Crop failures, pathogenic outbreaks and famine are serious problems
facing society. Understanding an organism's genome will help provide
the genetic tools needed to solve these complex problems in a shorter
time and with lesser effort. To efficiently study an organism's genome,
it can be partitioned into a permanent and stable collection of DNA
fragments, called a library. Such libraries provide convenient access
to a genome for both laboratory and breeding applications. Genomic
libraries can be used as substrates to physically map and sequence
entire genomes, clone agriculturally important genes and to investigate
gene expression patterns. Further, genomic libraries also provide
powerful tools and resources for evaluating germplasm conservation
stocks and biological diversity. Since the ongoing explosion of genetic
data and molecular clone resources has opened new scientific
possibilities with unfamiliar terms and acronyms to researchers
venturing across all avenues of applied science
Serious consideration must be given to the various kinds of libraries
that can be constructed with respect to different insert sizes,
fragmentation methods, vectors, coverage, downstream operations, etc.
Though downstream applications (see the Applications section of this
chapter) differ for types of libraries, fragmentation size is the first
consideration in genomic library construction. Genomic DNA libraries
are classified as shotgun libraries, medium-size insert libraries and
large insert libraries depending on insert sizes. A shotgun library,
with a smaller insert size (generally 1.5–10 kbp), is made using
high-copy plasmid vectors. It is the most common resource for complete
sequencing of large genomic DNA clones (e.g. cosmid, PAC, BAC and YAC
clones) and large DNA fragments (PCR products and restriction
fragments). With the introduction of capillary sequencers for
large-scale, high-throughput DNA sequencing, the shotgun library
approach is considered a standard method for generating sequence-ready
sublibraries for genome projects and positional cloning studies.
Cosmid and fosmid libraries contain medium-sized inserts (35–45 kbp). Cosmids are hybrids of λλ-phage
and plasmids. They can replicate in the cell like a plasmid due to the
plasmid replication origin and be packaged like a phage because of
existing cos sites. Since most of the λλ-phage
structure has been deleted, they can carry DNA inserts up to 45 kbp.
This type of library is useful for cloning studies because of its
simplicity and economic efficiency for construction in comparison with
large insert library construction. The fosmid cloning system is similar
in size to cosmids and the vector is derived from the endogenous E. coli
F1 factor that maintains inserts in a single-copy state adding to the
fidelity and stability of the DNA insert. The fosmid system is useful
for easy generation of medium-sized insert (45 kbp) genomic libraries
produced from small amounts of source DNA such as flow-sorted
chromosomal DNA (Gingrich et al., 1996).
Large-insert genomic DNA libraries are essential for
physical mapping, positional cloning and genome sequencing of complex
genomes. There are two principal large-insert cloning systems: yeast
and bacterial artificial chromosome systems (YACs and BACs). The YAC
cloning system was first developed in 1987 (Burke et al., 1987), and uses Saccharomyces cerevisiae
as the host and maintains large inserts (up to 1 Mb) as linear
molecules with a pair of yeast telomeres and a centromere. Although
used extensively in the late 1980s and early 1990s, this system has
several disadvantages (Anderson, 1993; Zhang and Wing, 1997).
The recombinant DNA in yeast can be unstable. DNA manipulation is
difficult and inefficient. Most importantly, a high level of
chimerism—cloning of two or more unlinked DNA fragments in a single
molecule—is inherent within the YAC cloning system. These disadvantages
impede the utility of YAC libraries and subsequently this system has
been gradually replaced by the BAC cloning system introduced in 1992
(Shizuya et al., 1992).
The BAC system uses a derivative of the E. coli F-factor as a vector and E. coli
as the host, making BAC cloning and subsequent down-stream procedures
efficient and easy to perform. Recombinant DNA inserts of up to 200 kb
can be efficiently cloned and stably maintained as single-copy plasmids
in E. coli. BAC libraries have been developed for essentially all major crop plants, as well as model plant species, such as Arabidopsis thaliana
and rice, and can be accessed from the laboratories that made the
libraries or through various stock centres (e.g. AGI, CUGI and ABRC).
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