•
Describe the structure and function of DNA and explain how
proteins are made
•
Differentiate between eukaryotic and prokaryotic cells and
chromosome structure and explain how this difference impacts
gene regulation in the two cell types
•
Differentiate between bacterial cultures grown in liquid and
solid media and explain how to prepare each type using sterile
technique
•
Discuss the characteristics of viruses and their importance in
genetic engineering
•
Explain the fundamental process of genetic engineering and
give examples of the following applications: recombinant DNA
technology, site
-
specific mutagenesis, and gene therapy
•
Describe the process of gel electrophoresis and discuss how the
characteristics of molecules affect their migration through a gel
The manipulation of genetic
information, specifically the
deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) codes, is the
center of most biotech research and
development.
The genetic information within cells is
stored in
DNA
Before we get into DNA lets review
Cells!
A Review of Cell Structure
Online activity on cells:
http://learn.genetics.utah.edu/content/begin/cells/
There are
two major types of cells
Prokaryotic Cells
Eukaryotic Cells
Key Terms to Remember!
Plasma Membrane
–
double
-
layer structure
of lipids and proteins that surrounds the
outer surface of cells
Cytoplasm
–
inner contents of a cell between
the nucleus and plasma membrane
Organelles
–
structures in the cell that
perform specific functions
•
Prokaryotic Cells
(include bacteria)
–
No nucleus and no organelles
Have a nucleus and many organelles
Organelles
Nucleus
Mitochondria
Endoplasmic reticulum
Golgi apparatus
Vero cells
are a cell line which is widely used to make
vaccines. The cell line was derived from kidney epithelial cells of
the African Green Monkey. The cell line was established in 1962 by
Japanese scientists. Vero cells are susceptible to a broad range of
viruses so they are used to develop vaccines diseases associated
with those viruses. One of the vaccines for which Vero cells have
been used in recent years is the vaccine against influenza ("the
flu").
HeLa Cells
cells are the first immortal cell line, or a cell line that
continues to reproduce and "live" outside the human body. These
cells have been used in cell research in projects that have benefited
mankind around the world. The original cells were taken from
cancerous cervical tumor from a poor African
-
American woman
named Henrietta Lacks who died from cervical cancer in 1951.
Escherichia coli
(commonly abbreviated
E. coli)
is a gram
negative, rod shaped bacteria that is commonly found in the lower
intestine of warm blooded organisms.
Most E. coli strains are harmless, but some can cause serious food
poisoning in humans, and are occasionally responsible for product
recalls. The harmless strains are part of the normal flora of the gut.
E. coli are not always confined to the intestine, and their ability to
survive for brief periods outside the body makes them an ideal
indicator organism to test environmental samples for fecal
contamination.
The bacteria can also be grown easily and its genetics are
comparatively simple and easily manipulated or duplicated
through a process of metagenetics, making it one of the best
-
studied prokaryotic model organisms, and an important species in
biotechnology and microbiology.
Chinese hamster ovary cells or CHO
cells
are cells that have been derived from the ovary of
the Chinese hamster. In year 1957, scientist T.
Puck at Dr. George Yerganian's laboratory at the
Boston Cancer Research Foundation used a female
Chinese hamster to extract this cell line. Today, it
is a widely used mammalian cell line in biological
research since its introduction in the year 1960.
One of the characteristics of CHO cells is that it
requires the amino acid “Proline” for its growth
and it’s an adherent monolayer cell line.
A rapid growing cell line with excellent ability to
express recombinant protein makes it the cell line
of choice in experiments relating to gene
expression, genetics, toxicity screening and
nutritional studies.
Viruses
are particles of
nucleic acid, protein, and in
some cases, lipids.
Viruses can reproduce
only
by infecting living cells
.
Viruses differ widely in
terms of size and structure.
As different as they are, all
viruses have one thing in
common: They enter living
cells and, once inside, use
the machinery of the infected
cell to produce more viruses.
A
virus's protein coat
is
called its
capsid
.
The capsid includes
proteins that enable a virus
to enter a host cell.
