1st SEMESTER | 2022-2023 SEPTEMBER 26, 2022

LESSON 6: DNA TECHNOLOGY

LECTURER: Mr. Stefan Paolo C. Jesalva

rDNA TECHNOLOGY PROCEDURE

TOPIC OUTLINE
➢ These are the procedures in the making
INTRODUCTION of recombinant DNA (rDNA):
rDNA TECHNOLOGY PROCEDURE
1. ISOLATING OF DNA
TECHNIQUES OF rDNA

APPLICATIONS OF rDNA

INTRODUCTION
What is Recombinant DNA technology?
➢ These are DNA molecules that are
extracted from different sources and
chemically joined together.
➢ E.g. DNA comprising an animal gene
may be recombined with DNA from a
bacterium.
DISCOVERY OF RECOMBINANT DNA
TECHNOLOGY
1953 Watson & Crick
DISCOVERY OF DNA STRUCTURE
1967 ISOLATION OF DNA LIGASE
1970 ISOLATION OF RESTRICTION IN THE
NUCLEASE (REase)
1972 Paul Berg
GENERATION OF rDNA TECHNOLOGY
1973 Cohen & Boyer
PRODUCTION OF THE FIRST PLASMID
VECTOR CAPABLE OF BEING
REPLICATED WITHIN A BACTERIAL
HOST
GOALS OF RECOMBINANT DNA TECHNOLOGY
o To isolate and characterize a gene
o To make desired alterations in one or
more isolated genes
o To return altered genes to living cells
o Artificially synthesize a new gene
o Alternating the genome of an organism
o Understanding the hereditary diseases
and their cure
o Improving human genome
- DNA can be isolated in several different
steps:
o Cells are broken using a
detergent which breaks open or
disrupts the cell or plasma
membrane. Cell contents such
as proteins and RNA are
degraded using protease for
proteins and RNAase or
ribonuclease for RNA. This
ensures that they do not
precipitate with the DNA at the
next step. This is very important
because we are just isolating or
extracting DNA. It should only
be purely DNA. Without a mix of
protein or RNA.
o The DNA is then turned into a
solid precipitate by adding cold
alcohol or ethanol.
o You can then isolate the solid
DNA using centrifugation to
collect the now solid DNA
which is found at the bottom of
the tube.
2. CUTTING OF DNA
- DNA can be cut into large fragments by
mechanical shearing.
- This is accomplished by the use of
restriction enzymes which are also

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 known as the “scissors of molecular - Over here you have your foreign DNA.
genetics.” This foreign DNA is what is going to be
- Restriction enzyme: inserted into your vector plasmid to form
o A special class the rDNA.
of sequence- - The cutting or the cleaving of the
specific enzyme. plasmid as well as the foreign DNA is
o It is found in catalyzed by the EcoR1 endonuclease
bacteria. which is a restriction enzyme that is
o These enzymes isolated by species of E.coli. Take note
are site-specific that in cleaving or cutting, there is only
meaning that they one restriction endonuclease enzyme
cleave DNA that is used for both the foreign DNA and
molecules only at specific nucleotide the plasmid vector.
sequence. - As you can see after the foreign DNA
o REases (restriction endonucleases) and REases have been cut, the
recognizes DNA base sequences that nucleotides of both match or are
are palindrome. complimentary with each other. That is
why our REases are site specific.
*palindrome – these are sites of the many
- After they have been cut, they will be
restriction enzymes that cut or restrict the DNA.
joined together through two processes:
o REases make staggered cuts with o Annealing
complementary base sequences for » This is a heat treatment
easy circulization. process that changes
the physical and
sometimes also the
chemical properties of
a material to increase
ductility and reduce
the hardness to make it
more workable.
» This is important
because it makes the
foreign DNA much
more ductile and
workable so that there
is easier and faster
circulization and
insertion into the
vector.
o DNA ligase
» Joined by this enzyme
in which this seals the
- This is an illustration showing the cleaving
foreign DNA fragment
or the cutting of the plasmid vector as
together with the
well as the foreign DNA and the joining
plasmid vector forming
to form the recombinant DNA.
your plasmid chimera
- Here is the plasmid vector. Vectors are
or hybrid DNA.
vehicles for delivering foreign DNA into
recipient cells. In this case, our vector is
a plasmid.
3. AMPLIFYING THE RECOMBINANAT DNA

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 - This is the step wherein the recombinant
DNA is added or inserted into a host cell.
- This is transforming the recombinant DNA
into a bacterial host strain.
- The host cells are treated with calcium
dichloride (CaCL2) and then the DNA is
added into the host cells.
- The cells are heat shocked at 42
degrees Celsius and the DNA goes into
the cell by a somewhat unknown
mechanism. VECTORS USED IN rDNA TECHNOLOGY
- Once the recombinant DNA is in the - A vector is an area of the DNA that can
cell, it will be replicated. When the cell
join another DNA but without losing the
divides, the replicated recombinant
limit for self-replication.
molecules will go to both the daughter
o it should be capable of
cells which themselves will divide later.
replicating in a host cell.
- Thus, the DNA is amplified.
o it should have convenient
OVERVIEW OF THE AMPLIFICATION:
restriction enzyme sites for
inserting DNA of interest.
o it should have a selectable
marker to indicate which host
cells received the recombinant
DNA molecule.
o it should be small and easy to
isolate
- There are a total of 6 different vectors
that are used:

