Recombinant DNA Technology

Biotechnology
• The use of microorganisms, cells, or cell components to make
a product
– Foods that are produced by the action of microorganisms
(bacteria and yeasts)
– Antibiotics
– Vitamins
– Enzymes

1. Mutation & Selection
– Genetic mutation and recombination provide a diversity of
organisms.
– The process of natural selection allows the growth of those
best adapted to a given environment.
2. Microorganisms can exchange genes in a process of natural DNA
recombination – genetic modification.
What do we know?

• Modern Biotechnology
– Selection
• Culture a naturally-occurring microbe that produces desired product (antibiotic producing
bacteria)
– Mutation and selection
• Mutagens cause mutations that might result in a microbe with a desirable trait (penicillin
produced by the fungus over 1000 times)
• Select and culture microbe with the desired mutation
– Gene modification
• Change a specific DNA bases ( change the corresponding codons) to change a protein
• Recombinant DNA Technology:
– Recombinant DNA - DNA that has been artificially manipulated to combine
genes from two different sources.
– Genes transferred - among unrelated species via laboratory manipulation.
– Genetic engineering - human manipulation of an organism's genetic material in
a way that does not occur under natural conditions
What could we do?

Figure 9.1.2
An Overview of Recombinant DNA Technologies
1. Gene of interest (DNA) is isolated
(DNA fragment)
2. A desired gene is inserted into a
DNA molecule - vector
(plasmid, bacteriophage or a viral genome)
3. The vector inserts the DNA into a
new cell, which is grown to form a
clone.
(bacteria, yeast, plant or animal cell)
4. Large quantities of the gene product
can be harvested from the clone.

Tools for Genetic engineering
1. Restriction Enzymes
• Naturally produced by bacteria – restriction endonucleases
– Natural function - destroy bacteriophage DNA in bacterial cells
– Cannot digest host DNA with methylated C (cytosine)
• A restriction enzyme
– Substrate –DNA -recognizes one particular nucleotide sequence in DNA
and cuts the DNA molecule (breaks down the bond between two
nucleotides)
• Prepackaged kits are available for rDNA techniques
sticky ends blunt ends

Table 9.1 Selected Restriction Enzymes Used in rDNA Technology

Figure 9.2
Restriction Enzymes
• Fragments of DNA produced by the same restriction enzyme will
spontaneously join by base pairing.
• Each of the DNA strands will have a break

2. Ligase
• DNA ligase is a enzyme that can link together DNA strands that
have double-strand breaks (a break in both complementary strands
of DNA).
– Naturally DNA ligase has applications in both DNA replication and DNA
repair .
– Needs ATP
• DNA ligase has extensive use in molecular biology laboratories
for genetic recombination experiments
ATP

3. Vectors
• Vectors - small pieces of DNA used for cloning (the gene to be
inserted into the genetically modified organism must be combined
with other genetic elements in order for it to work properly)
• Requirements of the Vector
1. Self-replication - able to replicate in the host (origin of
repliction)
2. Cloning site (site for recognition of restriction nucleases)
3. Promoter (and operator) - to support the gene (new DNA) expression in the
host
4. Selectable marker – antibiotic resistance
5. Proper size

Vectors
1. Plasmid vectors
– Plasmids are self-replicating circular molecules of DNA
– Encode antibiotic resistance ( selection marker)
2. Viral vectors - retroviruses, adenoviruses and herpes viruses
– Accept much larger pieces of DNA
– Mammalian hosts

1. Bacteria
- E. coli - used because is easily grown and its
genomics are well understood.
– Gene product is purified from host cells
2. Yeasts - Saccharomyces cerevisiae
– Used because it is easily grown and its genomics are known
– May express eukaryotic genes easily
– Continuously secrete the gene product.
– Easily collected and purified
3. Plant cells and whole plants
– Plants are easily grown - produce plants with new properties.
4. Mammalian cells
– Harder to grow
– Medical use.
Hosts for DNA recombinant technology

