Download Biotechnology and Genomics

Biotechnology and
Genomics
_________________ is the
production of identical copies
of DNA, cells or organisms
If you think about it, members of a
bacterial colony in a sample are
clones because they all came
from division of the same cell
____________ twins are even clones
Single embryo separate to become
two.
_______ ______ is production of many identical
copies of the same gene.
If the inserted gene is replicated and
expressed, we can recover the
_______________or ________________.
Cell containing gene
of interest
Bacterium
1 Gene inserted into
plasmid
How
It’s
Done
Bacterial
chromosome
Plasmid
Recombinant
DNA (plasmid)
Gene of
interest
2 Plasmid put into
bacterial cell
Recombinant
bacterium
DNA of
chromosome
•
•
•
•
Cuts DNA at specific points. Like…
Cleaves vector (plasmid) and foreign (human) DNA.
Cleaving DNA makes DNA fragments ending in short
single-stranded segments with “______________”
These allow for insertion of foreign DNA into vector
DNA.
A
G
T
C
A
G
A
A
T
T
C
G
C
T
C
T
T
A
A
G
C
G
A
A
T
T
T
T
A
Google: McGraw-Hill
“Restriction
Endonucleases”
C
G
C
G
C
G
A
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DNA Ligase
Remember its
function in DNA
replication?
• Job?
• DNA “_____________” has
now occurred
• The cell membranes of
bacteria can be made more
permeable to take up plasmids
at this point.
– Bacteria and plasmids will
reproduce.
– Many copies of the plasmid and
many copies of the foreign gene
Google: mcgraw-hill:
Go to www.sumanasinc.com
are now produced.
“Steps in Cloning a
Animations-molecular biology-plasmid cloning
gene”
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DNA Cloning: Polymerase Chain
Reaction (____)
• Amplifies a targeted sequence of DNA
– Create millions of copies of a single gene or a specific
piece of DNA in a test tube
• Requires:
– 1. ______ __________ from hot-springs resident
bacterium, Thermus aquaticus (called ______
_____________)
• Withstands the temperature necessary to separate
double-stranded DNA.
– 2. A supply of __________________________ for the
new, complementary strand
– 3. _________________: ~10-20 base sequences which
match the ends of the DNA sequence of interest
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PCR
A three-step cycle—______________, ________________,
____________ —brings about a chain reaction that produces an
exponentially growing population of identical DNA molecules
PCR
cycles
DNA
copies
first
1
second
2
third
4
fourth
8
fifth
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DNA double strand
new strand
new
old strand
old
old
new
and so forth
Go to www.sumanasinc.com
Animations-molecular biology-PCR
8
5
TECHNIQUE
3
Target
sequence
3
Genomic DNA
1
5
5
3
3
5
2
Cycle 1
yields
2
molecules
3
Advertisement for
thermocycler
(machine used
for PCR)
http://www.cnpg.c
om/video/flatfiles/
539/
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Applications of PCR: Analyzing DNA
Segments
• ________ ________________________ is the
technique of using DNA fragment lengths called
MCGRAW RFLP’s to match/investigate DNA samples. GOOGLE:
HILL “RESTRICTION
FRAGMENT LENGTH
• HOW?
POLYMORPHISM”
– By treating DNA samples with restriction
enzymes, a unique collection of different
length fragments is produced
– Electrophoresing these fragments separates
the fragments according to their
• __________________
– Usually used to measure number of repeats
of short sequences
– Produces distinctive banding pattern
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What is Electrophoresis?
Electrophoresis is a laboratory technique for
separating mixtures of charged molecules.
• __________________: a
material composed of two or
more elements or parts.
•___________________: a
molecule (such as a protein or
DNA) that has too many or too
few electrons.
Charged molecules are separated based on
their electrical charge and size.
Mixture of
Charged
Molecules
Essentials parts of GEL
ELECTROPHORESIS
• ___________________________– the flow
of electric charge
• Acts as a _____________, a device to
filter small particles out of a mixture of
larger particles.
How Separation Occurs
Many molecules (amino acids, peptides, proteins,
DNA, and RNA) have naturally occurring negative
and positive charges on them. The sum of these
charges determines the overall charge.
When introduced to an electrical current,
negatively charged molecules are attracted to the
positive electrode and positively charged
molecules are attracted to the negative electrode.
N+
O
N
Positively Charged
Amino Acid
-
- +
+
-
+
+
Positively Charged Peptide
+ - +
- + +
+ - -+
+
Negatively Charged Protein
How Separation Occurs
Molecule Size:
The porous material is made of microscopic particles suspended in
a gel. The microscopic particles attach to one another forming
tunnels that act as a sieve to separate the molecules. Small
molecules can move faster than large molecules.
