rDNA Technology

Recombinant DNA
•
Technology
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
requirement, within different species i.e. DNA sequences that
would not normally occur together
• Recombinant DNA technology procedures by which DNA
from different species can be isolated, cut and spliced
together -- new "recombinant " molecules are then multiplied
in quantity in populations of rapidly dividing cells (e.g.
bacteria, yeast).
 Plasmids
• These are double stranded extrachromosomal DNA that are usually
circular and mostly found inside certain bacterial species e.g. E. coli.
• However most plasmids are now commercially available, ready to be
used, providing specific fragment insertion sites.
• Plasmids in genetic engineering are also known as ‘vectors’.
• Vectors also include viruses known as bacteriophage that use
bacteria as their host to replicate.
• Hence a bacteriophage can be used to transfect and create several
copies of the DNA fragment of interest by replicating several times in
a bacteria.
 Structure of Plasmids
Every plasmid has certain essential elements. These are as follows
•Origin of replication (OR) – This refers to a specific location in the strand
where the replication process begins. In plasmids, this region is A=T rich
region as it is easier to separate the strands during replication. 
•Selectable marker site – This region consists of Antibiotic resistance
genes which are useful in the identification and selection of bacteria that
contain plasmids. 
•Promoter region – this is the region where the transcriptional machinery
is loaded.
•Primer binding site – this is the short sequence of single-strand DNA
which is useful in DNA amplification and DNA sequencing.
•Multiple cloning sites – This site contains various sequences where the
restriction enzymes can bind and cleave the double stranded structure.
 Types of Plasmids

Based on the presence of the TRA gene plasmids can be

classified into two types:
1.Conjugative plasmids – these plasmids contain TRA
(transfer) gene and are commonly seen in bacteria.
2.Non-conjugative plasmids – these types of plasmids lack
the TRA genes.
Based on functions the plasmids can be classified into
the following types:
1. F Plasmids (Fertility plasmids)
•They contain the TRA genes and hence can be transferred from one cell
to another.
•They can replicate inside the bacterial cell.
•They cause the synthesis of a pilus, which is a long protein-rich
structure that helps in cell-cell interaction. 
2. R plasmid (Resistance plasmids)
•These plasmids contain and transmit genes for Antibiotic resistance
from one cell to another. 
•The antibiotic resistance gene protects the bacteria from antibiotics in
human medicines and antibiotics naturally present in the soil. 
•These types of plasmids are usually large in size and present in low copy
numbers in the cell. 
3. Col Plasmids (Colicin plasmids)
•These are known as bacteriocinogenic plasmids because
they produce bacteriocins. 
•These proteins have the ability to kill the closely related
bacterial cells which lack Col plasmids. 
•These plasmids are observed in E. coli.
4. Degradative Plasmids
•These types of plasmids have the ability to digest unusual substances
such as toluene, camphor, salicylic acid, etc. 
•The presence of these plasmids in the organism enables the breakdown of
various chemicals and substances. 
5. Virulence Plasmids
•These plasmids produce virulence factors that enable the bacteria to infect
other cells. Bacteria containing virulence plasmids are able to infect the
plant, animal, and human cells.
•Example – Ti plasmid is the virulence plasmid present in Agrobacterium
tumefaciens which causes crown gall disease in plants. 
 Functions and Applications of Plasmids
1. The important use of plasmids is that they can be used as vectors to insert
a specific gene into other organisms due to their capacity to incorporate a
gene and replicate inside the cell. 
2. They are an important factor in bacteria as they carry antibiotic resistance
genes. 
3. Degradative plasmids can be used to degrade industrial chemicals which
are a threat to the environment. 
4. As plasmids are easy to manipulate, they are being used in gene therapy
as well. 
5. Because plasmids are good vectors (a vehicle/factor which is used to
transfer a gene from one organism to another) they are used in drug
delivery and for hormone production in other cells. 
6. Plasmids are an important source of horizontal gene transfer. 
 Restriction endonucleases
 Bacteria also produce enzymes called restriction endonucleases
that cut DNA molecules at specific places into many smaller
fragments called restriction fragments.
 There are many different kinds of restriction endonucleases
 A restriction enzyme cuts only double-helical segments that
contain a particular sequence, and it makes its incisions only
within that sequence--known as a "recognition sequence”
 Sticky end and blunt end are the two possible configurations
resulting from the breaking of double-stranded DNA
 Restriction Enzymes and plasmid
•If two complementary strands of DNA are of equal
length, then they will terminate in a blunt end, as in
the following example:

• 5'-CpTpGpApTpCpTpGpApCpTpGpApTpGpCpGpTpApTpGpCpTpApGpT-3'

• 3'-GpApCpTpApGpApCpTpGpApCpTpApCpGpCpApTpApCpGpApTpCpA-
5'
 Restriction Enzymes and plasmid
•However, if one strand extends beyond the
complementary region, then the DNA is said
to possess an overhang:

•5'-ApTpCpTpGpApCpT-3'
•3'-TpApGpApCpTpGpApCpTpApCpG-5'
 Recombinant DNA
Technology
Restriction Enzymes and plasmid
•If another DNA fragment exists with a
complementary overhang, then these two
overhangs will tend to associate with each other
and each strand is said to possess a sticky end:
 Recombinant DNA
Technology
Restriction Enzymes and plasmid
•5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-
3'

•3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-
5'

Becomes
•5'-ApTpCpTpGpApCpT pGpApTpGpCpGpTpApTpGpCpT-3'

•3'-TpApGpApCpTpGpApCpTpApCpGp CpApTpApCpGpA-5'
 Recombinant DNA
Technology
Restriction Enzymes and plasmid
•Restriction Enzymes are primarily found
in bacteria and are given abbreviations
based on genus and species of the
bacteria.
•One of the first restriction enzymes to be
isolated was from EcoRI
•EcoR1 is so named because it was
isolated from Escherichia coli strain called
RY13.
 Recombinant DNA
Technology
Digestion of DNA by EcoRI to produce
cohesive
ends
 Recombinant DNA
Technology
Creating Recombinant DNA (Fig 3.2):
Selecting a
Gene in a
plasmid and
Antibiotic
selection.
Granulocyte colony stimulating factor (G-CSF)
Granulocyte Macrophage colony stimulating factor (GM-CSF