The capsid proteins of a
typical virus bind to
receptors on the surface of
a cell and “trick” the cell
into allowing it inside.
Once inside, the viral genes
are expressed.
The cell transcribes and
translates the viral genetic
information into viral
capsid proteins.
Sometimes that genetic
program causes the host
cell to make copies of the
virus, and in the process
the host cell is destroyed
Because viruses must bind precisely to
proteins on the cell surface and then use a
host's genetic system, most
Viruses are highly specific to the cells they infect.
Plant viruses infect plant cells
Most animal viruses infect only certain related
species of animals
Bacterial viruses infect only certain types of
bacteria.
Viruses that infect Bacteria are called
b
acteriophages
.
To View how a virus attacks watch this video!
Learn the Basics Website
:
http://learn.genetics.utah.edu/content/begin/tour/
Building block of DNA is the
nucleotide
Each nucleotide is composed of
Pentose (5
-
carbon)
sugar
called deoxyribose
Phosphate
molecule
A
nitrogenous base
The nitrogenous bases are the interchangeable
component of a nucleotide
Each nucleotide contains one base
Adenine (A), thymine (T), guanine (G) or
cytosine (C)
–
Nucleotides are joined together to
form long strands of DNA and each
DNA molecule consists of two strands
that join together and wrap around
each other to form a
double helix
–
Nucleotides
in a strand
are held
together by
phosphodiester bonds
–
Each strand has a
polarity
–
a 5’ end
and a 3’ end
•
The two strands of a DNA molecule are held together by
hydrogen bonds
–
Formed between complementary base pairs
–
Adenine (A) pairs with thymine (T)
–
Guanine (G) pairs with cytosine (C)
–
The two strands are
antiparallel
because their polarity is
reversed relative to each other
Chromosome Structure
Chromosomes
–
highly coiled and tightly
condensed package of DNA and proteins
Occurs only during DNA replication
Chromatin
–
strings of DNA and DNA
-
binding
proteins called histones
State of DNA inside the nucleus when the cell is
NOT dividing
Most human cells have two sets (pairs) of
23 chromosomes, or 46 chromosomes total
Called
homologous pairs
Autosomes
–
chromosomes 1
-
22
Sex chromosomes
–
chromosome pair # 23
X and Y chromosomes
Gametes
(sex cells)
contain a single set of
23 chromosomes (haploid number, n)
DNA Replication
Cells divide by a process called
mitosis
Sex cells divide by a slightly different process called
meiosis
Mitosis
One cell divides to form two daughter cells, each with
an identical copy of the parent cell DNA
In order to accomplish this, the DNA of the parent cell
must be copied prior to mitosis
DNA undergoes
Semiconservative Replication
Replication occurs in such a manner that, after
replication, each helix contains one original
(parental) DNA strand and one newly synthesized
DNA strand
1.
Unwinding the DNA
Helicase
enzyme breaks the hydrogen bonds
holding the two DNA strands together;
“unzips” DNA
DNA binding proteins hold the strands apart
Separation of strands occurs in regions called
origins of replication
2.
Adding short segments of RNA
Primase
enzyme adds RNA primers
RNA primers start the replication process
3. Copying the DNA
DNA polymerase enzyme binds to the
RNA primers
Uses nucleotides to synthesize
complementary strands of DNA
Always works in one direction
–
5’ to 3’
direction
Occurs only in
genes
RNA polymerase
unwinds DNA helix and
copies one strand of DNA into RNA
Binds to a
promotor
region
Copies DNA in a 5’ to 3’ direction into RNA
Uses nucleotides
Adenine,
uracil
, guanine, and
At end of gene, RNA polymerase
encounters the
termination sequence
RNA polymerase and newly formed
strand of RNA are released from DNA
molecule
RNA strand is called a
messenger RNA
(mRNA)
Multiple copies of mRNA are transcribed
from each gene during transcription
The mRNA then gets processed
Initial mRNA produced is the primary
transcript
Immature and not fully functional
A series of modifications before
primary transcripts are ready for
protein synthesis
RNA splicing
Polyadenylation
Addition of a 5’ cap
mRNA is read during a process called
translation
Translation takes place in the cytoplasm
Works in three nucleotide units of mRNA
called
codons
Each codon codes for a single amino acid
One amino acid may be coded for by more
than one codon
Start codon (AUG)
Stop codons
There are three different types of RNA
involved in the translation process
•
mRNA
–
exact copy of the gene; carries
the genetic code from nucleus to the
cytoplasm
•
rRNA
–
component of ribosomes, the
organelles responsible for protein
synthesis
•
tRNA
–
transports amino acids to
ribosome
1.
