Download Control of Gene Expression

BIOTECHNOLOGY UNIT:
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•
•
•
•
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“The Immortal Life of Henrietta Lacks”
Dr. Wayne Grody- Guest Speaker
“GATTACA”
Ch18&19 Pro&Eukaryotic control of gene expression
Ch11 Cell Signaling Cell Communication
Ch20&21 Biotechnology…manipulating
microorganisms to do our bidding!!!! Tools/techniques
• LAB 6 MOLECULAR BIOLOGY
– Bacterial transformation
– Restriction Enzymes/DNA Digests
– Gel Electrophoresis (DNA fingerprinting)
• BIOTECH RESEARCH PAPER & SYMPOSIUM
Control of Gene Expression
Chapter 18
VOCABULARY
Heterochromatin
Euchromatin
Methylation
Transcrition
factors
Histones
nucleosomes
Operon/
Operator/
Promoter
Gene
experssion
Inducible/
Repressible
ATP
ADP
AMP cAMP
Jacob &
Manod
Galactosidas
e
Glucose
Galactose
Lactose
Transcription
factor
Negative
regulation
Positive
regulation
VOCABULARY
Inducer/
CRP
Corepressor
Regulatory
Gene
QUESTIONS TO ANSWER
Who?
What? Where? When? Which? Why? How?
Q&A
•
•
•
•
Q:What is gene expression?
A: Activating a gene to produce a protein.
Q: What is an operon?
A: The genes for creating an enzyme and the genes that
regulate their transcription.
• Q: What makes up an operon?
• Answer:
1) Promoter region
2) Operator region
3) Structural genes
OPERON
Promoter, Operator, Structural Genes
Control of gene expression
• The expression of genetic material controls cell
products, and these products determine the
metabolism and nature of the cell.
• Gene expression is regulated by both environmental
signals and developmental cascades or stages.
• Cell signaling mechanisms can also modulate and
control gene expression.
• Thus, structure and function in biology involve two
interacting aspects: the presence of necessary
genetic information and the correct and timely
expression of this information.
Gene expression
can be under:
a) Negative regulation:
When the operon is
turned off by
chemicals.
repressible or inducible
or
b) Positive regulation:
When gene
expression is
stimulated by
chemicals.
2 Types of Negative Regulation
Part 1: The trp operon
(repressible operon)
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•
•
•
What does repressible mean?
This operon is located in E. coli bacteria.
The purpose of the operon is to create the
enzymes that synthesize the amino acid
tryptophan.
When the operon is “ON:
1) RNA polymerase binds to the promoter region.
2) RNA polymerase crosses over the operator region because
the repressor is inactive and therefore does not bind to the operator.
3) The structural genes are transcribed and tryptophan is synthesized.
OPERON “ON”
Switching the trp operon “off”
•
Done by a repressor: a protein that binds to the
operator region.
• It blocks the attachment of RNA polymerase to the
promoter region.
1) A gene called a regulatory gene (located away from
the operon) produces the trp operon repressor.
2) The repressor protein is allosteric having an active and
inactive state/shape.
3) At first the repressor protein is in its inactive form.
4) When the amino acid tryptophan binds to the repressor
the repressor becomes its active form and binds to the
operator region… blocking transcription.
5) Since tryptophan assists in turning the operon off it is
called a co-repressor.
6) When the levels of tryptophan drop the repressor loses its
tryptophan, changes shape, and the repressor is released
from the operator , initiating transcription again.
TURNING THE OPERON OFF
regulatory gene
operator
Regulatory gene codes for the repressor which may bind to the operator
Types of Negative Regulation
Part 2: The lac operon
(inducible operon)
• What does inducible mean?
• This operon is also located in E. coli bacteria.
• It was first discovered by: Francois Jacob and Jacques
Monod (1961) called the “Jacob and Monod model”
• The purpose of the operon is to produce the enzyme
B-galactosidase that splits (via hydrolysis) lactose into
glucose and galactose.
• This operon is normally off because the repressor protein
is formed in its active shape, thus binds to the operator
region blocking RNA polymerase.
• If the RNA polymerase is being blocked
how does transcription ever occur???
Done by an inducer: a molecule that
binds to and inactivates the repressor.
