Recombinant DNA

- Recombinant DNA technology is one of the recent advances in biotechnology, which was developed b
y two scientists named Boyer and Cohen in 1973.

(rDNA) is a form of artificial DNA that is created by combining two or more sequences that would not
normally occur together through the process of gene splicing.
Recombinant DNA technology
is a technology which allows DNA to be produced via artificial means. The procedure has been used to
change DNA in living organisms and may have even more practical uses in the future.
Recombinant technology begins with the isolation of a gene of interest (target gene). The target gene is
then inserted into the plasmid or phage (vector) to form replicon. The replicon is then introduced into host
cells to be cloned and either express the protein or not.  The cloned replicon is referred to as recombinant
DNA. The procedure is called recombinant DNA technology. Cloning is necessary to produce numerous
copies of the DNA since the initial supply is inadequate to insert into host cells.
Six steps of Recombinant DNA
1. Isolating (vector and target gene)
2. Cutting (Cleavage)
3. Joining (Ligation)
4. Transforming
5. Cloning
6. Selecting (Screening)
1. Isolation and purification of DNA.

Both vector and target DNA molecules can be prepared by a variety of routine methods. In some cases,
the target DNA is synthesized in vitro.
2. Cleavage of DNA at particular sequences.
Cleaving DNA to generate fragments of defined length, or with specific endpoints, is crucial to
recombinant DNA technology. The DNA fragment of interest is called insert DNA. In the laboratory,
DNA is usually cleaved by treating it with commercially produced nucleases and restriction
endonuclease.
3. Ligation of DNA fragments.
A recombinant DNA molecule is usually formed by cleaving the DNA of interest to yield insert DNA and
then ligating the insert DNA to vector DNA (recombinant DNA or chimeric DNA). DNA fragments are
typically joined using DNA ligase (also commercially produced).
4. Introduction of recombinant DNA into compatible host cells.
In order to be propagated, the recombinant DNA molecule (insert DNA joined to vector DNA) must be
introduced into a compatible host cell where it can replicate. The direct uptake of foreign DNA by a host
cell is called genetic transformation (or transformation). Recombinant DNA can also be packaged into
virus particles and transferred to host cells by transfection.
5. Replication and expression of recombinant DNA in host cells.
Cloning vectors allow insert DNA to be replicated and, in some cases, expressed in a host cell. The ability
to clone and express DNA efficiently depends on the choice of appropriate vectors and hosts.
6. Identification of host cells that contain recombinant DNA of interest.
Vectors usually contain some genetic markers, or genes, that allow the selection of host cells that have
taken up foreign DNA. The identification of a particular DNA fragment usually involves an additional
step—screening a large number of recombinant DNA clones. This is almost always the most difficult
step.
Some methods are:
• Selection of plasmid DNA containing cells
• Selection of transformed cells with the desired gene
• PCR detection of plasmid DNA
• Genetically Modified Organisms (GMOs)
Ways in which these plasmids may be introduced into host organisms:
❖ Biolistic. In this technique, a “gene gun” is used to fire DNA-coated pellets on plant tissues. Cells
that survive the bombardment, and are able to take up the expression plasmid coated pellets and acquire
the ability to express the designed protein.
❖ Plasmid insertion by Heat Shock Treatment. Heat Shock Treatment is a process used to transfer
plasmid DNA into bacteria. The target cells are pre-treated before the procedure to increase the pore sizes
of their plasma membranes. This pretreatment (usually with CaCl2) is said to make the cells “competent”
for accepting the plasmid DNA. After the cells are made competent, they are incubated with the desired
plasmid at about 4°C for about 30min. The plasmids concentrate near the cells during this time.
Afterwards, a “Heat Shock” is done on the plasmid-cell solution by incubating it at 42°C for 1 minute
then back to 4°C for 2 minutes. The rapid rise and drop of temperature is believed to increase and
decrease the pore sizes in the membrane. The plasmid DNA near the membrane surface are taken into the
cells by this process. The cells that took up the plasmids acquire new traits and are said to be
“transformed”.
❖ Electroporation. This technique follows a similar methodology as Heat Shock Treatment, but, the
expansion of the membrane pores is done through an electric “shock”. This method is commonly used for
insertion of genes into mammalian cells.
Applications of Recombinant DNA
• 1. Analysis of Gene Structure and Expression
• Using specialized recombinant DNA techniques, researchers have determined vast amounts of
DNA sequence including the entire genomic sequence of humans and many key experimental
organisms. This enormous volume of data, which is growing at a rapid pace, has been stored and
organized in two primary data banks:
• GenBank at the National Institutes of Health, Bethesda, Maryland,  and the EMBL Sequence
• Data Base at the European Molecular Biology Laboratory in Heidelberg, Germany.
• 2. Pharmaceutical Products – Drugs – Vaccines
Some pharmaceutical applications of DNA technology:  Large-scale production of human hormones and
other proteins with therapeutic uses  Production of safer vaccines  A number of therapeutic gene
products — insulin, the interleukins, interferons, growth hormones, erythropoietin, and coagulation factor
VIII—are now produced commercially from cloned genes.
• 3. Genetically modified organisms (GMO) – Transgenic plants – Transgenic animal Use of
recombinant plasmids in agriculture – plants with genetically desirable traits
• herbicide or pesticide resistant corn & soybean – Decreases chemical insecticide use – Increases
production
• “Golden rice” with beta-carotene – Required to make vitamin A, which in deficiency causes
blindness.
• Crops have been developed that are better tasting, stay fresh longer, and are protected from
disease and insect infestations.
• Disease resistance
• viruses, fungi, bacteria that cause plant diseases
• “Super-shrimp”
• Cold tolerance
Antifreeze gene from cold water fish introduced to tobacco and potato plants
 • Drought tolerance & Salinity tolerance
• As populations expand, potential to grow crops in otherwise inhospitable environments
• Application in medicine
• Human Gene Therapy
● Diagnosis of genetic disorders
• ● Forensic Evidence