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(a) Describe and explain the observed results
Example:
Initially before the virus is introduced, the bacteria is exponentially growing. As the number of
bacteria reaches carrying capacity, the virus is added to the system. When the virus is added to
the culture of bacteria, the number of bacteria rapidly decreases as the number of viruses
increases. This is because the virus is going through a lytic cycle and is “attacking” the bacteria.
After the viral DNA enters the bacteria and creates more viruses, the bacteria lyses and viruses
spill out into the culture, which accounts for the drop in bacteria and the rise in number of
viruses. However, after the number of viruses peaks, numbers of bacteria rise again while virus
numbers drops, until both numbers appear to reach an equilibrium. This could occur if the
bacteria mutates and become resistant to the virus. A mutation could cause the cell receptors on
the surface of the bacteria to change, making the virus unable to attach to the bacteria, and
therefore infect it. Bacteria could also adapt new restriction enzymes that cut up viral DNA
1 point for each bullet
● description only of relative numbers of bacteria and viruses over time; must include
initial bacterial growth phase
● bacterial growth dynamics (exponential phase, carrying capacity)
● infection phase (virus “attacks” bacteria, bacteria decline while virus multiples)
● recovery phase (resistant mutant/immune bacteria survive to reproduce, virus number
drops)
● coexistence phase (viruses multiply only in nonresistant/”sensitive” cells or lysogenic
situation develops)
● exceptional description of a particular phase
● population reaches an equilibrium
(b) Discuss the infection cycle of a DNA virus from attachment to lysis.
Infection with a DNA virus begins with attachment of the virus to the cell membrane. This
occurs via cellular receptors that recognize each other between the virus and cell. The DNA then
can enter the cell by injection with a tail apparatus, while the protein capsid is left outside of the
cell. The sheath of the tail contracts, injecting the viral DNA into the cell, which is then
hydrolyzed. Once inside the cell, the viral genome replicates using the host cell’s energy and
cellular components. The viral DNA is also transcribed and translated to create proteins for new
capsids and glycoproteins for cell receptor molecules. At this point, assembly of new viruses can
occur with the replicated viral genomes and newly created capsids and glycoproteins. The phage
then direct creation of an enzyme that allows fluid to enter the cell, which causes the cell to swell
and eventually burst. This then releases the newly created phages, which can now infect more
bacterium's.
1 point for each bullet
● attachment to host cell (cell wall, membrane, etc., attachment to something)
● penetration/injection of DNA/nucleic acid
● synthesis of viral components (nucleic acids and/or proteins)
● assembly/packaging of viruses
● lysis (release, budding); needs details beyond simply cells burst/lysed
(c) Describe how the genome of a retrovirus like HIV become incorporated into the genome of
the host cell.
A retrovirus, like HIV, is surrounded by a viral envelope with glycoproteins that enable the virus
to bind to specific receptors on the cells it attacks. They then fuse with the plasma membrane,
with the capsid proteins being removed, releasing viral proteins and RNA. Reverse transcriptase
then creates a DNA strand complementary to the viral RNA. Finally, the reverse transcriptase
creates a second DNA strand complementary to the first, fulfilling a double stranded DNA. The
newly created double stranded DNA is then integrated into the cell’s DNA as a provirus. Proviral
genes are used to transcribe more RNA molecules and viral protein, with the viral DNA forever
in the cell.
1 point for each bullet
● retrovirus = RNA virus
● use of reverse transcriptase (enzyme) to create DNA “version”
● single strand to double strand conversion
● enzymatic incorporation into human genome, give at least one enzyme
● exceptional description of mechanism
2.
(a) Describe the essential features of two of the procedures/techniques shown below. For EACH
of the procedures/techniques you describe, explain how it’s application contributes to
understanding genetics.
● The Use of a bacterial plasmid to clone and sequence a human gene
● Polymerase Chain Reaction (PCR)
● Restriction fragment length polymorphism (RFLP) analysis
Plasmids : When one is able to find a gene of interest, it is inserted into a small circular piece of
DNA. This process (the inserting of a gene into a plasmid) is called transformation. Because
plasmid are small enough to insert into DNA with ease, it is a perfect tool for scientists to use.
