17 Genomic Libraries
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Future uses of the genomic clone
Many vectors have features incorporated into their design that facilitate
their use in subsequent studies. For example, one may wish to rapidly
map an insert, identify its ends and then cleave it free of vector sequences
for use in chromosome walking. The PWE and sCOSI series of cosmid vectors are examples of two vectors with features that are useful for such studies. They contain a Notl restriction site, which occurs infrequently in genomic DNA, flanking the BamHI cloning site. Between the Notl and BamHI
sites are short segments of known sequences for which primers are commonly available (T3, T7). Hybridization ofSouthern blots of digested clones
with radiolabeled T3 or T7 primers can rapidly identify the respective end
fragments and, when combined with partial digests, can assist in the construction of a restriction map of the clone.
On occasion, large-insert genomic clones are useful for in vivo expression studies. This approach often involves transfection of an intact genomic
clone into an eukaryotic cellline. The process is not very efficient, and transfectants will usually make up a small fraction of the final total cell population. The fraction will be greatly increased if a selectable marker such as
antibiotic resistance is encoded by the vector. While many cosmid vectors
contain these markers, most lambda vectors do not.
Complete vs. partial digest libraries
Intact human chromosomes are too large tobe easily manipulated and propagated in prokaryotic cell hosts. All the currently available hosts require
fragmentation of the source DNA. This can be accomplished by either random shearing or complete or partial digestion using a restriction endonuclease. Each of these methods has certain advantages and limitations.
Complete digest libraries
As the name implies, in this approach the source DNA is cleaved to completion with an enzyme that produces fragments with ends that are compatible with the cloning site of the vector. This is the most efficient method
for cloning a specific restriction fragment if its size is known tobe within the
acceptable range for the vector being used, since each chromosomal copy in
the source material is potentially clonable. If digestion is truly complete,
however, no clones with overlapping fragments could ever be isolated
from such a library, making it impossible to do sequential chromosome
265
Future uses of the genomic clone
Many vectors have features incorporated into their design that facilitate
their use in subsequent studies. For example, one may wish to rapidly
map an insert, identify its ends and then cleave it free of vector sequences
for use in chromosome walking. The PWE and sCOSI series of cosmid vectors are examples of two vectors with features that are useful for such studies. They contain a Notl restriction site, which occurs infrequently in genomic DNA, flanking the BamHI cloning site. Between the Notl and BamHI
sites are short segments of known sequences for which primers are commonly available (T3, T7). Hybridization ofSouthern blots of digested clones
with radiolabeled T3 or T7 primers can rapidly identify the respective end
fragments and, when combined with partial digests, can assist in the construction of a restriction map of the clone.
On occasion, large-insert genomic clones are useful for in vivo expression studies. This approach often involves transfection of an intact genomic
clone into an eukaryotic cellline. The process is not very efficient, and transfectants will usually make up a small fraction of the final total cell population. The fraction will be greatly increased if a selectable marker such as
antibiotic resistance is encoded by the vector. While many cosmid vectors
contain these markers, most lambda vectors do not.
Complete vs. partial digest libraries
Intact human chromosomes are too large tobe easily manipulated and propagated in prokaryotic cell hosts. All the currently available hosts require
fragmentation of the source DNA. This can be accomplished by either random shearing or complete or partial digestion using a restriction endonuclease. Each of these methods has certain advantages and limitations.
Complete digest libraries
As the name implies, in this approach the source DNA is cleaved to completion with an enzyme that produces fragments with ends that are compatible with the cloning site of the vector. This is the most efficient method
for cloning a specific restriction fragment if its size is known tobe within the
acceptable range for the vector being used, since each chromosomal copy in
the source material is potentially clonable. If digestion is truly complete,
however, no clones with overlapping fragments could ever be isolated
from such a library, making it impossible to do sequential chromosome