1)
2)
3)
4)
The molecule allolactose (an isomer of lactose) binds to the
repressor, inducing an allosteric change.
The repressor is released from the operator region.
The RNA polymerase can move along the template strand
catalyzing the synthesis of mRNA.
When the lactose levels decrease the repressor binds to the
operator region and transcription is shut down.
Figure 18.21a The lac operon: regulated synthesis of inducible enzymes
Figure 18.21b The lac operon: regulated synthesis of inducible enzymes
Compare and contrast:
• The enzymes produced by the trp operon
are called repressible enzymes and are
involved in anabolic pathways.
• The enzymes produced by the lac operon
are called inducible enzymes and are
involved in catabolic pathways.
Types of Positive Regulation:
A closer look at the lac operon
• In order for the lac operon to produce
enzymes in large quantities, a second
factor must exist…
• a low concentration of glucose.
How does E. coli sense the low levels of glucose
and how is this relayed to the lac operon?
By the molecule cyclic AMP or cAMP.
cAMP is present in large quantities when the glucose levels are low.
1) The cAMP binds to an allosteric protein called cAMP receptor
protein or CRP
2) The activated CRP binds to a site within the lac promoter adjacen
to the TATA box.
3) The attachment of CRP makes it easier for RNA polymerase to
bind to the promoter region.
Figure 18.22a Positive control: cAMP receptor protein
Figure 18.22b Positive control: cAMP receptor protein
CRP is known as an activator protein because it
activates transcription.
If the levels of glucose increase the levels of cAMP
decrease and the CRP is released from its binding site.
Figure 18-22x cAMP
Figure 18.20b The trp operon: regulated synthesis of repressible enzymes (Layer 2)
Figure 18.21b The lac operon: regulated synthesis of inducible enzymes
Figure 18.22a Positive control: cAMP receptor protein
Figure 18.22b Positive control: cAMP receptor protein
The Organization and Control of
Eukaryotic Genomes
Chapter 19
How is eukaryotic gene expression different
from prokaryotic gene expression?
1. Importance of cell specialization in multicelluar Euk’s.
2. Greater size of genome of Euk’s and chromatin structure:
-single, circular, chromosome in PROKARYOTES
-double, linear, protein enhanced in EUKARYOTES*
Histones/Nucleosomes = DNA is coiled around bundles of
8 or 9 histone proteins to form DNA-histone complexes
called nucleosomes.
1. Euchromatin = regions where DNA is loosely bound
to nucleosomes and is actively transcribed.
2. Heterochromatin = regions where nucleosomes are
more tightly compacted and DNA is
inactive. (stains darker)
* Compactly organized as chromosomes during cell division.
Figure 19.1 Levels of chromatin packing
DNA Packing is the first level
of control of eukaryotic gene expression
Figure 19.0 Chromatin in a developing salamander ovum
Only _3_% of eukaryotic
DNA is translated into
protein products,
compared to almost
_100_% of prokaryotic
DNA.
Sizewise…
several million nucleotide
pairs
vs.
2 x 10 8 pairs per
chromosome
(that’s x 46 in humans!)
Figure 19.x1a Chromatin
HETEROCHROMATIN
EUCHROMATIN
•
Repetitive DNA = noncoding segments not transcribed
within a gene
» CENTROMERE- center
» TELOMERE- ends (telomerase)
» PSEUDOGENES = not transcribed, almost identical to
a coding gene. May represent evolutionary precursor—
mutated over the years.
•
TRANSPOSONS or “jumping genes”= can move to a
new location on the same chromosome or to a different
chromosome. Discovered by Barbara McKlintock (maise)
Have the effect of a mutation… can change the expression
of a gene
1. Turn on or off its expression
2. Have no effect at all
Enduring understanding 3.B:
Expression of genetic information involves cellular
and molecular mechanisms.
• Essential knowledge 3.B.1:
• Gene regulation results in differential gene
expression, leading to cell specialization.
Enduring understanding 3.B:
Expression of genetic information involves cellular
and molecular mechanisms.
• Essential knowledge 3.B.1:
• Gene regulation results in differential gene
expression, leading to cell specialization.
Both DNA regulatory sequences,
regulatory genes, and small regulatory
RNAs are involved in gene expression.