After targeting the gene of interest, scientists will use restriction enzymes to cut genomic DNA
in specific places in the genome, thus generating small DNA fragments which can then be more
easily isolated. After inserting this cut out DNA fragment into the plasmid, it is fully sealed
together using DNA ligase. The ligated plasmid is then transmitted into a bacterial host cell
where it can be easier duplicated and grown. For sequencing purposes, one may grow a large
amount of bacteria to obtain more of the desired gene. The cells are then lysed (disintegrated),
and one can isolate the gene using DNA extraction methods (such as micropipetting). Once
isolated, the gene can be sequenced through the use of short primers to mark the beginning and
end of the target gene.
1 point for each bullet point:
Describe the use of plasmid for cloning/sequencing a human gene
● Cut plasmid with “restriction” enzyme
● Cut/isolate human sequence with the corresponding “restriction” enzyme
● Mix/anneal/ligate
● Introduce recombinant plasmid into bacteria
● Select recombinant bacteria (e.g., antibiotic resistance, fluorescence, reporter gene, etc.)
● Bacterial reproduction used to amplify the sequence
● Describe either degradative (MaxamGilbert) or dideoxy (Sanger) method to generate
fragments
● Electrophoresis to separate fragments
● Read the sequence (automated method is OK)
Explain the contribution of this procedure
● Source of the DNA is immaterial to cloning
● Used to produce transgenic organisms
● Used to make human proteins (e.g. insulin, HGH)
● Understanding gene structure/regulation
● comparative genomics
● Development of gene therapies
● Making gene library
● Amplifying a particular sequence
PCR : The polymerase chain reaction is a technique in which a piece of DNA is rapidly copied
and amplified. DNA is placed in a test tube with Taq polymerase, a DNA polymerase with
resistance to heat, nucleotides, and primers. The DNA is first heated until the two strands
denature, and afterward the primer allows hydrogen bonds to form at both ends of the strand.
Finally, DNA polymerase matches the nucleotide sequences along the 3’ end of the primer.
Cycle 1 produces 2 DNA strands, Cycle 2 ends with 4, and Cycle 3 finishes with 8, and each
consecutive cycle increases the percentage of DNA that match the target sequence. The DNA
produced from PCR will be studied or used in comparisons, as PCR is the primary method to
‘purify’ DNA from scant sources. PCR enables scientists to identify infectious and hereditary
diseases, analyze genetic fingerprints, and clone DNA for analysis or sequencing.
1 bullet point for:
Describe PCR
● Heat to separate strands
● Add primers
● Cool to anneal
● Add polymerase and/or nucleotides
● Specification of heat stable (Taq) polymerase
● Description of thermocycling process
● Repetition of process
Explain the contribution of this procedure
● Allows amplification of very small samples
● Replicates/amplifies a defined region
● Can be automated to allow for faster expansion of knowledge
● Can be used for forensics
● Can be used for diagnosis
● Evolutionary applications
● Other
RFLP:
(b) The Southern Blot protocol has become common practice for many biology related activities
(evolutionary history, paternity/maternity cases, crime scene investigation…). Describe the step
by step process associated with this protocol.
Southern blotting: is a method used by scientists to detect certain gene sequences on a strand of
DNA. This is extremely helpful because one strand of DNA consists up million of genes, so
being able to narrow down on one specific sequences makes the process a lot easier. The process
consists of several steps:
1) DNA is digested with a restriction enzyme and separated by gel electrophoresis. Because there
are so many different restriction fragments on the gel, it usually appears as a smear rather than
discrete bands. The DNA is then denatured into single strands through NaOH incubation.
2) The DNA is transferred to a membrane which is a sheet of special blotting paper. This
includes the use of an alkali buffer solution, as well as paper towels and weights to help sustain
the pattern during transfer. The DNA fragments retain the same pattern of separation they had on
the gel.
3) The blot is incubated with many copies of a singlestranded DNA probe. This probe will form
base pairs with its complementary DNA sequence and bind to form a doublestranded DNA
molecule.
4) The location of the probe is revealed by incubating it with a colorless substrate that the
attached enzyme converts to a colored product that can be seen or gives off light which will
expose Xray film.
Through examining the xray film, one is able to determine which strains contain the targeted
DNA.
