DNA Technology and
Genomics
What is biotechnology?
• Manipulation of organisms or their components to make
useful products
• Which of these are “biotechnology”?
• Use of microbes to make red wine or cheese
• Selective breeding of livestock
• Modify specific genes and move them between organisms
KWL
Write either something you know or something you want to know about biotechnology
on your sticky notes (with your initials) and place them on the board
• Know
• Want to Know
How does DNA control the cell??
You Must Know
• The terminology of biotechnology
• The steps in gene cloning with special attention to the
biotechnology tools that make cloning possible
• The key ideas that make PCR possible
• How gel electrophoresis can be used to separate DNA
fragments or protein molecules
Key Questions
1.
2.
3.
4.
5.
6.
7.
8.
What is biotechnology (or DNA technology), and what are some
practical applications that affect our lives?
How are restriction enzymes used to create recombinant DNA?
How are genes cloned? (hint: there are generally 5 steps)
What is PCR, what is it used for, and what are the steps of the
process?
What is gel electrophoresis, what is it used for, and what are the
steps of the process?
What are microarrays and what are they used for?
How is DNA technology used in diagnosis of disease, gene therapy,
pharmaceuticals, forensics, environmental work, and agriculture?
What important safety and ethical questions does DNA technology
raise?
Commonly Used Terms
o Genetic engineering – the process of manipulating genes and
genomes
o Biotechnology – the process of manipulating organisms or
their components for the purpose of making useful products
o Recombinant DNA – DNA that has been artificially made,
using DNA from different sources and often different species.
Example: introducing a human gene (like the insulin gene)
into an E. coli bacterium
o Gene cloning – the process by which scientists can produce
multiple copies of specific segments of DNA that they can
then work with in the lab
What is biotechnology?
• Manipulation of organisms or their
components to make useful products
• Modify specific genes and move them
between organisms
• We need to be able to cut out specific
genes and reinsert them somewhere else!
Restriction Enzymes
o Restriction enzymes – used to
cut strands of DNA at specific
locations called restriction sites;
derived from bacteria
 When DNA is cut by restriction
enzymes, the result will be a set
of restriction fragments which
will have at least one single
stranded end called a sticky end
 Sticky ends can form H-bonds
with complementary singlestranded pieces of DNA which
are sealed with DNA ligase
What is biotechnology?
• Manipulation of organisms or their
components to make useful products
• Modify specific genes and move them between
organisms
• Why would we want to modify, move, and clone
genes?
• Engineer bacteria to produce medicine or clean
oil spills; investigate the function of a gene (super
important in cancer research!); identify criminal
at crime scene; etc
Cloning Genes
Generally occurs in 5 steps
1.
Isolate the vector (plasmid) and gene-source DNA (ex: human gene).
The plasmid should include a gene for antibiotic resistance (for
example, ampicillin resistance denoted ampR).
Cloning Genes
2. Insert the DNA (human gene) into
the vector.
a)
Digest plasmid and human DNA with
same restriction enzyme (this
creates compatible sticky ends on
both the human DNA and the
plasmid)
b) Mix the fragments of human DNA
with the clipped plasmids; the sticky
ends of a plasmid base-pair with the
sticky ends of the human DNA
fragment
c) Join the DNA molecules using DNA
ligase
Cloning Genes
3. Introduce the cloning
vector (recombinant
plasmid) into the cells.
Plasmids are taken up by
bacteria by transformation
Cloning Genes
4. Clone the cells (and foreign genes)
and select for cells that have been
transformed.
• Transformed bacteria are plated on
solid nutrient medium containing the
antibiotic for which the plasmid
confers resistance (for example,
ampicillin) so that only bacteria that
have been transformed (those that
have taken up the plasmid with ampR)
can survive and reproduce.
• A reporter gene, such as green
fluorescent protein, may also be linked
to the plasmid to help identify
colonies of transformed bacteria.
• Each reproducing bacterium forms a
colony and in the process, the human
genes are also cloned.
Cloning Genes
5. Identification of cell clones carrying
the gene of interest (human gene).
The gene itself or the protein it
produces can be identified.
• Nucleic acid hybridization
• Nucleic acid probe
Cloning Genes
5. Identification of cell clones carrying
the gene of interest (human gene).
• Nucleic acid hybridization – the
method for detecting the DNA of a
gene directly using base pairing
between the gene and a
complementary sequence n another
nucleic acid molecule
• Nucleic acid probe – the short, single
stranded molecule of DNA or RNA that
is complementary to a portion of the
gene; making the probe radioactive or
fluorescent makes the gene easier to
track
Check for Understanding
Place the events involved in gene cloning in the correct order
A. Add DNA ligase to bond sticky ends
B. Isolate plasmid (vector) DNA and human DNA
C. Plate cells onto medium with antibiotic
D. Put plasmids into bacteria by transformation
E. Mix DNAs (plasmid and human gene)
F. Identify clone/colony containing gene of interest
G. Cut both DNAs with same restriction enzyme
Check for Understanding
The correct order is…
B. Isolate plasmid (vector) DNA and human DNA
G. Cut both DNAs with same restriction enzyme
E. Mix DNAs (plasmid and human gene)
A. Add DNA ligase to bond sticky ends
D. Put plasmids into bacteria by transformation
C. Plate cells onto medium with antibiotic
F. Identify clone/colony containing gene of interest
Key Questions
Working with your partner, discuss and write down answers to
the following questions:
1. What is biotechnology (or DNA technology), and what are
some practical applications that affect our lives?