• Regulatory sequences are stretches of DNA that
interact with regulatory proteins to control
transcription. (ex. promoter, terminator, enhancer)
• A regulatory gene is a sequence of DNA encoding
a regulatory protein (repressor & activator) or
RNA (miRNA & siRNA) blocks translation on a
transcribed mRNA by binding to it.
– Micro RNA
– Small Interfering RNA
– RNA interference (RNAi) is a biological
process in which RNA molecules inhibit gene
expression, typically by causing the
destruction of specific mRNA molecules.
– Two types of small ribonucleic acid (RNA)
molecules – microRNA (miRNA) and small
interfering RNA (siRNA) – are central to RNA
interference. RNAs are the direct products of
genes, and these small RNAs can bind to other
specific messenger RNA (mRNA) molecules and
either increase or decrease their activity, for
example by preventing an mRNA from producing
a protein. RNA interference has an important role
in defending cells against parasitic nucleotide
sequences – viruses and transposons – but also in
directing development as well as gene expression
in general.
In eukaryotes, gene expression is complex
and control involves regulatory genes,
regulatory elements and transcription
factors that act in concert.
• Transcription factors bind to specific DNA
sequences and/or other regulatory proteins.
• Some of these transcription factors are
activators (increase expression), while others
are repressors (decrease expression).
• The combination of transcription factors
binding to the regulatory regions at any one
time determines how much, if any, of the gene
product will be produced.
Controlling Eukaryotic Gene Expression
• Under positive control
• Transcription will not take
place without the assembly
of the transcription
complex
• Transcription Factors are
regulatory proteins that
bond to the enhancer
region, the promoter
• Once the transcription factors
(TATA box) and to each
have assembled around the
other.
promoter, they are called a
transcription complex.
Areas that regulate eukaryotic transcription:
a) The Enhancer Region: causes the chromosome to loop
and make contact with the Promoter regions.
• Located thousands of nucleotides away from the promoter.
• Activator proteins bind to the enhancer regions and then
to the transcription complex after the DNA loops.
• When the activator proteins (special transcription factors)
bind to the transcription complex, RNA polymerase is
positioned over the promoter region and the rate of
transcription increases.
b) The Silencer Region: a repressor region.
• Located close to the enhancer region.
• Repressor Proteins bind to the silencer
sites prevent the activator proteins from
binding to the enhancer region.
Turning On A Gene
Turning On A Eukaryotic Gene
Figure 19.8 A eukaryotic gene and its transcript
ALTERNATIVE RNA SPLICING
Control of Translation
Protein Processing
cancer
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•
•
•
What is cancer?
Unregulated cell growth and division.
What causes cancer?
Damage to the genes regulating the cell
division cycle.
• Usually by carcinogens (cancer causing agents).
• Tumor = a cluster of cancerous cells
• Metastases = When cells leave the tumor, spread,
grow new tumors.
• Sarcomas = Tumors of the cells in connective tissue,
muscle or bone.
• Carcinoma = Tumors of cells in epithelial tissue like
skin.
• The three deadliest human cancers:
• Lung …smoking
• Colorectal …diet
• Breast…causes is still unknown, however
some forms are inherited as the genes BRCA1
and BRCA 2.
• Genes that cause cancer are called oncogenes.
• The normal versions of these genes are called
proto-oncogenes and they code for proteins that
stimulate normal cell growth and division.
• How do proto-oncogenes become oncogenes?
• Translocation, or movement of fragments of
chromosomes(break off and attach somewhere else) that
result in being around an active promoter.
• Amplification, or increasing the number of copies of the gene
in the cell.
• Point mutations, change the sequence, creating mutant
proteins
• Two genes that are significant: ras gene and the
p53 gene.
• The ras gene creates a protein that influences the
cell cycle.
• The mutated ras protein is hyperactive, leading to
excessive cell division.
• The p53 gene becomes active when DNA is
damaged and creates a tumor suppressing protein.
• Mutating the p53 gene can lead to the formation of
tumors.
• Chemicals in cigarette smoke induce p53
mutations.
• 15% of the cancers worldwide are associated with
viral infections. Certain viruses can insert
oncogenes others may insert DNA into protooncogenes, turning them into oncogenes.
