BME 203 - Lecture No. 18-19

Lecture 18-19
Genetic Engineering
Recombinant DNA Technology
Maj Md. Ashrafuzzaman, PhD, EME

Copyright © 2010 Pearson Education, Inc.
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

What do we know?
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 could we do?
• 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
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.
Figure 9.1.2
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)
sticky ends blunt ends
• Prepackaged kits are available for rDNA techniques

Table: Selected Restriction Enzymes Used in rDNA Technology

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 NA
• Mammalian hosts

Hosts for DNA recombinant technology
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.
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. Protoplast fusion
– yeast, plants and algal cells
4. Microinjection
5. Gene gun

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
1. Plasmid vector contains the genes for:
ampR (ampicillin resistance)
Lac Z (β-galactosidase) gene
P O LacZ
cloning site
Cloning restriction site
• The cloning site (restriction enzymes site) is inserted into the β-galactosidase gene.
• Cloning the desired gene at that site destroys β-galactosidase gene.
P O Desired gene ~ Lac Z

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
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 30 cycles
…
….
Finished after 30 cycles – 1,073,741,842 molecules
Figure 9.4.1
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

Southern blotting

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

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
Site of insertion foreign DNA
Selection
GMO
• 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

Figure: Gene silencing could provide treatments for a wide range of diseases.
Nucleus
DNA
An abnormal gene,
cancer gene, or virus
gene is transcribed in a
host cell.
RNA
transcript
mRNA
siRNA
siRNA binds
mRNA.
Cytoplasm
RISC breaks
down the RNA
complex.
No protein
expression occurs.
Intro to DNA Cloning by Recombinant DNA
Methods
To study a gene, one must first prepare and purify its DNA in
relatively large amounts. This is accomplished via the
recombinant DNA (rDNA) technology method known as DNA
cloning. In cloning, a DNA molecule of interest is spliced into a
vector such as a bacterial plasmid or virus forming a rDNA
molecule which can be propagated in bacterial cells such as E.
coli. After replication and amplification of the rDNA in the
bacterium, it is purified for sequencing and other manipulations
used in gene characterization.

DNA Cleavage by Restriction Enzymes
Restriction enzymes are nucleases that are very important in rDNA
technology. These enzymes make double-stranded cuts in DNA molecules at
specific 4-8 bp palindromic (two-fold symmetrical) sequences called
restriction sites. Many restriction enzymes make staggered cuts in DNA
molecules resulting in single-stranded complementary sticky ends (Fig.).
Sticky-ended fragments can be readily joined together to synthesize rDNA
molecules (Fig.). In many cases, cleavage at the restriction site is blocked by
methylation of bases in the site.

Joining of DNA Molecules by Ligation
Plasmid vectors containing a
DNA of interest (e.g., genomic
DNA) can be readily constructed
by ligating restriction fragments to
vector DNA that has been
digested with the same restriction
enzyme (Fig.). Base-pairing
between the complementary
sequences of the sticky ends
aligns the fragments for covalent
linkage by a DNA ligase, typically
T4 DNA ligase. This enzyme uses
2 ATP to provide energy for
joining the 3'-hydroxyl and 5'-
phosphate groups of the base-
paired fragments together in 2
new 3'-5' phosphodiester bonds.
Note, all restriction enzymes
produce a 5'-phosphate and 3'-
hydroxyl group at the cut site.

E. coli Plasmid Cloning Vectors
Plasmids are autonomously replicating circular DNAs found in bacterial cells.
Naturally occurring plasmids contain an origin of replication (ori) for
propagation in the host cell and one or more genes that specify a trait that may
be useful to the host. Cloning vectors are plasmids that have been genetically
engineered to reduce unneeded DNA and to introduce selectable markers such as
antibiotic resistance genes (e.g., ampr) that are used to force cells to maintain the
plasmid. Polylinker sequences that encode several unique restriction sites for
cloning purposes also are engineered into these vectors (Fig.).

Cloning of DNA in Plasmid Vectors
An overview of the steps required for
DNA cloning in a plasmid vector is
presented in Figure. In Step 1, the DNA
1
of interest is ligated into a plasmid
cloning vector. In Step 2, the
recombinant plasmid is introduced into
E. coli host cells by transformation. In 2
Step 3, cells that have taken up the
plasmid are selected on antibiotic 3
(ampicillin) agar. In Step 4, the
transformed cells replicate their
chromosomal and plasmid DNA and
multiply to form a colony. Cells in the 4
colony contain the cloned DNA and are
themselves clones. The rDNA plasmid
then is harvested by growing a larger 4
culture of the cells.

Construction of cDNA Libraries (Part 1)
A genomic DNA library is a collection of cloned DNA fragments representing all
of the DNA of an organism. A cDNA library (complementary DNA), is a
collection of cloned DNA fragments corresponding to all mRNAs transcribed in a
certain tissue or organism. Libraries can be constructed using plasmid cloning
vectors. To construct a cDNA library, one begins by isolating mRNA from the cell
or tissue of interest (Fig.). Because many genes are transcribed at a low frequency,
it is best to start with a cell/tissue that expresses the
gene of interest at a relatively high

level. cDNAs are transcribed from a
mRNA template by a retroviral enzyme
known as reverse transcriptase (RT). In
Step 1, mRNA isolated by oligo-dT
affinity chromatography is hybridized
via its 3' poly(A) tail to an oligo-dT
primer. In Step 2, RT synthesizes the
first cDNA strand. In Step 3, RNA is
destroyed and a poly(dG) tail is added
by terminal transferase. In Step 4, the
cDNA is hybridized to an oligo-dC
primer. (Go to next slide).

In Step 5, a DNA polymerase
is used to synthesize the second
strand of the cDNA. In Step 6,
EcoRI sites that might be
present within the mRNA
coding region are protected by
methylation using EcoRI
methylase. In Step 7,
unmethylated EcoRI linkers,
that encode EcoRI restriction
sites, are ligated to the ends of
the fragment. In Step 8a, the
cDNA is cleaved with EcoRI
restriction enzyme, generating
sticky-ended cDNA fragments.
(See next slide).

In the last steps of cDNA library construction, the plasmid vector is cut with
EcoRI restriction enzyme (Step 8b), and then the EcoRI-cut cDNA and plasmid
are ligated together (Step 9). Finally, the E. coli host strain is transformed and
cells are plated (Step 10) on selective medium. To be complete, both genomic
and cDNA libraries for higher eukaryotes must contain on the order of a million
individual clones.

Screening cDNA Libraries
To screen a plasmid library (Fig.), colonies
representing each cloned DNA first are
plated on a number of petri plates. Library
DNA then is lifted onto nitrocellulose
membranes which serve as replicas of the
plates. Bound DNA is denatured and
hybridized with a radioactively-labeled
single-strand DNA probe (next slide). After
washing, spots corresponding to colonies
containing the DNA of interest are detected
by autoradiography. Because not all DNA
gets lifted onto the membranes, DNA for
the clone can be purified from the residual
colony on the original plate. Note, that
oligonucleotide probes must only be ~ 20
nucleotides long to recognize unique
sequences even in genomic DNA. The
probe sequence can be derived from
genome sequencing databases, or designed
based on the known sequence of a protein.

DNA Detection by
Membrane Hybridization
The general method for screening a
membrane-bound DNA sample for a
gene of interest is illustrated in Fig.
5.16. This involves fixation of single-
stranded DNA to the membrane,
hybridizing the fixed DNA to a
labeled DNA probe complementary
to the gene of interest, removal of un-
hybridized probe by washing, and
detection of the specifically
hybridized probe by
autoradiography, etc.

Construction of a Yeast
Genomic Library in a
Shuttle Vector
Plasmids known as E. coli-yeast shuttle
vectors (Fig.) can replicate in both
organisms. Shuttle vectors contain 1)
origins of replication for both species (ori,
E. coli; ARS, yeast), 2) markers for
selection in E. coli (ampr) and yeast
(URA3), and 3) a CEN sequence that
ensures stable replication and segregation
in yeast. The method for construction of a
yeast genomic library in a E. coli-yeast
shuttle vector is illustrated in Figure. A
total of ~105 clones is needed to include all
genes, if the genomic DNA is cut into
fragments of about 10 kb in length.

Screening by Functional Complementation
A yeast genomic library can be screened by the technique of
functional complementation to isolate the cloned version of a gene of
interest (Fig.). First, all recombinant plasmids from the library are
isolated from E. coli, pooled, and used to transform haploid ura3-
yeast that carry a conditional lethal ts copy of the gene of interest.
Transformants are selected by plating on uracil-deficient agar at the
permissive temperature. Second, transformants are replica plated onto
agar and incubated at the nonpermissive temperature to identify
colonies carrying a wild type version of the gene of interest. Only
cells containing the library copy of the wild type gene can survive at
high temperature.

Safety Issues and Ethics
• 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.

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Lecture 18-19

Maj Md. Ashrafuzzaman, PhD, EME

• The use of microorganisms, cells, or cell components to make

Copyright © 2010 Pearson Education, Inc.

• The process of natural selection allows the growth of those best

2. Microorganisms can exchange genes in a process of natural DNA

Copyright © 2010 Pearson Education, Inc.

• Mutation and selection

• Recombinant DNA Technology:

2. A desired gene is inserted into a DNA molecule

3. The vector inserts the DNA into a new cell,

4. Large quantities of the gene product can be

• Prepackaged kits are available for rDNA techniques

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

• DNA ligase has extensive use in molecular biology laboratories for

Copyright © 2010 Pearson Education, Inc.

4. Selectable marker – antibiotic resistance

Copyright © 2010 Pearson Education, Inc.

2. Viral vectors - retroviruses, adenoviruses and herpes viruses

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

5. Growing successfully the clone (transformed organisms).

Lac Z (β-galactosidase) gene

P O Desired gene ~ Lac Z

Copyright © 2010 Pearson Education, Inc.

3. The transformed cells are grown in the presence of:

• If the ligation was successful, the bacterial colony will be

1.Gene libraries are made of

3. Synthetic DNA is made by a

Copyright © 2010 Pearson Education, Inc.

• A short piece of labeled

• Polymerase Chain Reaction (PCR)

Copyright © 2010 Pearson Education, Inc.

1. Target DNA (template)

Cycle program in PCR machine:

• DNA sequencing - is the process of

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

• This provides tools for diagnosis and

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

• Natural genetic engineer

Copyright © 2010 Pearson Education, Inc.

• Genes for resistance to herbicide glyphosate, Bt toxin, and

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.

gene of interest at a relatively high

Copyright © 2010 Pearson Education, Inc.

Copyright © 2010 Pearson Education, Inc.