1 point for each bullet:
Describe RLFP analysis
● DNA sample cut with “Restriction” enzyme(s)
● Separation of fragments (electrophoresis)
● Description/elaboration of electrophoresis (charge/size/apparatus)
● Visualize fragments (probes, dyes, blots)
● Compare fragment sizes/mobility
● Compare single and double digests (two or more restriction enzymes)
● Compare individuals/species/organisms/tissue samples
Explain the contribution of RFLP analysis
● Trace RFLPs as genetic markers in families
● Diagnose disease/carriers/prenatal samples
● Prepare fingerprints (for forensics etc)
● Order fragments for physical mapping
● Compare genomes of different species/evolutionary relationships
● Locate the flanking regions of the gene/sequence
● Find mutations
● Individual bands can be used for further analysis
● Can determine presence of sequence without knowing its function
(c) All humans are nearly identical genetically in coding sequences and have many proteins that
are identical in structure and function. Nevertheless, each human has a unique DNA fingerprint.
Explain this apparent contradiction.
Although all DNA is composed of the same 4 nucleotides, G, C, A, and T, due to the extreme
length of one strand of DNA as well as the amount of DNA we have in our bodies, there is a lot
of room for differences. Variations in the DNA code sequence can alter the mRNA sequence
(with the exception of wobbling in the tRNA, where the resulting RNA is not changed) or
variations in protein folding and structure. The position of the DNA sequence difference also
affects a change in the phenotype. Since such a large percentage of the human genome are made
up of introns or noncoding DNA segments, variations in such areas will still lead to the same
enzyme function and structure. There are so many proteins and potential coding sequences that
lead to everyone having a unique DNA fingerprint/genetic profile.
1 point for each bullet:
Source of difference in DNA fingerprint
● Variation in noncoding material (introns, spacers, minisatellites, “junk,” transposable
elements)
● Point mutations, small deletions, SNPs (single NT polymorphisms)
● Variable number of tandem repeats (VNTRs/STRs)
Recognition of differences
● A small percentage difference of a very large genome results in a large number of
nucleotide differences
● PCRbased fingerprinting: differences found by where primers anneal
● Variation in restriction enzyme cutting sites
Similarities among proteins
● Redundancy in the code for amino acids
● Neutral/silent mutation does not alter the function of the protein
3.
Explain how you might
(a) Identify the location of the gene responsible for producing the neurotransmitter
In order to isolate the desired gene, we would use western blotting. This can be done in the
following steps:
1. Separate the proteins by size using gel electrophoresis.
2. Place a nitrocellulose membrane on the gel and, using electrophoresis, drive the protein
(polypeptide) bands onto the nitrocellulose membrane.This gives you a nitrocellulose membrane
that is imprinted with the same protein bands as the gel.
3. Incubate the nitrocellulose membrane with a primary antibody. The primary antibody, which
is a specific antibody, sticks to your protein and forms an antibodyprotein complex with the
protein of interest.
4. Incubate the nitrocellulose membrane with a secondary antibody. This antibody should be an
antibodyenzyme conjugate. The secondary antibody should be an antibody against the primary
antibody. This means the secondary antibody will "stick" to the primary antibody, just like the
primary antibody "stuck" to the protein. The conjugated enzyme is there to allow you to visualize
all of this. It's kind of like a molecular flare stuck on the antibodies so you can visualize what’s
going on.
5. To actually see your enzyme in action, you'll need to incubate it in a reaction mix that is
specific for your enzyme. If everything worked properly, you will see bands wherever there is a
proteinprimary antibodysecondary antibodyenzyme complex, or, in other words, wherever
your protein is.
6. Put xray film on your gel to detect a flash of light, which is given off by the enzyme.
(b) Identify brain cells in which the gene for the neurotransmitter is expressed
Use RTPCR. Since we know the dna sequence, cDNA can be synthesized. This can be used as a
probe. Add the probe to different cells in the brain. The probe will bind to the known dna
sequence. Add the probe to various cells in the brain.
(c) Create bacterial colonies needed to produce large quantities of the neurotransmitter for use in
treating a brain disorder
Put the gene into a plasmid with a strong promoter
4.
