Unit 3: Introduction to Studying DNA

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14 Δεκ 2012 (πριν από 4 χρόνια και 6 μήνες)

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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: