Genomic Libraries: Construction and Applications

DNA sequencing became possible in the late 1970s and has been one of the most
influential developments in biomedical research to date. This technology has rapidly
expanded since the publication of the human genome in 2003 and genomics is now
used in a wide variety of fields. The ability to sequence an organism’s genome has
helped to provide genetic tools that can be used to solve a range of complex
questions, from applications in medicine where it can be used to predict, diagnose,
and treat a range of diseases, to helping prevent crop failures by engineering plants
that can withstand environmental stressors.

Genomic Library Construction

The first step in any DNA sequencing project is constructing a genomic library. A
genomic library contains all the sequences that are present in the genome of an
organism; expressed genes, non-expressed genes, exons and introns, promoter and
terminator regions, intervening DNA sequences etc. To create the library, an
organism’s genomic DNA is extracted and then digested using a restriction enzyme.
Many recombinant DNA molecules must be produced to cover the entire genome.

There are five main steps in gene cloning:

1. Isolation of the genomic DNA and vector

 2. DNA fragment generation
1.
1.
1. Restriction digestion
2. Mechanical shearing
3. Ligation of DNA into a vector system
4. Transformation of the recombinant DNA into a host cell
5. Library screening

Genomic library construction ( Aluquette via Wikimedia Commons)

Let’s look at each in turn.

Isolation of the genomic DNA and vector

Genomic DNA isolation is an easy technique that can be done following standard
protocols or by using a ready-to-use DNA extraction kit. This process includes cell
lysis, removal of proteins and impurities, DNA precipitation, DNA purification, and
DNA elution. The quantity of the resulting isolated genomic DNA needs to be
around 100 micrograms or more and have a purity of around 1.80 at the 260/280
absorbance.

DNA Fragment Generation

Once extracted, the DNA must be broken down into small, random-sized fragments
which are carried out using two common techniques: restriction digestion and
mechanical shearing. Restriction digestion uses a restriction enzyme that cleaves the
DNA into fragments. The enzyme used binds to its specific recognition site, which
is present multiple times through the genome and produces fragments that have
either sticky or blunt ends. A cutter that produces sticky ends is preferred as these
are easy to insert into a vector. An alternative method for fragmentation is
mechanical shearing. DNA fragmentation is carried out artificially, producing blunt
end cuts. This technique requires an extra processing step to facilitate ligation into
the vector.

Ligation of DNA into a vector system

Next, the DNA fragments are incorporated into a suitable vector. Common vectors
used for genomic library preparation are plasmid, cosmid, phage, bacteriophage,
lambda phage, BAC, or YAC. The vector that you use will depend on the size of the
DNA fragment you are trying to clone. For example, a plasmid has a carrying
capacity of 15kb of DNA whereas a YAC system can be used for DNA fragments
between 250-2000 kb.

Once selected, the vector must be processed to enable the DNA fragment to be
inserted. Usually, the vector is digested using the sale restriction enzyme that was
used to cut the genomic DNA. The fragment and vector DNA can then be ligated
using a ligase enzyme before being sent for transformation. When preparing the
vector, it is also important to consider the use of a selectable or screenable marker
that can be used to validate the insert, such as an antibiotic-resistant gene.

Transformation of the recombinant DNA into a host cell

The transformation steps involve inserting the recombinant vector into a host cell,
such as E. coli or yeast cells, where it replicates to produce copies of the fragments
of genomic DNA. Once inserted, the cells are cultured on agar media that contains
an antibiotic. This means that only the transformed cells containing an antibiotic-
resistant gene can grow.

Library Screening
Library screening is used to identify the genes of interest. The two most common
screening methods are colony hybridization and plaque hybridization, and the
method you choose will depend on the cloning vector you used. Colony
hybridization is a method of selecting bacterial colonies containing the desired
genes. This method is used to identify genes in a plasmid- or cosmid-based library.
Plaque hybridization on the other hand is used to identify the desired genes in phage-
based libraries. Both methods use filter hybridization, enabling the primary
screening of libraries by in situ replication on a nitrocellulose membrane.

Applications

Genomic library construction is the first step in any DNA-sequencing project and is
vital to research genome structure and function. These libraries have a wide range
of applications including SNP analysis, mutational detection, copy number variation
studies and population-based genetic studies. Research in this area has helped to
identify new genes, find changes in the genome that contribute to disease
development, and in the generation of transgenic animals and plants through genetic
engineering. In addition, genomics is also frequently being used to study microbial
communities and in agriculture for improving the quality and quantity of crop yields.