Figure 19.13 Genetic changes that can turn proto-ocogenes into oncogenes
Figure 19.14 Signaling pathways that regulate cell growth (Layer 1)
Figure 19.14 Signaling pathways that regulate cell growth (Layer 2)
Figure 19.14 Signaling pathways that regulate cell growth (Layer 3)
Figure 19.15 A multi-step model for the development of colorectal cancer
• The basic structure of viruses includes a
protein capsid that surrounds and protects
the genetic information (genome) that can
be either DNA or RNA.
• Viruses have a mechanism of replication
that is dependent on the host metabolic
machinery to produce necessary viral
components and viral genetic material.
• Some classes of viruses use RNA without a
DNA intermediate; however, retroviruses,
such as HIV, use a DNA intermediate for
replication of their genetic material.
• Some viruses introduce variation into the
host genetic material.
– When the host is bacterial, it is referred to as
lysogenesis;
– whereas in eukaryotic cells, this is referred to as
transformation.
• Since viruses use the host metabolic pathways,
they experience the same potential as the host for
genetic variation that results from DNA
metabolism.
SUMMARY
Viral & Bacterial Control of
Gene Expression
CHECK FOR UNDERSTANDING
Both positive and negative control
mechanisms regulate gene expression
in bacteria and viruses.
1. The expression of specific genes can be turned on by the
presence of an _________.
2. The expression of specific genes can be inhibited by the
presence of a _________.
3. Inducers and repressors are small molecules that interact
with ____________ and/or regulatory sequences.
4. Regulatory proteins inhibit gene expression by binding to
_________and blocking transcription (negative control).
5. Regulatory proteins stimulate gene expression by binding
to DNA and stimulating transcription (___________) or
binding to _________ to inactivate repressor function.
6. Certain genes are continuously expressed; that is, they are
always turned “on,” e.g., the _________ genes.
Both positive and negative control
mechanisms regulate gene expression
in bacteria and viruses.
• The expression of specific genes can be turned on by the
presence of an inducer.
• The expression of specific genes can be inhibited by the
presence of a repressor.
• Inducers and repressors are small molecules that interact
with regulatory proteins and/or regulatory sequences.
• Regulatory proteins inhibit gene expression by binding to
DNA and blocking transcription (negative control).
• Regulatory proteins stimulate gene expression by binding to
DNA and stimulating transcription (positive control) or
binding to repressors to inactivate repressor function.
• Certain genes are continuously expressed; that is, they are
always turned “on,” e.g., the ribosomal genes.
Gene regulation accounts for some of the
phenotypic differences between organisms
with similar genes.
QUESTIONS:
1. describe the connection between the regulation of gene
expression and observed differences between different kinds of
organisms.
2. describe the connection between the regulation of gene
expression and observed differences between individuals in a
population.
3. explain how the regulation of gene expression is essential for the
processes and structures that support efficient cell function.
4. use representations to describe how gene regulation influences
cell products and function.
Gene regulation accounts for some of the
phenotypic differences between organisms
with similar genes.
QUESTIONS:
1. describe the connection between the regulation of gene expression and observed differences
between different kinds of organisms. Structure and function in biology result from the presence
of genetic information and the correct expression of this information.
2. describe the connection between the regulation of gene expression and observed differences
between individuals in a population. The expression of the genetic material controls cell products,
and these products determine the metabolism and nature of the cell. Most cells within an organism
contain the same set of genetic instructions, but the differential expression of specific genes
determines the specialization of cells.
3. explain how the regulation of gene expression is essential for the processes and structures that
support efficient cell function. Some genes are continually expressed, while the expression of most
is regulated; regulation allows more efficient energy utilization, resulting in increased metabolic
fitness.
4. use representations to describe how gene regulation influences cell products and function.
Gene expression is controlled by environmental signals and developmental cascades that involve
both regulatory and structural genes. A variety of different gene regulatory systems are found in
nature. Two of the best studied are the inducible and the repressible regulatory systems (i.e.,
operons) in bacteria, and several regulatory pathways that are conserved across phyla use a
combination of positive and negative regulatory motifs. In eukaryotes, gene regulation and
expression are more complex and involve many factors, including a suite of regulatory molecules.
