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Recombinant DNA
A recombinant DNA molecule is produced by joining together two or more DNA segments
usually from different organisms. More specifically, a recombinant DNA molecule is a vector
(e.g. plasmid, phage or virus) into which the desired DNA fragment has been inserted to enable
its cloning in an appropriate host. Genetically engineered DNA prepared by splicing genes from
one species into the cells of a host organism of a different species. Such DNA becomes part of
the host's genetic makeup and is replicated.
The idea for recombinant DNA was first proposed by Peter Lobban, a graduate student of Prof.
Dale Kaiser in the Biochemistry Department at Stanford University Medical School. The first
publications describing the successful production and intracellular replication of recombinant
DNA appeared in 1972 and 1973.
Recombinant DNA molecules are produced with one of the following three objectives:
1) To obtain a large number of copies of specific DNA fragments.
2) To recover large quantities of the protein produced by concerned gene.
3) To integrate the gene in question into the chromosome of a target organism where it
express itself.
Recombinant DNA technology: A series of procedures that are used to join together
(recombine) DNA segments. A recombinant DNA molecule is constructed from segments of
two or more different DNA molecules. Under certain conditions, a recombinant DNA molecule
can enter a cell and replicate there, either on its own or after it has been integrated into a
chromosome.
Vector
In molecular cloning, a vector is a DNA molecule used as a vehicle to artificially carry foreign
genetic material into another cell, where it can be replicated and/or expressed. A vector
containing foreign DNA is termed recombinant DNA.
Fig. The pGEX-3x plasmid cloning vector
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The four major types of vectors are i. Plasmids: Plasmids are double-stranded generally circular DNA sequences that are
capable of automatically replicating in a host cell. Plasmid vectors minimalistically
consist of an origin of replication. Modern plasmids generally have many more
features, notably including a "multiple cloning site" which includes nucleotide
overhangs for insertion of an insert, and multiple restriction enzyme consensus sites
to either side of the insert. Plasmids may be conjugative/transmissible and nonconjugative.

Conjugative: mediate DNA transfer through conjugation and therefore spread rapidly
among the bacterial cells of a population; e.g., F plasmid, many R and some col
plasmids.

Nonconjugative: do not mediate DNA through conjugation, e.g., many R and col
plasmids.
ii. Viral vectors: Viral vectors are generally genetically-engineered viruses carrying
modified viral DNA or RNA. Viral vectors frequently are lacking infectious
sequences, they require helper viruses or packaging lines for large-scale transfection.
Viral vectors are often designed for permanent incorporation of the insert into the
host genome, and thus leave distinct genetic markers in the host genome after
incorporating the transgene. For example, retroviruses leave a characteristic retroviral
integration pattern after insertion that is detectable and indicates that the viral vector
has incorporated into the host genome.
iii. Cosmid: A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid
plasmid (often used as a cloning vector) that contains a Lambda phage cos sequence.
Cosmids' (cos sites + plasmid = cosmid) DNA sequences are originally from the
lambda phage. Cosmids can be used to build genomic libraries.
Characteristics of cosmid:
a) Cosmids are able to contain 37 to 52 kb of DNA, while normal plasmids are able to carry
only 1–20 kb.
b) They can replicate as plasmids if they have a suitable origin of replication, for example
SV40 ori in mammalian cells.
c) They frequently also contain a gene for selection such as antibiotic resistance. These
cells which did not take up the cosmid would be unable to grow.
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iv. Bacterial artificial chromosome: It is a DNA constructing, based on a functional
fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria,
usually E. coli. F-plasmids play a crucial role because they contain partition genes
that promote the even distribution of plasmids after bacterial cell division. The
bacterial artificial chromosome's usual insert size is 150-350 kbp. A similar cloning
vector called a PAC has also been produced from the bacterial P1-plasmid.
Properties of a good vector:
i.
Origin of replication: The origin of replication (also called the replication origin) is a
particular sequence in a genome at which replication is initiated. This can either involve
the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of
DNA or RNA in viruses, such as double-stranded RNA viruses.
ii.
Promoter: In genetics, a promoter is a region of DNA that initiates transcription of a
particular gene. Promoters are located near the genes they transcribe, on the same strand
and upstream on the DNA. Promoters can be about 100–1000 base pairs long.
iii.