Initiation
–
small ribosome subunit binds to 5’
end of mRNA
Moves along the mRNA until the start codon is
found (AUG)
2.
Elongation
–
tRNAs, carrying the correct
amino acid, enter the ribosome, one at a time,
as the mRNA code is read
3.
Termination
–
ribosome encounters the stop
codon
Newly formed protein is released
Many Biotech efforts modify DNA
molecules with the goal of affecting
protein production
In humans, about 40,000 genes are needed
for an organism to function
A typical cell synthesizes more than 2000
different kinds of proteins and hundreds
or thousands of copies of these proteins are
usually needed at any given time
If you multiply these numbers by the
hundreds of types of cells we have in our
body, the numbers reach into the millions
The entire sum of DNA in a cell is variable
from organism to organism, but every cell
within an organism, except sex cells, has
the same genome.
Even though there are different
quantities of DNA in cells from different
organisms, the DNA itself is virtually
identical.
Organism
Haemophilus influenza: childhood ear infections
Mycoplasma genitalium: Free living bacterium
Caenorhabditis elegans: Free
-
living roundworm
Homo sapiens: humas
#of Genes
1,749
470
19,899
40,000
Size (bp)
1,830,137
580,070
97,000,000
3,000,000,000
All DNA molecules are made of A,T,C,G
Virtually all DNA molecules form a double
helix
The nucleotides connect to one another via
phosphodiester bonds between the sugar and
phosphate
Hydrogen bonds hold each base to its
complementary base creating the two sides of the
DNA molecule
The amount of A is always equal to the
amount of T
The amount of G is always equal to the
amount of C
In prokaryotic cells, the DNA is floating in
the cytoplasm.
It typically contains only one, long, circular
DNA molecule (chromosome)
It is usually folded in on itself and only contains
several thousand genes (compared to a human
who has around 40,000 genes)
Some bacteria
contain small rings of
DNA that are outside
of the “chromosome”
floating in the
cytoplasm called
plasmids
Plasmids only
contain a few genes
(5 to 10) that
usually code for
proteins that offer
some additional
characteristics that
may be needed only
under extreme
conditions.
The most common type of plasmids are
R plasmids
R plasmids contain antibiotic resistance genes
These genes allow the bacteria to survive exposure to
antibiotics that would normally kill them
Bacteria can transfer genetic information between
themselves by a process called
conjugation.
Transferring plasmids give bacteria a way of
“evolving” by gaining new and better characteristics
Because plasmids are pieces of DNA that
can accept, carry, and replicate (clone)
other pieces of DNA, they are often used as
vectors.
A Vector is a vehicle used to transfer the genetic
material such as DNA sequences from the
donor organism to the target cell of the
recipient organism.
http://www.phschool.com/science/biology_place/labbench/lab6/enzwork.html
•
Just like other enzymes, restriction enzymes show specificity
for certain substrates (DNA)
•
They work by cutting the phosphodiester bond (in the sugar
-
phosphate backbone) that joins adjacent nucleotides in a
DNA strand.
•
The cut DNA within a specific sequence of bases called a
recognition sequence
or
restriction site
Modifications of DNA can be as simple as
changing a single base
Changing DNA sequences may alter the
production of some proteins in a cell or
organism
New proteins may also be created
Genetic Engineering:
the production of
rDNA molecules and their insertion into
cells
rDNA: (recombinant DNA) :
pieces of
DNA that have been cut and then pasted
back together
These technologies are the methods
used
to create new DNA molecules by piecing
together different DNA molecules
When cells accept the
rDNA
and start
expressing the new genes (by making the
new proteins) they are considered
genetically engineered.
