264
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
Tolerance of 'difficult to clone' sequences
Some hosts have restriction systems that cleave methylated DNA or DNA
containing unusual restriction sites, thereby preventing their propagation.
A library grown in such a host would be incomplete because genomic fragments having susceptible restriction sites would not be represented. Fortunately, a nurober of mutant strains have been isolated that allow cloning
and stable propagation of these previously unclonable segments. Genomic
fragments with unusual sequence properties such as inverted repeats, long
stretches of repetitive DNA, or sequences with homology to host or vector
sequences, may also be unclonable or undergo partial deletion or rearrangement in some hosts. Lastly, there appear tobe segments that are repeatedly unclonable in cosmid libraries and yet can be reproducibly isolated in
lambda, YAC or P llibraries ( and vice versa). Thus, failure to isolate a clone
from one library does not mean that the segment is unclonable, and the use
of additional libraries increases the likelihood of success.
Quantity and complexity of starting material
Single cos site vectors (i.e., pcos2EMBL, PWE series) are less efficient in
packaging than newer double cos site vectors such as sCosl which can
be used with extremely small quantities of starting DNA. Likewise, YAC
transformation efficiencies are orders of magnitude lower than those
achieved with plasmids or bacteriophage/cosmid transfections. Vectors
with the highest cloning efficiencies (bacteriophage and/or plasmid) are
preferred when the quantity of source DNA is limiting.
Occasionally, only small and reasonably well-defined DNA fragments
are required. The source of fragments could be an entire or partial human
chromosome (propagated in a rodent cellline) or a YAC, PI, cosmid or
bacteriophage insert. Virtually any fragment can be subdivided and propagated as smaller subunits. These libraries, constructed from only part of the
genome, are known as subgenomic libraries. They are useful in the isolation
and characterization of segments present in multiple copies within the genome. Subgenomic libraries, however, are most commonly constructed for
use in large scale genomic sequencing projects. Y ACs or cosmids are more
easily sequenced after being subcloned into plasmids which contain much
smaller inserts.
LUIZ F. ONUCHIC AND GREGORY G. GERMINO
Tolerance of 'difficult to clone' sequences
Some hosts have restriction systems that cleave methylated DNA or DNA
containing unusual restriction sites, thereby preventing their propagation.
A library grown in such a host would be incomplete because genomic fragments having susceptible restriction sites would not be represented. Fortunately, a nurober of mutant strains have been isolated that allow cloning
and stable propagation of these previously unclonable segments. Genomic
fragments with unusual sequence properties such as inverted repeats, long
stretches of repetitive DNA, or sequences with homology to host or vector
sequences, may also be unclonable or undergo partial deletion or rearrangement in some hosts. Lastly, there appear tobe segments that are repeatedly unclonable in cosmid libraries and yet can be reproducibly isolated in
lambda, YAC or P llibraries ( and vice versa). Thus, failure to isolate a clone
from one library does not mean that the segment is unclonable, and the use
of additional libraries increases the likelihood of success.
Quantity and complexity of starting material
Single cos site vectors (i.e., pcos2EMBL, PWE series) are less efficient in
packaging than newer double cos site vectors such as sCosl which can
be used with extremely small quantities of starting DNA. Likewise, YAC
transformation efficiencies are orders of magnitude lower than those
achieved with plasmids or bacteriophage/cosmid transfections. Vectors
with the highest cloning efficiencies (bacteriophage and/or plasmid) are
preferred when the quantity of source DNA is limiting.
Occasionally, only small and reasonably well-defined DNA fragments
are required. The source of fragments could be an entire or partial human
chromosome (propagated in a rodent cellline) or a YAC, PI, cosmid or
bacteriophage insert. Virtually any fragment can be subdivided and propagated as smaller subunits. These libraries, constructed from only part of the
genome, are known as subgenomic libraries. They are useful in the isolation
and characterization of segments present in multiple copies within the genome. Subgenomic libraries, however, are most commonly constructed for
use in large scale genomic sequencing projects. Y ACs or cosmids are more
easily sequenced after being subcloned into plasmids which contain much
smaller inserts.