DNA TECHNOLOGY AND
GENOMICS
Chapter 20
Ch 20 & 21 VOCABULARY
put a + by the terms you know and – by the ones you don’t.
GMO
Clone
Vaccine
biotechnology
Genetic
engineering
GFP
ligation
plasmid
endonuclease
technology
PCR
insulin
Gene
expression
Transgenic
Gel electrophoresis
DNA
Gene therapy
Restriction
enzyme
Somatic cell
Nuclear transfer
DNA Fingerprint
HGH
Dolly
vector
Biomedical
agriculture
RFLP
interleukin
ethical
interferon
transformation
GENERATE YOUR OWN QUESTIONS:
1pt question words: Who? What? Where? When?
2pt question words: Which? How?
3pt question words: Why?
Genetic Engineering is the application of molecular
genetics for practical purposes.
Uses:
1) Identify genes for specific traits
2) Transfer genes for a specific trait from one
organism to another.
Tools for manipulating genes:
1) Restriction enzymes (endonucleases)
2) Cloning vector (bacterial plasmid)
Transgenic/Recombinant organisms contain DNA
that was not part of their original genome.
Green fluorescent protein
(GFP) is responsible for
the green bioluminescence
of the jellyfish Aequorea
victoria.
This is a GM mouse!
5. The genetic composition of cells can be altered by
incorporation of exogenous DNA into the cells. As a
basis for understanding this concept:
a.Students know the general structures and functions of
DNA, RNA, and protein.
b. Students know how to apply base-pairing rules to
explain precise copying of DNA during
semiconservative replication and transcription of
information from DNA into mRNA.
5. The genetic composition of cells can be altered by
incorporation of exogenous DNA into the cells. As a
basis for understanding this concept:
c. Students know how genetic engineering (biotechnology) is used to
produce novel biomedical and agricultural products.
d.* Students know how basic DNA technology (restriction digestion
by endonucleases, gel electrophoresis, ligation, and
transformation) is used to construct recombinant DNA molecules.
e.* Students know how exogenous DNA can be inserted into
bacterial cells to alter their genetic makeup and support expression
of new protein products.
Recombinant organisms contain DNA
that was not part of their original
genome.
The green fluorescent
protein (GFP) created
by these transgenic
mice is responsible for
the green
bioluminescence of
the jellyfish Aequorea
victoria.
This GMO is a GM mouse!
Practical Uses of DNA
Technology:
• Gene Therapy
• Pharmaceuticals- HGH, Interferons,
Interleukins etc.
• Vaccines- solution that contains a harmless
version of a virus or bacterium to stimulate
an immune response & formation of
“memory” cells.
• Increased Agricultural Yields- ex. crops that
don’t need fertilizer.
Ethical Issues
• Describe two potential safety and
environmental problems that could result
from genetic engineering.
Golden rice contains beta-carotene, which our bodies use to make vitamin A…
normal rice does not. Vitamin A deficiency results in blindness & lowered immunity.
Figure 20.x2 Injecting DNA
Somatic
Cell
Nuclear
Transfer
What does that mean?
“Pharm” animals create other species proteins secreted in their milk, ex. spider’s silk.
Figure 20.16 One type of gene therapy procedure
We owe the field of Genetic Engineering
to the bacterial cell.
1) Plasmids: small circular pieces
of DNA.
Plasmids often contain genes
for antibiotic
resistance, thus
protection from fungi.
Ex. Penicillin
Ampicillin, Amoxycillin
2) Conjugation:
• Bacteria exchanging
plasmids.
• Pili = cytoplasmic
extensions used to
contact other bacteria.
• Plasmids are
exchanged through the
pili.
3) Restriction endonucleases
• Molecular “scissors” that
cut DNA at specific
sequences.
• Provide protection for
bacteria against viruses.
* More details to follow.
4)Transformation:
•
bacteria can incorporate new
DNA into their genome from the
external environment.
We will do this in the next lab
• Give E. Coli a plasmid that
contains:
1) Gene for Ampicillin resistance
protein
2) Gene for B galactosidase
enzyme to digest xgal sugar to
make blue protein.
How restriction endonucleases
work
• The enzyme will
recognize a specific
sequence of DNA &
cut it at that specific
place in a specific
way.