(a) Explain how the principles of gel electrophoresis allow for the separation of DNA fragments
Example:
Gel electrophoresis allows for the separation of DNA fragments by running DNA
fragments through a gel, with a DC current moving the gel along at different speeds due to their
molecular weight difference and charge difference. DNA is negatively charged, and a constant
voltage will cause the molecules with greater charge and lower molecular weight to migrate a
longer distance per unit time. With the weight of certain stock dyes known, the weight and
charge of the fragments could be then calculated.
(4 Point Max)
● Electricity Electrical potential (charge, field) moves fragments
● Charge negatively charged fragments/move (toward (+) anode) through gel/() charge
due to phosphate groups
● Rate/Size Smaller fragments move faster (farther) relative to large fragments/Describe
logarithmic relationship
● Calibration DNA’s of known molecular weights are used as markers/standards
● Resolution Depends on concentration of gel; is determined by pore size
● Apparatus DNA is stained for visualization of bands/explains use of wells, gel material,
tracking dye, buffers
(b) Describe the results you would expect from the electrophoretic separation of fragments from
the following treatments of the DNA segment above. Assume that the digestions occurred under
appropriate conditions and went to completion.
I. DNA digested with only enzyme X
II. DNA digested with only enzyme Y
III. DNA digested with only enzyme X and enzyme Y combined
IV. Undigested DNA
I. DNA digested with only enzyme X would create four restriction fragments. The lengths of
these fragments would be 400 bp, 1300 bp, 1500 bp, and 1700 bp. The shortest fragments travel
the fastest and furthest while the longest fragments travel the slowest and least distance.
II. DNA digested with only enzyme Y would create two restriction fragments, with lengths of
900 bp and 4000 bp.
III. DNA digested with both enzyme X and enzyme Y would create five restriction fragments,
with lengths of 400 bp, 500 bp, 1200 bp, 1300 bp, and 1500 bp.
IV. Undigested DNA will only lead to one band with a length of 4900 bp.
(4 point max)
● Treatment I Describe 400, 1300, 1500, 1700 bp fragments or 4 bands or correct
diagram with explanation
● Treatment II Describe 900, 4000 bp fragments or 2 bands or correct diagram with
explanation
● Treatment III Describe 400, 500, 1200, 1300, 1500 bp fragments or 5 bands or
correct diagram with explanation
● Treatment IV Describe 4900 bp fragment or 1 band or correct diagram with
explanation
(c) Explain both of the following
(1) The mechanism of action of restriction enzymes.
(2) The different results you would expect if a mutation occurred at the recognition site for
enzyme Y.
Example:
Restriction enzymes are found in bacteria as a defense against phage viruses. When DNA
is identified as foreign, restriction enzymes cut DNA at specific sequence locations, hence, each
cut corresponds to a particular response. Restriction enzymes recognize a specific short DNA
sequence, and cut both DNA strands at precise points within the site. The enzyme will cut at
every recognized location of the sequence, unless the sequence has been modified in a way that
the restriction enzyme can no longer recognize the sequence, such as methylation.
If a mutation occurred at the recognition site of enzyme Y, then enzyme Y will either not
function at all, producing the results given if Y does no cutting, or cut the DNA based on a
different sequence, providing strands of different lengths and hence different migration distances
per unit of time.
Part C1: (4 pt max for C1 & C2)
● Recognition binding of enzyme to target sequence/specific short bp sequences of double
stranded DNA are targeted/Recognizes specific targets 48 bp long/Site may be
palindromic
● Cutting enzyme cuts at every target location/may cut frequently or rarely
Cuts but does not alter the sequence
Alternate...
● Detail Point fragment lengths correspond to lengths between cutting sites/may generate
blunt or sticky ends
Methylation or modification
Breaks the phosphodiester bond/Describe mechanism in living systems
Restriction site may function as a genetic marker
Part C2:
● Change in II Uncut/1 band (looks like IV)
● Change in III Like I: 4 bands
Alternate…
● Detail Point Describes that RFLPs (markers) might correlate with phenotypic variation
Y site might become an X site
Deletion/Insertion at Y site changes fragment length
Silent alteration (pyrimidine > pyrimidine or purine > purine) in some target
sequences
5.