Porous
Material
Fig. 20-9a
TECHNIQUE
Mixture of
DNA molecules of
different
sizes
Power
source
Anode
– Cathode
+
Gel
1
Power
source
–
2
+
Homework: Do a virtual DNA fingerprinting exercise
GOOGLE: NOVA + CREATE A DNA FINGERPRINT
Restriction fragment analysis is useful for comparing two
different DNA molecules, such as two alleles for a gene
Normal -globin allele
175 bp
DdeI
Sickle-cell
allele
Large fragment
201 bp
DdeI
Normal
allele
DdeI
DdeI
Large
fragment
Sickle-cell mutant -globin allele
376 bp
DdeI
201 bp
175 bp
Large fragment
376 bp
DdeI
DdeI restriction sites in normal and
sickle-cell alleles of -globin gene
DdeI
Electrophoresis of restriction fragments
from normal and sickle-cell alleles
DNA Fingerprinting & Paternity
Mother
Child
Male 1
few
Male 2
Base repeat units
Who is the father?
Male 1 or 2?
many
DNA Band patterns
Fluorescence units
a.
Increasing size
GOOGLE:
MCGRAW-HILL
“DNA fingerprinting”
b. Automated DNA fingerprinting
Go to www.sumanasinc.com
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Animations-biotechnology-paternity
testing
STR’s: short tandem repeats
A method of profiling called
STR analysis uses loci in
DNA where the same
sequences of bases may
occur a number of times.
For example the sequence
GACT may be repeated 5x
in one individual and 20x in
another individual. The
number of repeats varies
from person to person
This can help identify
individuals from each other
or biological evidence
This is the FBI’s 13 Loci standard for matching
individuals to evidence. The possibility of two
individual’s coding the same in STR’s for the 13 loci
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is greater than 1 in a billion
Biotechnology Products
• Genetically engineered organisms can
produce biotechnology products. They are
called “genetically modified organisms” or
(_______’s)
• Organisms that have had a foreign gene
inserted into them are ______________.
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Transgenic Bacteria
• Genes of interest are inserted
into bacteria.
• Bacteria are grown in large
vats called
________________ and
product is harvested.
– Products on the market include
insulin, hepatitis B vaccine, tPA, and human growth
hormone.
• Some genetic
modifications can
promote the health of
plants and other can
degrade harmful
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Transgenic Plants
• Agricultural Crops
– Foreign genes now give cotton, corn, and potato
strains the ability to produce an insect toxin
– Soybeans are now resistant to a common herbicide
– Weed-killer resisting crops are now common
(“Roundup” ready)
Human Hormone
Production:
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Transgenic Animals
• Vortex Mixing:
– Many types of animal
eggs have taken up the
gene for bovine growth
hormone (bGH) through
a procedure that uses
agitation, tiny needles
and foreign DNA to insert
a gene into the egg.
– The procedure has been
used to produce larger
livestock animals
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Gene Pharming:
microinjection of human gene
-Use of
transgenic farm
animals to
produce
pharmaceuticals
.
Genes coding for
therapeutic &
diagnostic proteins
are incorporated
into an animal’s
DNA
-The proteins
appear in the
animal’s
milk
.
Plans are to produce drugs to treat:
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Treating Disorders
◦
 __________ ______ involves procedures
to give patients healthy genes to make up
for a faulty gene.
 It also includes the use of genes to treat
genetic disorders and various human
illnesses.
 There are __________ (outside body) and
Ex Vivo
__________ (inside body)
of
◦ Inmethods
Vivo
Children with Severe Combined
gene therapy.
Immunodeficiency
(SCID)
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Human Genome Project
_________________ is the study of genomes of humans and other
organisms.
_____________- All the genetic information of an individual (or species)
Goals of Human Genome Project
Did it ever get done? How many base pairs?
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Findings of the Human Genome Project
• Humans have between ____________ genes (still
being researched)
– Most genes code for proteins
– Much of the other DNA was formerly described as
“junk”
• “Junk” did not specify the order of amino acids in a
polypeptide
Recent observations suggest that
between 74% and 93% of the genome
is transcribed into RNA but not all
translated into proteins
Therefore, the “junk” (nontranslated
RNA) may code for regulatory gene
expression necessary for proper
cellular events.
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Gene Structure
 Historically, genes were
defined as discrete units
of heredity that
corresponded to a locus
on a chromosome.
 Prokaryotes typically
possess a single circular
chromosome without
____________ and have
less DNA.
 Eukaryotic chromosomes
with much more DNA are
much more complex.
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Eukaryotic Gene Structure
• ___________________________ are DNA sequences
that occur between genes
• __________________ DNA elements occur when the
same sequence of two or more nucleotides are
repeated many times along the length of one or more
chromosomes.
Remember
STR’s?... Can
also be referred to
as
______________
• __________________ are specific DNA sequences
that have the remarkable ability to move within and
between chromosomes. AKA: jumping genes!
YouTube: Transposons: Shifting segments of the
genome
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• The study of the structure, function, and interaction of cellular
proteins
• At least 20-25K of our genes are translated into proteins
• The sum total of these
is called the human
__________
• Understanding protein function is essential to the development
of better drugs. How?
• Once the primary structure of these protein is known
– It should be possible to predict their tertiary structure
– Computer modeling of the tertiary of these proteins is an important part
of proteomics
Worth a look -one last link for biotech products from BIO-RAD:
http://bio-rad.cnpg.com/Video/flatFiles/799/
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Current understanding of what a
“Gene” is…
• A genomic sequence (either DNA or RNA)
directly encoding functional products, either
RNA or protein.
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