intro

•Recombinant DNA technology refers to the joining

together of DNA molecules from two different species that
are inserted into a host organism to produce new genetic
combinations that are of value to science, medicine,
agriculture, and industry.
•Recombinant DNA (rDNA), on the other hand is the
general name for a piece of DNA that has been created by
the combination of at least two strands.
•They are DNA molecules formed by laboratory methods
of genetic recombination (such as molecular cloning) to bring
together genetic material from multiple sources,
creating sequences that would not otherwise be found in
the genome.
 RECOMBINENT DNA TECH
• Recombinant DNA in a living organism was first achieved in 1973
by Herbert Boyer, of the University of California at San
Francisco, and Stanley Cohen, at Stanford University, who
used E.coli restriction enzymes to insert foreign DNA into
plasmids.
• A technique mainly used to change the phenotype of an organism (host)
when a genetically altered vector is introduced and integrated into the
genome of the organism. So, basically, this process involves the introduction
of a foreign piece of DNA structure into the genome which contains our
gene of interest. This gene which is introduced is the recombinant gene and
the technique is called the recombinant DNA technology.
• The recombinant DNA technology emerged with the discovery of
restriction enzymes in the year 1968 by Swiss microbiologist
Werner Arber,
 RECOMBINENT DNA TECH contd…
• Inserting the desired gene into the genome of the host is not as
easy as it sounds. It involves the selection of the desired gene for
administration into the host followed by a selection of the perfect
vector with which the gene has to be integrated and recombinant
DNA formed.
• Thus the recombinant DNA has to be introduced into the host. And
at last, it has to be maintained in the host and carried forward to
the offspring.
 Tools of recombinant DNA tech
• The enzymes which include the restriction enzymes help to cut, the polymerases-
help to synthesize and the ligases- help to bind. The restriction enzymes used in
recombinant DNA technology play a major role in determining the location at
which the desired gene is inserted into the vector genome. They are two types,
namely Endonucleases and Exonucleases
• The Endonucleases cut within the DNA strand whereas the Exonucleases remove
the nucleotides from the ends of the strands. The restriction endonucleases are
sequence-specific which are usually palindrome sequences and cut the DNA at
specific points. They scrutinize the length of DNA and make the cut at the
specific site called the restriction site. This gives rise to sticky ends in the
sequence. The desired genes and the vectors are cut by the same restriction
enzymes to obtain the complementary sticky notes, thus making the work of the
ligases easy to bind the desired gene to the vector.
Tools contd/…
• The vectors – help in carrying and integrating the desired gene. These form a very important part of
the tools of recombinant DNA technology as they are the ultimate vehicles that carry forward the
desired gene into the host organism. Plasmids and bacteriophages are the most common vectors in
recombinant DNA technology that are used as they have a very high copy number. The vectors are
made up of an origin of replication- This is a sequence of nucleotides from where the replication starts,
a selectable marker – constitute genes which show resistance to certain antibiotics like ampicillin; and
cloning sites – the sites recognized by the restriction enzymes where desired DNAs are inserted.
• Host organism – into which the recombinant DNA is introduced. The host is the ultimate tool of
recombinant DNA technology which takes in the vector engineered with the desired DNA with the help
of the enzymes.
• There are a number of ways in which these recombinant DNAs are inserted into the host, namely –
microinjection, biolistics or gene gun, alternate cooling and heating, use of calcium ions, etc.
Process /stpes of recombination
1.ISOLATION Of gentic material:-
• The first step in rDNA technology is to isolate the desired DNA
in its pure form i.e. free from other macromolecules.
• Since DNA exists within the cell membrane along with other
macromolecules such as RNA, polysaccharides, proteins, and lipids,
it must be separated and purified which involves enzymes such as
lysozymes, cellulase, chitinase, ribonuclease, proteases etc.
• Other macromolecules are removable with other enzymes or
treatments. Ultimately, the addition of ethanol causes the DNA
to precipitate out as fine threads. This is then spooled out to
give purified DNA.
2.Restriction enzyme digestion
• Restriction enzymes act as molecular scissors that cut DNA at
specific locations. These reactions are called ‘restriction enzyme
digestions’.
• They involve the incubation of the purified DNA with the selected
restriction enzyme, at conditions optimal for that specific enzyme.
• The technique ‘Agarose Gel Electrophoresis’ reveals the progress of
the restriction enzyme digestion.
• This technique involves running out the DNA on an agarose gel. On
the application of current, the negatively charged DNA travels to
the positive electrode and is separated out based on size. This allows
separating and cutting out the digested DNA fragments.
• The vector DNA is also processed using the same procedure.
3.Amplification using PCR
• Polymerase Chain Reaction or PCR is a method of making multiple copies of
a DNA sequence using the enzyme – DNA polymerase in vitro.
• It helps to amplify a single copy or a few copies of DNA into thousands to
millions of copies.
• PCR reactions are run on ‘thermal cyclers’ using the following components:
1. Template – DNA to be amplified
2.Primers – small, chemically synthesized oligonucleotides that are
complementary to a region of the DNA.
3.Enzyme – DNA polymerase
4.Nucleotides – needed to extend the primers by the enzyme.
• The cut fragments of DNA can be amplified using PCR and then ligated with
the cut vector.
4.Ligation of dna molecules
• The purified DNA and the vector of interest are cut with the same
restriction enzyme.
• This gives us the cut fragment of DNA and the cut vector, that is
now open.
• The process of joining these two pieces together using the enzyme
‘DNA ligase’ is ‘ligation’.
• The result­ing DNA molecule is a hybrid of two DNA molecules – the
interest molecule and the vector. In the ter­minology of genetics this
intermixing of dif­ferent DNA strands is called recombination.
• Hence, this new hybrid DNA molecule is also called a recombinant
DNA molecule and the technology is referred to as the recom­binant
DNA technology.
5.Insertion of Recombinant DNA Into Host
• In this step, the recombinant DNA is introduced into a recipient
host cell mostly, a bacterial cell. This process is ‘Transformation’.
• Bacterial cells do not accept foreign DNA easily. Therefore, they are
treated to make them ‘competent’ to accept new DNA. The
processes used may be thermal shock, Ca++ ion treatment,
electroporation etc.
6.Isolation of Recombinant Cells

