was previously explained. Each genome equivalent is expected to have one copy of a chromosome locus. These calculations, however, are much more difficult for cDNA
libraries since not all genes are expressed in equal abundance in a cell. Some
cells have a large number of copies of mRNA ( -1500/cell) derived from a
handful of genes whereas other mRNA molecules are present at less than 10
copies/cell. Consider a chicken red blood cell (RBC) library: 3 mRNA species
( -1500 copies/cell) comprise over 90% ofthe total mRNA molecules present
in chicken RBCs. One plate ofless than 100 clones has a probability >95% of
having at least one copy of each of the most common genes. Alternatively,
over 1000 fold more plaques would be required to find a message that is
rarely expressed in this cell type. The vast majority of chicken geneswill
never be found no matter how many millions of plaques one screens.
Only approx. 1000 genes, or ~ 1 - 5% of the total, are expressed in this
cell type. Thus, even a perfect cDNA library derived from chicken RBCs
willlack copies of most genes.
In sum, there are four major principles that determine the number of
clones that must be screened.
• The relative abundance of the transcript in the tissue or cell type that is
the source of the RNA is one of the most important factors. N orthern blot
analysis or a quantitative RNA protection assay can help one estimate
expression levels.