• The recombinant vector carrying foreign
DNA needs to be transferred into the
suitable host cells. Several methods have
been developed for introduction of
recombinant DNA molecule into host cells.
• According to types of vectors and host
cells, the methods are adopted. Some of
the methods of gene transfer into host
cells are briefly discussed below:

Insert the naked DNA into a host cell
1.Transformation
treatment make cells competent to accept foreign DNA (CaCl2 make
pores in cell membrane)
2. Electroporation
use electrical current to form microscopic pores in the membranes of
cell
3. Microinjection
4. Gene gun
Figure 9.5b

1. Transformation :
• Introduction of rDNA molecules into a
living cell is called transformation. The
DNA molecule comes in the contact of cell
surface. Then it is taken up by the host
cells.
• However, in nature the frequency of
transformation of many cells (e.g. yeast
and mammalian cells) is very less.
Secondly, all the time host cells do not
undergo transformation. Because they are
not prepared for it.

• A temperature sock of either low
temperature (0-5°C) or high temperature
(37- 45°C) is required for transformation.
Mandel and Higa (1970) reported that E.
coli cells become competent to uptake
DNA by treating cells with ice cold
CaCl2 and exposing the cells to 42°C for
about 90 seconds.

2. Transduction :
• The action or process
of transducing; especially : the transfer of
genetic material from one microorganism
to another by a viral agent (such as a
bacteriophage)

3. Electro oration (Electric
Field-mediated Membrane
Permeation) :
• In electroporation an electric current at high
voltage (about 350 V) is applied in a solution
containing foreign DNA and fragile host cells.
This creates transient microscopic pores in cell
membrane of naked protoplasts.
• Consequently foreign DNA enters into the
protoplast through these pores. The transformed
protoplasts are cultured in vitro which
regenerate respective cell walls.

4. Microinjection :
• In this technique foreign DNA is directly and forcibly
injected into the nucleus of animal and plant cells
through a glass micropipette containing very fine tip of
about 0.5 mm diameter.
• It resembles with injection needle. In 1982, for the first
time Rubin and Spradling introduced Drosophila gene
into P-element and microinjected into embryo. In 1982,
R.D. Palmiter and R.L.
• Brinter introduced rabbit recombinant pBR322 containing
a growth hormone gene into mature embryo of mouse in
vitro. The microinjected embryos were transferred into
uterus of mice which gave birth to transgenic mice.

5. Particle Bombardment Gun
(Biolistics) :
• This technique was developed by Stanford
in 1987. In this method macroscopic gold
or tungusten particles are coated with
desired DNA. A plastic micro-carrier
containing DNA coated gold/tungusten
particles are placed near rupture disc.
• The particles are bombarded onto target
cells by the bombardment apparatus.
Consequently foreign DNA is forcibly
delivered into the host cells.

CALCIUM PHOSPHATE MEDIATED DNA TRANSFER
• The process of transfection involves the admixture of isolated DNA
(10-100ug) with solution of calcium chloride and potassium
phosphate under condition which allow the precipitate of calcium
phosphate to be formed.
• Cells are then incubated with precipitated DNA either in solution or
in tissue culture dish. A fraction of cells will take up the calcium
phosphate DNA precipitate by endocytosis.
• Transfection efficiencies using calcium phosphate can be quite low,
in the range of 1-2 %. It can be increased if very high purity DNA is
used and the precipitate allowed to form slowly.

Limitations of calcium phosphate mediated DNA transfer
•Frequency is very low.
•Integrated genes undergo substantial modification.
•Many cells do not like having the solid precipitate adhering
to them and the surface of their culture vessel.
•Due to above limitations transfection applied to somatic gene
therapy is limited.