The names of proteins produced in this
way are written with an “r” in front of
them.
Example : rInsulin
Site
-
specific mutagenesis
refers to the process
of inducing changes (mutagenesis) in certain
sections (site
-
specific) of a particular DNA
code.
The changes in the DNA code are usually
accomplished through the use of chemicals,
radiation, or viruses.
Sometimes site
-
specific mutagenesis is “directed”
meaning a scientist is trying to make certain changes
in a protein’s structures that will translate into an
improved function.
Example: Subtilisin is an enzyme that is added to laundry
detergent to remove proteinacous stains (such as blood or
gravy) This protein was made from altering the protein
subilisin found in fungi. The alteration made the protein
able to function in an alkaline soln. such as laundry det.
Gene Therapy
is the process of correcting
faulty DNA codes that cause genetic
diseases and disorders.
The most common way to conduct gene
therapy is to use a virus to carry a normal gene
into cells containing defective genes (basically
gene replacement)
This therapy is currently being used for Cystic
Fibrosis, Parkinson's, diabetes, and some cancers
Gel Electrophoresis uses electricity to
separate
molecules using a gel slab.
By using electrophoresis, researchers can easily
separate and visualize charged molecules, such as
DNA fragments, RNA and proteins
The two most common types of gels are
agarose
(isolated from sea weed) and
polyacrylamide
(these
are used to separate smaller molecules such as
proteins and very small pieces of DNA or RNA)
Agarose gels are commonly made with concentrations
ranging from 0.6% to 3% agarose in buffer (the more
concentrated the more “straining effect” the gel will
have
Most common gel for DNA fragment separation is .8%
http://www.dnalc.org/view/15921
-
Gel
-
electrophoresis.html
http://learn.genetics.utah.edu/content/labs/gel/
The two types of stain that can be used to
visualize fragments on a gel are
Ethidium Bromide (EtBr): this is the most
common DNA gel stain; it glows orange when
mixed with DNA and exposed to UV light.
EtBr is a mutagen!!! (This means it causes cancer!)
Methylene blue: will bond with the nucleic acid
molecules, turing them a dark blue color
Methylene blue is not as sensitive as EtBr so the
banding pattern will be harder to see!
The body possesses three lines of
defense to prevent and fight off
intrusions by pathogens
The first two lines are non
-
specific
The third layer is the body’s specific
immune system.
Specific immune responses are
tailored to the type of invading
pathogen
Specific immune responses are triggered by
antigen
molecules
Antigen (Antibody Generator): proteins or other
molecules produced by pathogens
The key players in the specific immune
response are the
dendritic cells, macrophages,
and small white blood cells called B
lymphocytes (B cells) and T lymphocytes (T
cells)
Phagocytic macrophages and dendritic cells break
down pathogens and display antigenic fragments
from the pathogens on the surface of their cell
membrane
The B cells and T cells circulate through the body in
the blood and lymph looking for these displayed
antigenic fragments.
When the T cells see the displayed
antigenic fragment, they stimulate specific
B cells to reproduce and generate
antibodies
designed against the specific
structure of the antigen that was
encountered.
Antibodies (immunoglobulins):
a group of
serum proteins that are found in the
bloodstream that have a y
-
shaped structure but
have different antigen binding sites at their
ends.
Antigen binding sites are designed to fit the
shape of specific antigens
Antibodies bind to antigens like a lock
and key forming an antigen
-
antibody
complex
When the Ab/Ag complex forms it marks
the invading organism/antigen for
destruction by phagocytic cells
It also stimulates additional immune
responses
ELISA: Enzyme Linked Immunosorbant Assay
This assay is based on the principle that antibodies
are produced in the response to pathogens
The antibodies attach to their antigen targets with
great specificity to form Ag/Ab complexes.
There are two types of ELISA tests
Indirect ELISA:
Direct ELISA:
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