• For example E. coli bacteria have a restriction endonuclease
called EcoRI.
• It will recognize the site: 5’-- GAATTC--3’
3’--CTTAAG--5’
• Many recognition sites are palindrome sequences- read the
same forwards/backwards- see 2 strands.
• It will cut the DNA between: G AATTC
CTTAA G
• The results are “sticky ends” or short, single strands of bases.
• If two complementary sticky ends pair up, they can be joined by
DNA ligase.
Restriction Enzymes
RECOMBINANT PLASMIDS
• Stanley Cohen and
Herbert Boyer created the
first recombinant plasmids
in 1973.
• In one of the first
recombinant experiments
with animal DNA, Cohen
and Boyer spliced an
amphibian gene into a
bacterial plasmid.
1972
UCSF & Stanford scientists met at a conference in
Hawaii on bacterial plasmids. Over lunch of hot pastrami
sandwiches they decided to pool their resources. Within four
the joint labs succeeded in cloning predetermined
segments of DNA. This paved the way for a new, huge,
International industry that has created products such as:
Human growth hormome(HGH), human insulin, and a heart
medication to remove blood clots.
How to create a
recombinant plasmid
1) Treat plasmid and
amphibian DNA with the
same restriction
endonuclease (they used
EcoRI)
2) This creates the same sticky
ends on plasmid and
amphibian DNA.
3) Place both together with DNA
ligase to join the amphibian
gene with the plasmid.
The recombinant plasmid
is inserted (transformed)
into bacterial cells and the
bacteria made amphibian
mRNA.
But not the protein… we’ll
see why in a minute.
You can clone the gene using
the bacterial cells
When scientists create plasmids one
problem they encounter is that
prokaryotes cannot modify mRNA
by removing the eukaryotic intron
sequences.
Introns often prevent translation.
To overcome this problem, a DNA gene is
created using the modified mRNA as a
template.
1.
2.
3.
4.
5.
Modified mRNA is isolated from the cytoplasm.
An enzyme called reverse transcriptase creates a strand of DNA
from the mRNA template.
The newly synthesized strand of DNA can act as a template for the
complementary strand.
This type of DNA, synthesized without the intron sequence is called
complementary DNA or cDNA.
The cDNA can be successfully inserted into plasmids, transcribed
and translated by bacteria.
Figure 20.5 Making complementary DNA (cDNA) for a eukaryotic gene
Cloning A Gene
Screening for the Recombinant Plasmid
(How to find the few bacteria transformed
w/ recombinant plasmid from the rest!!!!)
• A recombinant plasmid should contain:
• 1) A gene for antibiotic resistance.
• 2) A functional gene containing a restriction site.
– Ex. THE GFP gene
• Why?
You can do this two ways:
1. They are grown on a media containing an antibiotic.
Only the bacteria that took up the plasmid will survive.
2. Knock out a gene- this let’s you know if you
successfully spliced your “gene of interest” into the
plasmid in the first place.
If a gene on the plasmid contains a restriction site, then
that gene will be rendered useless when the foreign
gene of interest is inserted.
Ex. The organisms that don’t “Glow” have the
recombinant plasmid and do… ex. make HGH.
For Example:
• The "Z gene" on a plasmid produces an enzyme that
metabolizes the sugar X-Gal.
• When the gene is functioning X-Gal is broken down
into a blue product.
• If the restriction site is within the "Z gene" and the
gene of interest is inserted there, the bacteria that have
this plasmid cannot metabolize X-gal.
• When they are cultured on a petri dish, using an X-gal
media, these bacteria will appear white.
• Other bacteria that have a plasmid without the gene of
interest will appear blue.
Other techniques:
• X Antibody staining
• X Radioactive DNA probe
• Restriction Digest of DNA/Gel
Electrophoresis
DNA FINGERPRINT/ RFLP analysis
Restriction Digest of DNA
Restriction Fragments separated by Gel Electrophoresis
DNA FINGERPRINT
Cut DNA at specific sequences w/ restriction enzymes.
Each different sample is cut at different locations- makes different sized fragments
Loaded onto gel. Electric current runs through. Pulls - DNA to + charge.
