Test Document (GGG)

Gene Libraries
The step following DNA extraction of an organism is the
construction of a library to organize the DNA. A gene library can
be defined as a collection of living bacteria colonies that have
been transformed with different pieces of DNA from the organism
that is the source of the gene of interest. If a library is to have a
colony of bacteria for every gene, it will consist of tens of
thousands of colonies or clones.
Library Construction
Constructing a gene library requires not only the extracted DNA,
but also restriction enzymes and a plasmid.

Step 1: DNA extracted from an organism,

with the gene of interest, is cut into genesize pieces with restriction enzymes. These
enzymes read the nucleotide sequence of
the DNA and recognize specific sequences.
The enzymes then cut the DNA sequence by
breaking the bonds between nucleotides in a
DNA strand.
Step 2: Bacterial plasmids are cut with the
same restriction enzyme. Plasmids are small circles of DNA in
bacterial cells that are naturally present in addition to the
bacteria's other DNA.

Step 3: The gene-sized DNA and cut plasmids

are combined into one test tube. Some of the
enzyme cut DNA pieces will combine with the
cut plasmids and form recombinant DNA (or
DNA in a 'new combination').
Step 4: The recombinant plasmids are then
transferred into bacteria using either
electroporation or heat shock. Electroporation
uses mild pulses of electricity to disrupt the cell
walls of the bacterium and create small holes.
The plasmids are small enough to pass through
the holes into the cell. Heat shock works in a
similar fashion. However, rather than using
electricity to create holes in the bacterium, it is done by
alternating the temperature between hot and cold.
Step 5: The bacteria is grown on a culture dish and allowed to
grow into colonies. All the colonies on all the plates (cultures) are
called a gene library.
Step 6: The gene library is then screened in order to discover
which bacterial colony is making copies of the one gene they are
interested in. Library screening identifies colonies, which have
that particular gene. Screening can be based on detecting the
DNA sequence of the cloned gene, detecting a protein that the
gene encodes, or the use of linked DNA markers. Therefore before
library screening can be done, the scientist must know either the
DNA sequence of the gene, or a very similar gene, the protein
that the gene produces, or a DNA marker that has been mapped
very close to the gene. When the bacteria multiply and replicate
the recombinant DNA, the number of gene copies also increases,
making gene or protein detection easier.

When the bacteria colony containing the desired

gene is located, the bacteria can be propagated
to make millions of copies of the recombinant
plasmid that contains the gene.
The plasmids can be extracted for the next steps
of genetic engineering, gene modification, and
transformation. Gene cloning is also important
because copies of a gene are needed for these procedures.
Controversies
Bacterial plasmids used in gene cloning naturally contain genes that encode some form of antibiotic
resistance. As a result of the gene cloning process, the cloned genes that are put into plant cells to
make a transgenic plant may also have an antibiotic resistance gene. The antibiotic resistance trait
is often used to help scientists determine which bacteria have been transformed. Those that have
antibiotic resistance have been transformed and are kept. Some critics of genetic engineering claim
that the potential risk of providing an opportunity for organisms in nature to gain antibiotic
resistance by taking up the plasmid outweighs the potential benefits of this technology.