- It starts with the cleaving or cutting of the o BACS (bacterial artificial chromosomes)
foreign DNA and its insertion into the » These are
plasmid vector forming the hybrid DNA bacterial plasmids
or recombinant DNA. derived from the F
- This rDNA is inserted into the host cell or plasmid which is a
host bacteria which is also known as conjugative plasmid
transformation of that cell or bacteria. that carries all the
Once that is completed, it follows the genes necessary for its
process of amplification which consists own transfer to another
of replication as well as cell division. bacteria.
- In the replication stage, the rDNA which » They are capable of
has been inserted will replicate followed carrying up to 300 kb of
by the division of that host cell or host DNA.
bacteria.
ENZYMES USED IN rDNA TECHNOLOGY

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 o YACS (yeast artificial chromosomes) o Lambda phage
» Lambda phage
vectors are
recombinant
infections, containing
the phage
chromosome in
addition to embedded
“outside” DNA.
» All in all, phage vectors
can convey bigger
» These are yeast vectors DNA groupings than
that have been plasmid vectors.
engineered to contain o Plasmid – commonly used vector
a centromere, » Plasmids are small,
telomere, origin of circular DNA molecules
replication, and a that are separate from
selectable marker. the rest of the
» they can carry up to chromosome.
1000 kb of DNA. » they replicate
» they are useful for independently of the
cloning eukaryotic bacterial chromosome
genes that contain » it is useful for cloning
introns. DNA inserts less than 20
o Expression vectors kb (kilobase pairs). This
» Expression is because inserts that
vectors are vectors are larger then 20 kb or
that carry host lost easily in the
signals that facilitate bacterial cell.
the transcription » If the DNA inserts are
and translation of an larger than 20 kb,
inserted gene. another type of vector
» they are very useful for should be used.
expressing new
eukaryotic genes in
bacteria TECHNIQUES
o Cosmid vectors - The following are techniques used in
» Cosmids are hybrids of rDNA technology aside from the
phages and plasmids procedure itself:
that can carry DNA ❖ Gel Electrophoresis
fragments up to 45 kb. » DNA fragments of
» they can replicate like different sizes can be
plasmids but can be separated by an
packaged like phage electrical field applied
Lambda. to a “gel.”
» the negatively
charged DNA migrates
away from the
negative electrode
and to the positive
electrode.

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 » the smaller the » This is accomplished by
fragment, the faster it digestion of the gene
migrates. singly with several
» The purpose of this enzymes and then in
technique is to detect combinations.
DNA and separate it by » The fragments are
its different sizes. subjected two gel
❖ Cloning libraries electrophoresis to
» A collection of the total separate the fragments
genomic DNA from a by size and the size are
single organism in a deduced based on the
certain vector. sizes of the fragments.
» the goal is to have ❖ PCR (Polymerase Chain
each gene Reaction)
represented in the » This allows the isolation
library at least once. of a specific segment
» The DNA is stored in a of DNA from a small
population of identical DNA or cell sample
vectors each using DNA primers at
containing a different the ends of the
insert of the DNA. segment of interest.
» It is genomic which ❖ Nucleic Acid Hybridization
means it is made from » Identify specific DNA
rDNA fragments of total sequences.
genomic DNA. » A southern blot allows
» It is also cDNA the detection of a
(complementary DNA) gene of interest by
which means it is made probing DNA fragments
from DNA synthesized that have been
from mRNA in a separated by
reaction that is electrophoresis with a
catalyzed by an “labeled” probe.
enzyme known as » the northern blot
reverse transcriptase. probes RNA on a gel
» The production of with a DNA probe.
cDNA involves the » the western blot probes
extraction and proteins on a gel with
purification of mRNA. an antibody.
These mRNA’s are used *blots – are the different types of formats that are
as a template for the used for this technique.
synthesis of cDNA by
the process of reverse ❖ DNA Microarrays
transcription. » This detects
❖ Restriction enzyme mapping expressions of
» Frequently it is thousands of genes all
important to have a at the same time and to
restriction enzyme site also determine the DNA
map of a cloned gene of a particular individual
for further or organism that contains imitations in its
manipulations of the genes.
gene.

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 » This attaches vast
majority of the protein-
encoding qualities
onto a microarray chip,
utilizing innovation in
light of the DNA silicon
chip industry.
» the chip can be utilized
to hybridize cell RNA
and measure the
statement rates of a
substantial number of
qualities in a cell.

APPLICATIONS OF rDNA
- 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.

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