Small fragments move fastest, Large fragments left at the top.
Unique banding pattern forms
Making a DNA Fingerprint
• A DNA sample is extracted from
nucleated cells.
• The DNA is amplified using
P.C.R.
• The DNA is cut into fragments by
restriction enzymes.
• The stained fragments are placed
into a gel, and are moved by an
electrical current.
• Comparison is made between
DNA samples.
Paternity Testing: Who’s the daddy?
A
or
B
Making a DNA Fingerprint… cont.
• Smaller fragments migrate the
farthest and the result is a column
of dark DNA bands that extend
across the gel.
• The amount of DNA between
restriction sites varies from
individual to individual of the
same species. The differences are
called restriction fragment
length polymorphisms or
RFLP’s. RFLP’s result in unique
restriction fragment patterns on a
gel.
Using the circle provided, construct a labled diagram of the restriction map of the
plasmid. Explain how you developed your map.
b) Describe how: recombinant DNA technology could
be used to insert a gene of interest into a bacterium.
Recombinant bacteria could be identified. Expression
of the gene of interest could be ensured.
b) Describe how: recombinant DNA technology could be used to insert a gene of
interest into a bacterium. Recombinant bacteria could be identified. Expression of the
gene of interest could be ensured.
c) Discuss how a specific genetically modified organism might provide
a benefit for humans and at the same time, pose a threat to a
population or ecosystem.
c) Discuss how a specific genetically modified organism might provide
a benefit for humans and at the same time, pose a threat to a
population or ecosystem.
Polymerase Chain Reaction
a way to make millions of copies of DNA!!!
What you need:
1. DNA sample
2. Free nucleotides
– A heat resistant DNA polymerase
– Example: Taq polymerase
3. Primers: short segments(20-30bases) of
DNA complementary to the ends of the
DNA being copied.
What to do:
1)
2)
3)
4)
Denature the original strand of
DNA with heat.
Cool the mixture, allowing the
primers to bind (anneal) to the
DNA.
The DNA polymerase binds
free nucleotides to the primer
using the original DNA strand
as a template. This creates two
copies of the DNA sample.
Repeat.
Gel Electrophoresis
• Technique used to separate restriction
fragments.
• DNA fragments of different lengths are
separated as they diffuse through a
gelatinous material under the influence
of an electric field.
• Since DNA is negatively charged
(phosphate groups), it moves toward the
positive electrode.
• Shorter fragments move further/faster
than longer ones so a pattern is made.
Figure 20.15 RFLP markers close to a gene
Figure 20.x1a Laboratory worker reviewing DNA band pattern
Figure 20.x1b DNA study in CDC laboratory
APPLICATIONS
of
Gel Electrophoresis
1. Compare DNA fragments of closely related species to
determine evolutionary relationships.
2. CSI. Compare restriction fragments between
individuals of the same species- murder, rape.
Fragments differ in length because of polymorphisms, slight
differences in DNA sequences. These fragments are called
restriction fragment length polymorphisms, or RFLP’s.
Figure 20.6 Genomic libraries
Figure 20.13 Alternative strategies for sequencing an entire genome
Table 20.1 Genome Sizes and Numbers of Genes
Figure 20.14a DNA microarray assay for gene expression
Figure 20.14b DNA microarray assay for gene expression
Figure 20.19 Using the Ti plasmid as a vector for genetic engineering in plants
DNA, and in some cases RNA, is the
primary source of heritable information.
CHECKING FOR UNDERSTANDING:
• Genetic engineering techniques can manipulate the
heritable information of DNA and, in special cases,
RNA.
Q: What are three genetic engineering techniques?
• Illustrative examples of products of genetic
engineering include:
Q: What are three examples of products of Genetic
Engineering?
DNA, and in some cases RNA, is the
primary source of heritable information.
• Genetic engineering techniques can manipulate the
heritable information of DNA and, in special cases,
RNA.
– ex.Electrophoresis , Plasmid-based transformation ,
Restriction enzyme analysis of DNA , Polymerase Chain
Reaction (PCR)
• Illustrative examples of products of genetic
engineering include:
– Genetically modified foods, Transgenic animals, Cloned
animals, Pharmaceuticals, such as human insulin or factor X