18 cDNA Libraries
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hosts. Second, expression cloning is an inefficient process. Half of the clones
will have inserts cloned in the wrong orientation ( 3'- 5'), which is unsuitable
for expression. Moreover, the cDNA will be inserted "in frame" with the
gene encoded by the vector and thereby expressed only one out of three
times. Taken together this means that six times as many clones must be
screened if one is searching for a gene based on its expressed product rather
than on its DNA sequence.
The inefficiency of this process can be halved by directionally cloning the
insert into the vector. This is achieved by synthesizing the cDNA with a
modified oligo(dT) primer that includes the sequence of a unique restriction site. A second unique restriction site is added to the 5' end by the ligation of a linker-adapter (Figure lA). The asymmetric sites allow insertion
of the sequence in the vector in the correct orientation, thus preserving the
5'- 3' orientation of the gene. N onetheless, a minimum of three tim es more
primary recombinants arestill required for expression screening than are
necessary for nucleic hybridizations.
A number of expression vectors have also been designed that allow expression in eukaryotic hosts. The requirements for regulating expression in
prokaryotic and eukaryotic hosts are different, and the expression cassettes
included in eukaryotic expression vectors reflect these differences. Another
feature of eukaryotic expression vectors is that they often encode markers
that allow selection of successfully transfected cells.
Non-Expression Libraries. Nucleic acid hybridization is the method
most commonly used to identify cDNA clones of interest in gene cloning
projects. There are numerous suitable bacteriophage and plasmid vectors
available. Distinguishing features include:
• limits of acceptable insert size;
• variety of cloning sites;
• ease of recovering inserts;
• positive selection for recombinants; and
• flanking RNA promoter sequences that allow generation of sense and
anti-sense transcripts. Lambda gtlO traditionally has been one of the
most commonly used lambda vectors since it accepts inserts from
0- 5.0 kb, yields large and uniform plaques with high titers, and has a
positive selection system.
The cloning site has been placed in the ci gene (lambda repressor) and successfulligation of an insert disrupts its expression. ci+ bacteriophage will
not form plaques whereas insert-containing bacteriophages form plaques
283
hosts. Second, expression cloning is an inefficient process. Half of the clones
will have inserts cloned in the wrong orientation ( 3'- 5'), which is unsuitable
for expression. Moreover, the cDNA will be inserted "in frame" with the
gene encoded by the vector and thereby expressed only one out of three
times. Taken together this means that six times as many clones must be
screened if one is searching for a gene based on its expressed product rather
than on its DNA sequence.
The inefficiency of this process can be halved by directionally cloning the
insert into the vector. This is achieved by synthesizing the cDNA with a
modified oligo(dT) primer that includes the sequence of a unique restriction site. A second unique restriction site is added to the 5' end by the ligation of a linker-adapter (Figure lA). The asymmetric sites allow insertion
of the sequence in the vector in the correct orientation, thus preserving the
5'- 3' orientation of the gene. N onetheless, a minimum of three tim es more
primary recombinants arestill required for expression screening than are
necessary for nucleic hybridizations.
A number of expression vectors have also been designed that allow expression in eukaryotic hosts. The requirements for regulating expression in
prokaryotic and eukaryotic hosts are different, and the expression cassettes
included in eukaryotic expression vectors reflect these differences. Another
feature of eukaryotic expression vectors is that they often encode markers
that allow selection of successfully transfected cells.
Non-Expression Libraries. Nucleic acid hybridization is the method
most commonly used to identify cDNA clones of interest in gene cloning
projects. There are numerous suitable bacteriophage and plasmid vectors
available. Distinguishing features include:
• limits of acceptable insert size;
• variety of cloning sites;
• ease of recovering inserts;
• positive selection for recombinants; and
• flanking RNA promoter sequences that allow generation of sense and
anti-sense transcripts. Lambda gtlO traditionally has been one of the
most commonly used lambda vectors since it accepts inserts from
0- 5.0 kb, yields large and uniform plaques with high titers, and has a
positive selection system.
The cloning site has been placed in the ci gene (lambda repressor) and successfulligation of an insert disrupts its expression. ci+ bacteriophage will
not form plaques whereas insert-containing bacteriophages form plaques