18 cDNA Libraries
277
Previous chapters have discussed the relative cloning efficiencies of cohesive vs. blunt ends. Double-stranded cDNA molecules synthesized using
DNA polymerase I have blunt ends which ligate very inefficiently. A large
fraction of the cDNA molecules would be lost from a library if one tried to
ligate them to a blunt-ended vector. Therefore, virtually all protocols for
synthesis of cDNA libraries include a step that adds cohesive ends which
are compatible with the cloning site of a vector. This is accomplished by
ligating short, double-stranded DNA molecules, called linkers. A unique
feature of these synthetic oligonucleotides is that they contain restriction
sites which are compatible with the vector's cloning sites. A very high molar
ratio oflinkers to cDNA molecules is easily achieved, and this compensates
for the inefficiency ofblunt end ligations. Excess linkers are removed by size
fractionation prior to cloning. The preparation of cDNA described under
Procedure as shown in Figure lA presents one of the many approaches in
current use (a modification of Gubler and Hoffman, 1983).
As previously discussed, there are methodologic problems common to
both genomic and cDNA libraries. cDNA libraries also have several unique
features that require consideration:
• Most cDNA libraries are constructed with the goal of obtaining fulllength cDNA copies of mRNAs. Careful selection of the enzymes (eg.
Superscript ( GibcoBRL, Gaithersburg, MD) for the reverse transcriptase,
etc.) and reaction conditions are important but byfar the most important
determinant of success is the quality of the original mRNA. Libraries
generated from low quality mRNA (i.e., partially degraded) will lack
full-length copies of larger mRNAs as well as rare transcripts.
• A high frequency of chimerk clones results when the concentration of
linkers used to construct the library is too low.
• After packaging or transformation, most libraries are amplified for storage and re-use. Ideally, each clone present in an originallibrary is replicated to the same degree so that its fractional representation remains
Fig. 1. A Full preparation of a cDNA insert. B Cloning method using lambda bacteriophage
vector. The vector is digested with restriction enzymes that produce ends compatible with the
cDNA linker adaptors. Size fractionation is used to remove the stuffer fragments. The phage
arms are allowed to reanneal at their cos sites prior to ligation to cDNA inserts. This results in
long concatamers of alternating phage arms and inserts. Included in packaging extract are
proteins that cleave at cos sites and then package the 38- 50 kb DNA fragments into phage
heads. Phage tails, also included in packaging extract, self-attach to the fllled heads. The
resultant particles are infectious and are incubated with bacteria for propagation.
277
Previous chapters have discussed the relative cloning efficiencies of cohesive vs. blunt ends. Double-stranded cDNA molecules synthesized using
DNA polymerase I have blunt ends which ligate very inefficiently. A large
fraction of the cDNA molecules would be lost from a library if one tried to
ligate them to a blunt-ended vector. Therefore, virtually all protocols for
synthesis of cDNA libraries include a step that adds cohesive ends which
are compatible with the cloning site of a vector. This is accomplished by
ligating short, double-stranded DNA molecules, called linkers. A unique
feature of these synthetic oligonucleotides is that they contain restriction
sites which are compatible with the vector's cloning sites. A very high molar
ratio oflinkers to cDNA molecules is easily achieved, and this compensates
for the inefficiency ofblunt end ligations. Excess linkers are removed by size
fractionation prior to cloning. The preparation of cDNA described under
Procedure as shown in Figure lA presents one of the many approaches in
current use (a modification of Gubler and Hoffman, 1983).
As previously discussed, there are methodologic problems common to
both genomic and cDNA libraries. cDNA libraries also have several unique
features that require consideration:
• Most cDNA libraries are constructed with the goal of obtaining fulllength cDNA copies of mRNAs. Careful selection of the enzymes (eg.
Superscript ( GibcoBRL, Gaithersburg, MD) for the reverse transcriptase,
etc.) and reaction conditions are important but byfar the most important
determinant of success is the quality of the original mRNA. Libraries
generated from low quality mRNA (i.e., partially degraded) will lack
full-length copies of larger mRNAs as well as rare transcripts.
• A high frequency of chimerk clones results when the concentration of
linkers used to construct the library is too low.
• After packaging or transformation, most libraries are amplified for storage and re-use. Ideally, each clone present in an originallibrary is replicated to the same degree so that its fractional representation remains
Fig. 1. A Full preparation of a cDNA insert. B Cloning method using lambda bacteriophage
vector. The vector is digested with restriction enzymes that produce ends compatible with the
cDNA linker adaptors. Size fractionation is used to remove the stuffer fragments. The phage
arms are allowed to reanneal at their cos sites prior to ligation to cDNA inserts. This results in
long concatamers of alternating phage arms and inserts. Included in packaging extract are
proteins that cleave at cos sites and then package the 38- 50 kb DNA fragments into phage
heads. Phage tails, also included in packaging extract, self-attach to the fllled heads. The
resultant particles are infectious and are incubated with bacteria for propagation.