Recombinant DNA technology - Cloning
A process of producing genetically modified organisms
A multi-step process.
1. Isolating and copying the genetic material of interest (DNA
fragment ).
2. Building a construct (recombinant DNA - vector and desired gene)
containing all the genetic elements for correct expression.
3. Inserting the vector into the host organism, directly through
injection or transformation.
4. Selecting the cells expressing that gene by growing under positive
selection (of an antibiotic or chemical) – clone .
5. Growing successfully the clone (transformed organisms).

Blue-white screening system
2. The vector is then transformed into host competent cell
(bacteria).
• Host is sensitive to ampicillin
• Host is β-galactosidase negative (do not carry LacZ gene)
3. The transformed cells are grown in the presence of:
• ampicillin.
• X-gal – substrate for β-galactosidase
– a colourless modified galactose sugar
– When metabolized by β-galactosidase form an insoluble
product (5-bromo-4 chloroindole) which is bright blue, and
thus functions as an indicator
4. Results
– Clones lacking the vector will not grow.
– Clones containing the vector without the new gene will be resistant to
ampicillin, able to metabolized X-gal and will be blue.
– Clones containing the recombinant vector will be resistant to
ampicillin and unable to hydrolyze X-gal (white colonies).
• If the ligation was successful, the bacterial colony will be
white; if not, the colony will be blue.

Obtaining DNA – gene of interest
1.Gene libraries are made of pieces of
an entire genome stored in plasmids
or phages
2. cDNA (complementary DNA) is made
from mRNA by reverse transcriptase
(enzyme found in retroviruses)
- Intron free DNA
3. Synthetic DNA is made by a DNA synthesis machine

Screening for the desired Gene
• Identify the particular cell that contains the specific gene of interest
(Presence of the vector with correct gene of interest)
• A short piece of labeled
DNA called a DNA probe
can be used to identify clones
carrying the desired gene.
• Radioactive labeled
• Fluorescent labeled
Labeled DNA probe ( 32P or fluorescence)
5’ *AGGCTTGTACTTTGGCGG 3’

Copying the genetic material of interest - PCR
• Polymerase Chain Reaction (PCR)
– A reaction to make multiple copies of a piece of DNA enzymatically
• Polymerase – enzyme is DNA polymerase from Thermus aquaticus – Taq
polymerase
– Taq's optimum temperature for activity is 75-80°C
– Can replicate a 1000 base pair strand of DNA in less than 10 seconds at
72°C
• Chain – chain of cycles of multiplication
DNA-
Dissociation temperature
Hybridization temperature

PCR amplify DNA to detectable levels
Figure 9.4.1
PCR reaction mixture :
1. Target DNA (template)
2. Short primers- to hybridize to the 5’ end
of each DNA strand
3. four NTP – ATP, GTP, TTP, and CTP
4. Buffer
5. DNA Taq Polymerase (enzyme)
Cycle program in PCR machine:
1. Denaturation -95ºC
2. Annealing (hybridization)- 60-65 ºC
3. Polymerase reaction -72 ºC
Product
Start with 1 molecule
First cycle – 2 molecules
Second cycle – 4 molecules
…
….
Finished after 30 cycles – 1,073,741,842 molecules
30 cycles

DNA sequencing
• DNA sequencing - is the process of
determining the precise order of nucleotides
within a DNA molecule
( A, G, C and T in a molecule of DNA)

Applications of recombinant DNA technology
1. Scientific applications
– Many copies of DNA can be produced
– Increase understanding of DNA
– Identify mutations in DNA
– Alter the phenotype of an organism
– Bioinformatics is the use of computer applications to study
genetic data;
– Proteomics – proteomics is the study of a cell’s proteins.
• determination of all the proteins expressing in the cell

• Shotgun sequencing - Recombinant DNA techniques were used
to map the human genome through the Human Genome Project
- has 24 distinct chromosomes (22 autosomal + X + Y)
- with a total of approximately 3 billion DNA base pairs
– containing an estimated 20,000–25,000 genes
– with only about 1.5-2% coding for proteins
– the rest comprised by RNA genes, regulatory
sequences, introns and controversially so-called
junk DNA
– This provides tools for diagnosis and
possibly the repair of genetic diseases

