Biotechnology and DNA

Technology
 Biotechnology
Use of microorganisms, cells, or cell components to
make a product
Uses
Food
Vaccines
Antibiotics
Vitamins
Mining
Typical genetic modification procedure
 Recombinant DNA Technology
Genetic engineering
Recombination of DNA occurs naturally in microbes
In the lab
DNA from vertebrate animals can be combined with DNA from
bacteria or viral gene in yeast
Uses
Recipient can express gene to make protein
Insulin production
Vaccine for hepatitis virus
Molecules of DNA can be amplified
Generating sufficient DNA for experimentation, analysis, and
identification of organisms
 Biotech tools: selection
Artificial selection is used to pick desirable traits
Animals
Plants
Microorganisms
 Biotech tools: mutation
Antibiotic resistance and mutations
New strains of antibiotic-producing microbes could
be created using mutagens
Random mutations were introduced to penicillin-
producing Penicillium via radiation
Able to increase penicillin production by over 1000
times
Site-directed mutagenesis
Targeted mutations used to make a specific change
in a gene
 Biotech tools: restriction enzymes
Restriction enzymes: special class of DNA-
cutting enzymes that exist in many
bacteria
Used to limit bacteriophages by
hydrolyzing phage DNA
Bacterial DNA is protected from
digestion by methylation of some
cytosines in DNA
Types of cuts
Blunt ends: cuts both strands in the
same place
Sticky ends: cuts strands to form
staggered ends
Need to digest one particular sequence of
nucleotide bases in DNA
Restriction enzymes recognize a
unique 4-8 nucleotide base sequence
Restriction enzymes and rDNA
 Biotech tools: vectors
A DNA molecule that transport foreign DNA into a cell
Must be self-replicating once in a cell
Smaller vectors are more easily manipulated than large vectors
Circular vectors allow it to be preserved instead of degraded in a recipient cell
Virus vectors can insert themselves quickly into the hosts chromosome
Shuttle vectors can exist in several different species
Can be used to move cloned DNA sequences among organisms
To identify a cell containing a vector, selectable marker genes can be used
Antibiotic resistance
Enzymes that perform reactions
Genes that express visible proteins
Types of vectors
Plasmids are one of the primary vectors used
Beneficial for modifying multicellular organisms
Viral DNA vectors can usually accept much larger foreign DNA than plasmids
Retroviruses, adenoviruses, and herpesviruses are being used to insert
corrective genes into human cells as gene therapy
 Biotech tools: polymerase chain
reaction (PCR)
Technique by which small samples of DNA can be quickly amplified thereby
increasing quantities so theres a large enough amount of DNA for analysis
Process
Each strand of target DNA serves as a template
Nucleotides and DNA polymerase (from Thermus aquaticus) are added
to facilitate synthesis
Primers (short pieces of nucleic acid) are used to start the reaction by
binding to complementary portions of the target DNA/gene
After each cycle of synthesis, DNA is heated to denature the strands
Each new strand can now serve as a template
Thermal cycler will repeat approximately 30 cycles of denaturing,
annealing, and elongation to yield more than a billion times more DNA
Products can be visualized by gel electrophoresis
PCR
 Biotech tools: polymerase chain
reaction (PCR)
Real-time PCR or quantitative PCR
Newly made DNA is tagged with fluorescent dye
Levels of fluorescence is measured after every PCR cycle
Quantitates amount of DNA after every cycle
Reverse-transcription PCR
Uses viral RNA or cells mRNA to make template
Can use reverse transcriptase to synthesize complementary DNA (cDNA)
mRNA contains exons and lacks introns so specific protein product can be made
 Genetic modification: insertion of foreign
DNA into cells

