CHAPTER 6

RECOMBINANT DNA TECHNOLOGY

Definition
Recombinant DNA technology involves using enzymes and various laboratory techniques to
manipulate and isolate DNA segments of interest. This method can be used to combine (or
splice) DNA from different species or to create genes with new functions. The resulting copies
are often referred to as recombinant DNA. Such work typically involves propagating the
recombinant DNA in a bacterial or yeast cell, whose cellular machinery copies the engineered
DNA along with its own. Recombinant DNA technology comprises altering genetic
material outside an organism to obtain enhanced and desired characteristics in living
organisms or as their products. This technology involves the insertion of DNA fragments
from a variety of sources, having a desirable gene sequence via appropriate vector
Recombinant DNA refers to the creation of new combinations of DNA segments that are not
found together in nature. The isolation and manipulation of genes allows for more precise
genetic analysis as well as practical applications in medicine, agriculture, and industry.
Fundamental changes in our society are occurring as a result of genetic engineering.
Making recombinant DNA
Overview: Isolate DNA --- Cut with restriction enzymes ---- Ligate into cloning vector ---
transform recombinant DNA molecule into host cell ---- each transformed cell will divide many,
many times to form a colony of millions of cells, each of which carries the recombinant DNA
molecule (DNA clone). Recombinant DNA is a form of artificial DNA that is made through the
combination or insertion of one or more DNA strands, therefore combining DNA sequences as
per your requirement, within different species i.e. DNA sequences that would not normally occur
together
In genetics recombinant DNA technology has many uses e.g: Agriculture: growing crops of
your choice (GM food), pesticide resistant crops, fruits with attractive colors, all being grown in
artificial conditions. Pharmacology: artificial insulin production, drug delivery to target sites
Medicine: gene therapy, antiviral therapy, vaccination, synthesizing clotting factors
Other uses: fluorescent fishes, glowing plants etc
 CHAPTER 7

DNA Damage and Repair

DNA DAMAGE AND REPAIR MECHANISMS
Damage to cellular DNA is involved in mutagenesis and the development of cancer. The
DNA in a human cell undergoes several thousand to a million damaging events per day,
generated by both external (exogenous) and internal metabolic (endogenous)
processes. Changes to the cellular genome can generate errors in the transcription of
DNA and ensuing translation into proteins necessary for signaling and cellular function.
Genomic mutations can also be carried over into daughter generations of cells if the
mutation is not repaired prior to mitosis. Once cells lose their ability to effectively repair
damaged DNA, there are three possible responses (Figure 1).
1. The cell may become senescent, i.e., irreversibly dormant. In 2005, multiple
laboratories reported that senescence could occur in cancer cells in vivo as well as in
vitro, stopping mitosis and preventing the cell from evolving further. 1-4
2. The cell may become apoptotic. Sufficient DNA damage may trigger an apoptotic
signaling cascade, forcing the cell into programmed cell death.
3. The cell may become malignant, i.e., develop immortal characteristics and begin
uncontrolled division.

SOURCES OF DNA DAMAGE

For many years, exogenous sources of damage have been thought to be the primary
cause of DNA mutations leading to cancer. DNA damage also contribute significantly to
mutations that lead to malignancy. 5 Both environmental and cellular sources can result
in similar types of DNA damage.
There are two types DNA damages
 physical mutagens
 chemical mutagens
DNA can be attacked by physical and chemical mutagens. Physical mutagens are
primarily radiation sources, including UV (200-300 nm wave length) radiation from the
sun. UV radiation produces covalent bonds that crosslink adjacent pyrimidine (cytosine
and thymine) bases in the DNA strand. Ionizing radiation (X-rays) initiates DNA
mutations by generating free radicals within the cell that create reactive oxygen species
(ROS) and result in single-strand and double-strand breaks in the double helix.
Chemical mutagens can attach alkyl groups covalently to DNA bases;
 DNA repair,
DNA repair any of several mechanisms by which a cell maintains the integrity of
its genetic code. DNA repair ensures the survival of a species by enabling
parental DNA to be inherited as faithfully as possible by offspring. It also preserves the
health of an individual. Mutations in the genetic code can lead to cancer and other
genetic diseases. Successful DNA replication requires that the
two purine bases, adenine (A) and guanine (G), pair with
their pyrimidine counterparts, thymine (T) and cytosine (C). Different types of damage,