2. Diagnose genetic disease
– RFLP analysis (Restriction fragment
length polymorphism)
• DNA profiling involved restriction
enzyme digestion, followed by
Southern blot analysis
– Southern blotting is used for
detection of a specific DNA sequence
in DNA sample
• DNA probes can be used to quickly
identify a pathogen in body tissue or
food.
– PCR analysis with specific primers

3. Recombinant DNA techniques can be used to for genetic
fingerprinting identification
• Forensic microbiology - use
DNA fingerprinting to identify
the source of bacterial or viral
pathogens.
– bioterrorism attacks (Anthrax in U.S. Mail)
– medical negligence (Tracing HIV to
a physician who injected it)
– outbreaks of foodborne diseases
Figure 9.16

4. Agricultural Applications
– Cells from plants with desirable characteristics can be cloned to
produce many identical cells, then can be used to produce whole plants
from which seeds can be harvested.
– Some bacteria can transfer genes to unrelated species
• Agrobacterium tumefaciens - a plant pathogen
• Cause tumors in plants
• Natural genetic engineer

Genetic engineering manipulation
GMO
Site of insertion foreign DNA
Selection
• Genes for resistance to herbicide glyphosate, Bt toxin, and
pectinase suppression have been engineered into crop plants.
• Genetically modified Rhizobium has enhanced nitrogen
fixation.
• Genetically modified Pseudomonas is a biological insecticide
that produces Bacillus thuringiensis toxin.

5. Nanotechnology
• Bacteria can make molecule-sized particles
– Bacillus cells growing on selenium form chains of elemental selenium

6. Therapeutic Applications
– Produce human proteins – hormones and enzymes
• Insulin
• hGH
• INFα, INFβ and INFγ
– Vaccines
• Cells and viruses can be modified to produce a pathogen’s surface protein
– Influenza
– Hepatitis B
– Cervical cancer vaccine
• Nonpathogenic viruses carrying genes for pathogen’s antigens as DNA
vaccines
• DNA vaccines consist of circular rDNA
– Gene therapy can be used to cure genetic diseases by replacing
the defective or missing gene.
– Gene silencing – RNA interference - siRNA or microRNA

• Accidental release - strict safety standards are used to avoid it
– Some microbes used in recombinant DNA cloning have been altered so
that they cannot survive outside the laboratory.
– Microorganisms intended for use in the environment may be modified to
contain suicide genes so that the organisms do not persist in the
environment.
• Ethical questions
– Should employers and insurance companies have access to a person’s
genetic records?
– Will some people be targeted for either breeding or sterilization?
– Will genetic counseling be available to everyone?
• GMO - Genetically modified crops must be safe for consumption
and for release in the environment.
Safety Issues and Ethics

• Compare and contrast biotechnology, genetic modification, and recombinant DNA.
• Compare selection and mutation.
• Identify the roles of a clone and a vector in making recombined DNA.
• Define restriction enzymes, and outline how they are used to make recombinant DNA.
• Outline the steps in PCR and provide an example of its use.
• Describe how a gene library is made
• Differentiate cDNA from synthetic DNA.
• List the properties of vectors.
• Describe the use of plasmid and viral vectors.
• Describe five ways of getting DNA into a cell.
• Explain how each of the following are used to locate a clone: antibiotic-resistance
genes, DNA probes.
• List one advantage of engineering the following: E. coli, Saccharomyces cerevisiae,
mammalian cells, plant cells.
• Discuss the value of the Human Genome Project.
• Define the following terms: random shotgun sequencing, bioinformatics, proteomics.
• Diagram the Southern blot procedure and provide an example of its use.
• Diagram DNA fingerprinting and provide an example of its use.
• List the advantages of, and problems associated with, the use of genetic modification
techniques.
Learning objectives

Recombinant DNA Technology

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