2. How are restriction enzymes used to create recombinant
DNA?
3. How are genes cloned? (hint: there are generally 5 steps)
E. Coli Insulin Factory
• In this activity we will simulate the process of inserting a
human gene (insulin) into a bacterial plasmid which can then
be taken up by a bacteria, causing the bacteria to produce the
human gene product
Key Questions
1.
2.
3.
4.
5.
6.
7.
8.
What is biotechnology (or DNA technology), and what are some
practical applications that affect our lives?
How are restriction enzymes used to create recombinant DNA?
How are genes cloned? (hint: there are generally 5 steps)
What is PCR, what is it used for, and what are the steps of the
process?
What is gel electrophoresis, what is it used for, and what are the
steps of the process?
What are microarrays and what are they used for?
How is DNA technology used in diagnosis of disease, gene therapy,
pharmaceuticals, forensics, environmental work, and agriculture?
What important safety and ethical questions does DNA technology
raise?
PCR
• PCR (or polymerase chain reaction) is a
method used to greatly amplify a particular
piece of DNA in vitro without the use of cells
• PCR is used when the source of DNA is scanty
or impure
• crime scene
• ancient artifacts
PCR
• Starting material
• double stranded DNA containing
sequence that is “targeted” for
copying
• heat-resistant DNA polymerase
• supply of all 4 nucleotides
• supply of primers specific to
target sequences
PCR
1. Heat solution briefly to separate DNA strands
PCR
2. Cool to allow specific primers to H-bond
PCR
3. DNA polymerase adds nucleotides to 3’ of each primer
• Repeat – solution is
again heated, cooled
to allow primer
binding, and DNA
polymerase adds
nucleotides to 3’ of
each primer…
PCR
Key Questions
Working with your partner, discuss and write down answers to
the following question:
4. What is PCR, what is it used for, and what are the steps of
the process?
20.2 – DNA technology allows us to study the
sequence, expression, and function of a gene
• Genomics – the study of whole sets of genes and their
interactions
• Gel electrophoresis
• Southern blotting
• DNA microarray assays
Gel Electrophoresis
• Gel electrophoresis – a lab technique that is used to separate
macromolecules, primarily DNA and proteins, on the basis of
their size and charge with the use of an electric current
Gel Electrophoresis
• In separating DNA, the negative charges on the phosphates in the
molecule cause DNA to move toward the positive pole of the
electrophoresis set-up
• The gel allows smaller molecules to move more easily than larger
fragments of DNA, thus the fragments are separated by size
Gel Electrophoresis
1. Samples containing a mixture of DNA molecules of different sizes
are placed in wells near one end of the gel; electrodes are attached
to both ends and voltage is applied
2. The negatively charged DNA molecules migrate toward the positive
electrode (the anode); longer molecules travel slower, shorter
molecules travel more quickly and thus farther
3. When the current is turned off, the DNA molecules are arrayed in
“bands” along a “lane” according to their size
Southern Blotting
• Southern blotting – combines gel electrophoresis and
nucleic acid hybridization to allow researchers to find a
specific human gene; specific enough to differentiate
between alleles
• Ex: southern blotting can distinguish between a normal hemoglobin
allele and a sickle-cell allele
Southern Blotting
DNA Microarray Assays
• DNA microarray assays – used to detect and study the
expression of thousands of genes at one time
• ex – used to see differences in gene expression of a breast cancer
tumor cell and a noncancerous breast cell
DNA Microarray Assays
1. Small amounts of single-stranded DNA fragments representing
different genes are fixed to a glass slide in a tight grid which is
termed a DNA chip
DNA Microarray Assays
2. All mRNA molecules from the cell being
tested are isolated and converted to
cDNA by reverse transcriptase, then
tagged with a fluorescent dye
3. The cDNA bonds to the ssDNA (singlestranded DNA) on the chip, indicating
which genes are “on” in the cell (actively
producing mRNA); the dye alerts the
researcher
DNA Microarray Assays
20.3 – The practical applications of DNA
technology affect our lives in many ways
For the following applications, give at least one example of how the
technology is being used.
1. Diagnosis of disease
2. Gene therapy
3. Pharmaceuticals
4. Forensic applications
5. Environmental cleanup
6. Agricultural applications
Safety and Ethical Concerns
• List at least 3 major safety measures and concerns and at least 3
major ethical questions that arise from use of DNA technology.