BIOTECHNOLOGY
01
DNA FINGERPRINTING
GEL ELECTROPHORESIS
Micropipette: Measures solutions by micrometer (L = 1
millionth of a liter)
• Use clean pipette tip each time!
1. Calibrate micropipette to 5 L
2. Each person aliquot 5 L of blue colored water (in
beaker) onto white paper
3. Calibrate micropipette to 10 L
4. Each person aliquot 10 L of blue colored water (in
beaker) onto white paper
DNA FINGERPRINTING
GEL ELECTROPHORESIS
Gel Electrophoresis Machine: used to filter DNA Fragments by length of
strand
1. Calibrate micropipette to 10 L
2. Each person load two gel “wells” with loading dye in the small tube
in the green foam
3. Put the top on the machine and plug machine into purple power
source
4. When two machines are plugged in, turn the machine to 150V and
go back to your desks
Wednesday, February 10
Today:
3rd/6th: Finish GATTACA
Review Genetic Engineering Techniques 13-2
DNA Fingerprinting Lab
• Read Pre-Lab
• Pre-Lab Questions/Practice
Tomorrow we will load the machines and run the Gel. We will analyze our
results Tuesday. Have a great weekend!
Next Week’s Homework:
Read/Notes 13-1 by Wednesday
Read/Notes 13-3 by Thursday
BIOTECHNOLOGY
The manipulation of living organisms or their
components to produce useful, usually
commercial, products (examples: pest
resistant crops, new bacterial strains, or
novel pharmaceuticals
Topics we will cover: DNA Fingerprinting,
Genetic Engineering, Stem Cells,
Therapeutic and Reproductive Cloning
13-2 Manipulating DNA
New Technologies are used to
• Extract DNA from cells
• Cut DNA into smaller pieces
• Identify the sequence of bases in a DNA
molecule
• Make unlimited copies of DNA
Genetic Engineering: making changes
in the genetic code of a living organism
13-2 Manipulating DNA
DNA Extraction:
Chemical procedure to remove the cell
membrane and nuclear membrane. Then the
DNA is release from the histones that keep it
wound in the nucleus.
13-2 Manipulating DNA
Cutting DNA:
DNA can be cut into smaller pieces by
restriction enzymes.
Restriction Enzymes break the covalent
bonds between nucleotides at specific
locations.
13-2 Manipulating DNA
Separating DNA:
Gel Electrophoresis filters DNA fragments
(DNA that has been cut up) through a gel
from the negative end to the positive end.
Smaller fragments move faster and further
through the gel.
13-2 Manipulating DNA
Making Copies:
PCR (Polymerase Chain Reaction) is a
controlled DNA Replication process which
can make millions of identical copies of a
DNA sample.
13-2 Manipulating DNA
Cutting and Pasting:
Restriction enzymes can be used to cut
portions of DNA out of a sample and a new
DNA portion can be added. This combined
DNA sample is called recombinant DNA.
DNA Fingerprinting
How do biologists get DNA out of a cell?
Break down cell membrane and nuclear
membrane then unwind DNA from histones.
What is a restriction enzyme?
A protein that breaks the covalent bonds
between nucleotides of DNA at specific
locations.
You will be EcoRI, the restriction enzyme that looks
CTTAAG
for
. When it finds this sequence, it
cuts between the last A and G. Use your pencil to
show these cuts.
DNA Sample 1
C T T A A G C T T A A G C T T A A G A A G C T T A A G G G C
G AA T T C G AA T T C G AA T T C T T C G AA T T C C C G
DNA Sample 2
C T T A A G T A C T A C C T T A A G C T T A A G C T T A A G
G AA T T C A T G A T G GA AT T C G A A T T C G A A T T C
Separating DNA
1. Fill in the blanks to outline the steps for gel electrophoresis
• A mixture of DNA fragments are placed at one end of a porous gel.
• An electrical current is applied to the gel.
• When the power is turned on, DNA molecules (fragments) which are
negatively charged move toward the positive end of the gel.
• The smaller fragments move faster.
Gel Electrophoresis
Length of DNA Sample 1
Fragment
11
10
9
8
7
6
5
4
3
2
1
DNA Sample 2
Lane 1: A
Lane 2: B
Lane 3: C
Lane 4: D
Lane 5: E
Lane 6: F
Crime Scene
Crime Scene
Suspect 1
Suspect 1
Suspect 2
Suspect 2
Locus
D3S1358
vWA
FGA
D8S1179
D21S11
D18S51
D5S818
Genotype
15, 18
16, 16
19, 24
12, 13
29, 31
12, 13
11, 13
Frequenc
y
8.2%
4.4%
1.7%
9.9%
2.3%
4.3%
13%
Locus
D13S317
D7S820
D16S539
THO1
TPOX
CSF1PO
AMEL
Genotype
11, 11
10, 10
11, 11
9, 9.3
8, 8
11, 11
XY
Frequenc
y
1.2%
6.3%
9.5%
9.6%
3.52%
7.2%
(Male)
Honors Biology
Thursday, February 18
Today you will need 13-1&13-3 notes and
your Genetic Engineering Packet.
• Explain Selective Breeding and how it is
used.
• Explain the process of cell transformation
and provide examples of how it is used.
• Prepare for Transformation Lab.
Mrs. Coleman: Room 414
Which trait would you want?
13-1 Changing the Living World
Selective Breeding: only allowing organisms
with desirable traits to produce the next
generation
13-1 Changing the Living World
Hybridization: crossing different organisms
to combine the desirable traits of each;
chromosomes must be close enough for
reproduction to take place
13-1 Changing the Living World
Inbreeding: continued breeding of organisms
with similar desirable traits
13-1 Changing the Living World
Genetic variation can be created within
a population by inducing mutations.