Transformation: cells can take up DNA from the surrounding environment

Competent cells: calcium carbonate treatment and heat shock
Electroporation: electrical current used to form microscopic pores that allow
DNA to enter
For cells with cell walls, they are usually converted to protoplasts (cells
with enzymatically removed cell walls) before electroporation
Protoplast fusion: fusion of protoplasts to form hybrid cells
Gene gun: shoot microscopic particles of DNA-coated metal particles using
helium
Microinjection: uses a glass micropipette to puncture the plasma membrane
and inject DNA into the cell
 Genetic modification: obtaining DNA
Genomic libraries: collection of clones
containing different DNA fragments
 Genetic modification: obtaining DNA
Synthetic DNA
Sequence of nucleotides is typed into a DNA
synthesizing machine
You must know the sequence
Already known
Predicted by generating the nucleotide sequence
based on the amino acids in the protein
Rare to clone a gene by synthesis
 Genetic modification: selecting a clone
Blue-white screening
Uses an ampicillin resistance gene
Host bacterium cannot grow on test
medium if the cell has not acquired
the vector
Uses -galactosidase enzyme (lacZ)
Host bacterium is grown on medium
with X-gal, which is the substrate for
-galactosidase
Results
Ampicillin-resistant bacteria will grow
Bacteria with the recombinant plasmid
will have the gene of interest inserted
into the lacZ gene
White colonies: have recombinant
plasmid: cannot hydrolyze X-gal (no
functional lacZ)
Blue colonies: dont have
recombinant plasmid: hydrolyze X-
gal (functional lacZ)
 Genetic modification: selecting a clone
Colony hybridization: common method of identifying cells that carry a
specific cloned gene
DNA probes: short segments of single-stranded DNA that are
complementary to the desired gene
DNA probes are labeled with enzyme or fluorescent dye
 Genetic modification: making a gene
product
Bacteria can be used to synthesize gene products
Easy to grow
Example : E.coli
Disadvantages
Endotoxins
Some bacteria (like Gram negatives) dont usually secrete
protein products
Yeast: may be more successful in expressing foreign eukaryotic
genes
Mammalian cells: best for producing proteins for medical use
Plant cells: can generate genetically modified plants
Can make therapeutic agents like painkillers and vaccines
Used to make synthetic rubber and melanin for sunscreens
 Applications: therapeutic
Proteins as products
Insulin: using rDNA to synthesize two short
polypeptide chains and chemically joined
together
Somatostatin: made in E. coli
Subunit vaccine
Protein portion of a pathogen made by
genetically modifying yeast
Vaccine cant cause infection
Animal viruses (like vaccinia virus) genetically
modified to carry a gene for another microbes
surface protein
DNA vaccines: usually circular plasmids that
include genes encoding a viral protein, controlled
by a human promoter region, and cloned in
bacteria
Acts as vaccines against HIV, influenza,
hepatitis, dengue
 Applications: therapeutic
Gene therapy: transforming human cells with a
normal gene to replace defective/ mutated gene
Can use adenovirus or retrovirus to deliver
genes
Treat hemophilia and severe combined
immunodeficiency
Gene silencing: natural process that occurs in
many eukaryotes and is used to defend against
viruses and transposons
Small interfering RNAs (siRNA)
Processed by the enzyme dicer
siRNA bind to mRNA
mRNA then can be destroyed by RNA-
induced silencing complex (RISC)
RNA interference (RNAi): small DNA insert
encoding siRNA against a gene of interest
Allows cells to produce own siRNA
Used to treat macular degeneration and
melanoma
 Applications: genome studies
Shotgun sequencing
Small pieces of genome of a free living cell
are sequenced to find the order of
nucleotides in the DNA
Sequences are assembled using a computer
Any gaps between pieces have to be found
and sequenced
Metagenomics: the study of genetic
material taken directly from environmental
samples
Human genome project
Started in 1990 and ended in 2003
Sequenced the entire human genome (~ 3
billion nucleotide pairs coding for 20,000 to
25,000 genes)
Less than 2% of the genome codes for
functional products
98% codes for miRNA, viral remnants, short
tandem repeats, introns, telomeres, and
transposons
 Applications: scientific
Bioinformatics: understanding the function of genes through computer-assisted
analysis
Proteomics: determining all of the proteins expressed in a cell
Reverse genetics: discovering the function of a gene from genetic sequencing by
mutating or blocking a gene to determine function
Genetic testing: used to screen for several hundred genetic diseases
Southern blotting: sequence of a mutated gene can be used as a diagnostic tool
using this technique
RFLPs or restriction fragment length polymorphism: DNA digested with
restriction enzymes
Fragments are separated by gel electrophoresis
RFLPs are transferred onto a filter by blotting
Exposed to a labeled probe made from the cloned gene of interest
Probe will hybridize to mutant but not normal gene
Fragments to which the probe bins are identified by a colored dye
Application: scientific- southern blotting
 Applications: scientific
DNA fingerprinting: identify bacterial or viral pathogens
DNA chips or PCR microarrays are used
22 primers from different microorganisms can be used in PCR
Forensics microbiology
Used to identify when microorganisms are used as weapons
Used in hospitals and food manufacturers
Nanotechnology: design and manufacture of extremely small
electronic circuits and mechanical devices built at the molecular level
Molecule-sized robots or computers can be used to detect
contamination in food, diseases in plants, and biological weapons
Bacteria used to produce nanospheres for potential drug targeting
and delivery
 Applications: agricultural
Ti plasmid from Agrobacterium tumefaciens is introduced into a plant
Ti plasmid cause the formation of crown gall (tumorlike growth)
T-DNA (part of the Ti plasmid) stimulates cellular growth and the production
of certain products
Can be used to expressed recombinant DNA
Uses
Herbicide resistance
Pesticide resistance
Weather resistance
Antisense DNA
DNA complementary to mRNA is synthesized
Antisense DNA is taken up by the cell and binds to mRNA to inhibit
translation
DNA-RNA hybrid is broken down
Agricultural uses
Increase nitrogen production in crops for growth
Animal husbandry
 Ethics
Safety
Control
Suicide genes
Toxicity
Genetic counseling
Other effects on human life?
 Self quiz 1
The DNA probe, 3-GGCTTA, will hybridize with
which of the following?
A. 5 CCGUUA
B. 5 CCGAAT
C. 5 GGCTTA
D. 3 CCGAAT
E. 3 GGCAAU

B
 Self quiz 2
When selecting for a clone, if you successfully
transformed bacteria with recombinant plasmids
the bacteria cannot hydrolyze X-gal, hence your
colony will be:
A. White
B. Blue
C. Clear
D. Purple
E. Green
A
 Self quiz 3
________ is a technique to determine the order
of nucleotides in a fragment of DNA.

A. PCR
B. Sequencing
C. Electrophoresis
D. Nucleic acid hybridization