• The transformation process generates a mixed population of

transformed and non-trans- formed host cells.
• The selection process involves filtering the transformed host cells
only.
• For isolation of recombinant cell from non-recombinant cell, marker
gene of plasmid vector is employed.
• For examples, PBR322 plasmid vector contains different marker gene
(Ampicillin resistant gene and Tetracycline resistant gene. When pst1
RE is used it knock out Ampicillin resistant gene from the plasmid,
so that the recombinant cell become sensitive to Ampicillin.
 APPLICATIONS
• Recombinant DNA is widely used in biotechnology, medicine and research.
• The most common application of recombinant DNA is in basic research, in which the
technology is important to most current work in the biological and biomedical sciences.
• Recombinant DNA is used to identify, map and sequence genes, and to determine their
function.
• Recombinant proteins are widely used as reagents in laboratory experiments and to generate
antibody probes for examining protein synthesis within cells and organisms.
• Many additional practical applications of recombinant DNA are found in industry, food
production, human and veterinary medicine, agriculture, and bioengineering.
1. DNA technology is also used to detect the presence of HIV in a person.
2.Application of recombinant DNA technology in Agriculture – For example, manufacture of Bt-
Cotton to protect the plant against ball worms.
3.Application of medicines – Insulin production by DNA recombinant technology is a classic
example.
4.Gene Therapy – It is used as an attempt to correct the gene defects which give rise to
heredity diseases.
5.Clinical diagnosis – ELISA is an example where the application of recombinant DNA is
possible.
LIMITATIONS
• Destruction of native species in the environment the genetically modified species are introduced in.
• Resilient plants can theoretically give rise to resilient weeds which can be difficult to control.
• Cross contamination and migration of proprietary DNA between organisms.
• Recombinant organisms contaminating the natural environment.
• The recombinant organisms are population of clones, vulnerable in exact same ways. A single disease or
pest can wipe out the entire population quickly.
• Creation of superbug is hypothesized.
• Ethical concern about humans trying to play God and mess with the nature’s way of selection. It is
exaggerated by the fear of unknown of what all can be created using the technology and how is it
going to impact the civilization.
• Such a system might lead to people having their genetic information stolen and used without
permission.
• Many people worry about the safety of modifying food and medicines using recombinant DNA
technology.