(a) Draw a circle and construct a labeled diagram of the restriction map of the plasmid. Explain
how you developed your map.
I developed my map by first looking at the
restriction fragments created by only the EcoRI.
That restriction enzyme cut the original 100 kb
DNA into 30 kb and 70 kb based on the gel
electrophoresis. Then, I looked at the restriction
fragments created by a combination of EcoRI +
HaeIII. Looking at the gel electrophoresis, we
can see that the DNA was cut to 40 kb, 30 kb,
20 kb, and 10 kb. Hence, it is clear that after
EcoRI, there are restriction sites in the larger
fragments. The HaeIII split the 30 kb into 20 kb
+ 10 kb and the 70 kb into 30 kb + 40 kb. This
can be found through trial and error
(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
1. Alright, so we will extract a plasmid from the bacteria. Using restriction enzymes, we’ll
cut it which will form sticky ends. Then, we’ll take our gene of interest and cut it with the
same restriction enzyme which also forms the same sticky ends. Now, with these sticky
ends, we can splice our gene of interest into that plasmid. Ligase would permanently join
the pieces together. Next, we will expose the bacteria into a solution with a plasmid in it
which some bacterias will take up in transduction. Then, that plasmid & gene of interest
will be in that bacteria!
2. We can identify the recombinant bacteria by testing for the phenotype we hope to get. For
example, say our gene of interest was ampicillin resistance. We’ll test to see which
bacteria has the plasmid by spreading them out on an agar plate with ampicillin. The
bacteria that can live and form colonies on that plate have the ampicillin resistant gene
and are the recombinant bacteria.
3. Expression of gene of interest could be ensured by removing the introns on our gene of
interest for the bacterial expression. Since bacteria are unable to remove the specific
introns (noncoding regions) which can prevent correct expression that eukaryotes do, it
is necessary to provide an intronfree DNA strand to them if the gene is to be expressed.
To do this, we can make it into cDNA by using mRNA and transcribe with reverse
transcriptase to create a complementary DNA. With the addition of a primer, we’ll get
our cDNA without introns! Then, the bacteria should be able to activate that gene and
express it.
1 point for each bullet point:
(1) Insert gene of interest (4 points max)
● Cut gene of interest from source and/or cut plasmid with restriction enzyme
● use SAME restriction enzyme on both
● Anneal/ligate/mix/combine gene of interest with vector (plasmid/virus/phage)
● “sticky ends”/bp matches/complementary
● Treatment for competent cells (CaCl /heat shock); incubate together
2
● Chemical modification can prevent restriction enzyme activity (methylation)
● Gene = cDNA (without intron) to fit into plasmid
(2) Identify recombinant bacteria (1 point)
● Phenotypic selection (antibiotic resistance/bluewhite colony selection/ “glo” gene,
product produced (e.g. insulin)
● Radioactively/fluorescently labeled (tag/dye)/mRNA
● Electrophoresis of cut recombinant vs. original (gene/plasmid) OR with sequence
comparison of recombinant vs. original (gene/plasmid) ( Not bacterial genome)
(3) Ensure expression of gene of interest (1 point)
● Promoter (for prokaryote)
● cDNA/removal of introns for prokaryotic expression
● Operon (e.g. nutrient/arabinose induced)
(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
POSSIBLE EXAMPLE:
● genetically modified cows given the bovine growth hormone will increase the amount of
milk produced at farms and dairy centers. With this, farmers can sell more milk to
supermarkets, and in turn the supermarkets have more supply of milk to sell to consumers
are grocery markets, ultimately increasing the profits of supermarket corporations and
dairy farmers. however, research seems to show that cows treated with BGH tend to form
udder infections; therefore, dairy farmers give those cows more doses of antibiotics.
repeated exposure to these antibiotics will foster a more rapid growth of resistance,
because a slight mutation could render the antibiotic ineffective against the bacteria. this
would as a result pose a threat to the cow population of the farm as these infections may
or may not be infectious.
1 point for each bullet point:
Discuss GM, benefit to humans, and threat to population/ecosystem:
● Nonhuman organism with specific, heritable GM trait
● Plausible benefit to humans related to the GM trait
● Plausible or unknown threat to population/ecosystem related to GM trait/modified
organism