Cloning site: A multiple cloning site (MCS), also called a polylinker, is a short segment
of DNA which contains many (up to ~ 20) restriction sites - a standard feature of
engineered plasmids. Restriction sites within an MCS are typically unique, occurring
only once within a given plasmid. MCSs are commonly used during procedures
involving molecular cloning or subcloning. Extremely useful in biotechnology,
bioengineering, and molecular genetics, MCSs let a microbiologist insert a piece of DNA
or several pieces of DNA into the region of the MCS. This can be used to create
transgenic organisms, also known as genetically modified organisms (GMOs).
iv.
Genetic markers: A genetic marker is a gene or DNA sequence with a known location on
a chromosome that can be used to identify individuals or species. It can be described as a
variation (which may arise due to mutation or alteration in the genomic loci) that can be
observed. A genetic marker may be a short DNA sequence, such as a sequence
surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long
one, like minisatellites.
v.
Antibiotic resistance: Antibiotic resistance is a form of drug resistance whereby some
(or, less commonly, all) sub-populations of a microorganism, usually a bacterial species,
are able to survive after exposure to one or more antibiotics; pathogens resistant to
multiple antibiotics are considered multidrug resistant (MDR) or, more colloquially,
superbugs. Microbes, rather than people, develop resistance to antibiotics.
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vi.
Epitope: An epitope, also known as antigenic determinant, is the part of an antigen that is
recognized by the immune system, specifically by antibodies, B cells, or T cells. The part
of an antibody that recognizes the epitope is called a paratope. Although epitopes are
usually non-self proteins, sequences derived from the host that can be recognized are also
epitopes.
vii.
Reporter genes: In molecular biology, a reporter gene (often simply reporter) is a gene
that researchers attach to a regulatory sequence of another gene of interest in bacteria,
cell culture, animals or plants.
viii. Targeting
sequence: Protein targeting or protein sorting is the biological mechanism by
which proteins are transported to the appropriate destinations in the cell or outside of it.
ix.
Protein purification tags: Protein tags are peptide sequences genetically grafted onto a
recombinant protein. Often these tags are removable by chemical agents or by enzymatic
means, such as proteolysis. Tags are attached to proteins for various purposes.
Applications of recombinant DNA technology
Recombinant DNA is widely used in biotechnology, medicine and research. Many additional
practical applications of recombinant DNA are found in industry, food production, human
and veterinary medicine, in agriculture, and in bioengineering. Some specific examples are
identified below:
1. Recombinant chymosin: Found in rennet, is an enzyme required to manufacture cheese.
It was the first genetically engineered food additive to be used commercially.
Traditionally, processors obtained chymosin from rennet, a preparation derived from the
fourth stomach of milk-fed calves.
2. Recombinant human insulin: Almost completely replaced insulin obtained from animal
sources (e.g. pigs and cattle) for the treatment of insulin-dependent diabetes.
Recombinant insulin is synthesized by inserting the human insulin gene into E. coli,
which then produces insulin for human use.
3. Recombinant human growth hormone (HGH, somatotropin): Administered to patients
whose pituitary glands generate insufficient quantities to support normal growth and
development.
4. Recombinant blood clotting factor VIII: A blood-clotting protein that is administered to
patients with forms of the bleeding disorder hemophilia, who are unable to produce
factor VIII in quantities sufficient to support normal blood coagulation.
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5. Recombinant hepatitis B vaccine: Prevention of hepatitis B infection is controlled
through the use of a recombinant hepatitis B vaccine. It contains a form of the hepatitis B
virus surface antigen that is produced in yeast cells.
6. Diagnosis of infection with HIV: Each of the three widely-used methods for diagnosing
HIV infection has been developed using recombinant DNA. The antibody test (ELISA or
western blot) uses a recombinant HIV protein to test for the presence of antibodies that
the body has produced in response to an HIV infection.
7. Golden rice: A recombinant variety of rice that has been engineered to express the
enzymes responsible for β-carotene biosynthesis. This variety of rice holds substantial
promise for reducing the incidence of vitamin A deficiency in the world's population.
Golden rice is not currently in use, pending the resolution of intellectual property,
environmental and nutritional issues.
8. Herbicide-resistant crops: Commercial varieties of important agricultural crops
(including soy, maize/corn, sorghum, canola, alfalfa and cotton) have been developed
which incorporate a recombinant gene that results in resistance to the herbicide
glyphosate (trade name Roundup), and simplifies weed control by glyphosate
application. These crops are in common commercial use in several countries.
9. Insect-resistant crops: Bacillus thuringeiensis is a bacterium that naturally produces a
protein (Bt toxin) with insecticidal properties. The bacterium has been applied to crops as
an insect-control strategy for many years, and this practice has been widely adopted in
agriculture and gardening.
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