• Radiation
• Chemicals
Why would we ever
want to do this?
13-1 Changing the Living World
…Because sometimes mutants have
favorable traits!
13-1 Changing the Living World
…Because sometimes mutants have
favorable traits!
Bacteria that can digest oil.
Plants with multiple copies of chromosomes which enhance size and durability.
(Polyploidy)
13-3 Cell Transformation
Please have 13-3 Notes out in case you need to add anything. Also have the
Section 13-3 Review open in your packet. You can probably complete this
while we are reviewing!
What did Griffith
discover?
What did Avery
discover?
13-3 Cell Transformation
During transformation, a cell
takes in DNA from outside the
cell, which then becomes part of
the cell’s DNA.
This “new” DNA is called
recombinant DNA.
13-3 Cell Transformation
Transformation of Bacteria (Prokaryote)
• Desired gene is located and removed using restriction enzymes.
• The same enzyme is used to cut bacterial DNA (plasmid) to make
room for the new gene.
• In this example, the gene for human growth hormone is inserted in
the bacterial plasmid.
• The plasmid containing recombinant DNA is returned to the bacteria.
**The bacteria’s ribosomes begin expressing the human gene!
13-3 Cell Transformation
About plasmids…
A plasmid is a circular piece of DNA found in
bacteria (prokaryotes). This DNA contains genes
necessary for survival.
When “new” genes are added to plasmids during
transformation, often genetic markers are also
added. Genetic markers are used to determine if
transformation has occurred or not.
Note: The genetic marker we will use in our lab is the gene
that should make the bacteria resistant to antibiotics. If this
gene is properly transformed into the bacteria, antibiotics will
not be able to kill the bacteria…scary!
13-3 Cell Transformation
Transformation of Plant Cells (Eukaryote)
Examples: genes for insect resistance, frost resistance,
and/or drought resistance added to most “edible” plants.
13-3 Cell Transformation
Transformation of Animal Cell (Eukaryote)
Animal cells may be transformed using a bacteria similar to plant
transformation, OR most commonly, desired genes are inserted directly
into the nucleus of an egg cell prior to fertilization.
The gene for “bioluminescence” is often used as a genetic
marker to see if other genes have been transformed into
organisms.
13-3 Cell Transformation
So how can transformation be
used to help humans?
Next I will be giving you a couple of minutes to complete the
Section 13-3 Review in your packet.
Genetic Transformation Lab
pGlo
Objectives:
1. Transform Escherichia coli using the
gene for bioluminescence which is
naturally found in the jellyfish Aequoria
victoria.
2. Determine if transformation was
successful using a genetic marker for
antibiotic resistance.
3. Observe newly expressed proteins in the
transgenic bacteria.
Genetic Transformation
The Genetic Creation of Glowing Bacteria
Genetic Transformation: Change
caused by
genes; inserting the genes from one
organism into another organism
Green Flourescent Protein (GFP):
The protein created in the jellyfish when
the gene for bioluminescence is
expressed
What is bioluminescence?
Plasmid:
Circular piece of DNA in a bacterial cell; can be
passed from one bacteria to another easily;
usually contains genes for traits beneficial to
survival.
Antibiotic: A
compound that blocks the
growth and reproduction of bacteria;
ampicillin is the antibiotic that should
work on E.coli under normal
conditions.
pGLO:
The plasmid that contains the green
fluorescent protein gene AND the gene
for antibiotic resistance
Note: This is a patented product. The company
that makes the pGLO plasmid has the legal rights
to the product!
Protein Synthesis
as it applies to this lab.
1. DNA  RNA  Protein  Genetic Characteristic (Trait)
2. DNA  RNA  Green Fluorescent Protein  Bioluminescence
Lab Materials & Safety
Lab Abbreviations
LB (Luria Broth) – Food for the bacteria – carbs!
Amp (ampicillin) – antibiotic that kills E.coli under normal
conditions
Ara (arabinose) – sugar that “turns on” the GFP gene;
positive transcription factor
pGLO (recombinant plasmid) – contains the antibiotic
resistance gene and the gene for bioluminescence
More Lab Materials &
Procedures
Genetic
Transformation
Lab
Genetic
Transformation
Lab
DNA
RNA
→ Protein
→ Trait→ Trait
DNA→→
RNA
→ Protein
Blue Tube
-pGlo (regular
bacteria)
Orange Tube
+pGlo (bacteria with
GFP gene and
antibiotic resistance
gene)
Teacher Has:
E.Coli starter
plate and pGlo
TS: Transformation Solution
(slightly acidic water)
LB: Luria Broth (food)
More Lab Materials &
Procedures
TS
TS
E.coli
E.Coli
pGLO
LB
LB
+pGLO
LB/AMP
+pGLO
LB/AMP/ARA
-pGLO
LB/AMP
-pGLO
LB
Petri Dish
+pGlo/LB/Amp
+pGlo/LB/Amp/Ara
-pGlo/LB/Amp
-pGLO/LB
Explanation of
Conditions (Petri Dish
Contents)
GFP & Antibiotic
Resistance Gene
Luria Broth
(bacteria food)
Ampicillin
(antibiotic)
GFP & Antibiotic
Resistance Gene
Luria Broth
(bacteria food)
Ampicillin
(antibiotic)
Arabinose
Regular Bacteria
Luria Broth
(bacterial food)
Ampicillin
(antibiotic)
Regular Bacteria
Luria Broth
(bacterial food)
Hypothesize growth on
Petri dishes
Grow?
Glow?
Observation of Petri
dishes after several days
Grow?
Glow?