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BIOCHEMISTRY 461
Dr. Bourque Chapter 5 Study Questions Key Fall 2010
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[10 pts] Restriction enzymes recognize DNA sequences that have 2-fold symmetry. The following
DNA contains at least 5 restriction enzyme recognition sites. Find and draw a box around 5 possible
recognition sequences that are either 4 or 6 base pairs in length.
ATTGGATCCTTGGCCATCTAGAGCAGAATTCCGCGC
TAACCTAGGAACCGGTAGATCTCGTCTTAAGGCGCG
[12 pts] Shown are the DNA sequences recognized by the enzymes Bam HI, Hha III, and Hae I.
Enzymes and cleavage sites:
Bam HI
5'-G↓GATCC-3'
3'-CCTAG↑G-5'
Hha I
Hae III
5'-GCG↓C-3' 5'-GG↓CC-3'
3'-C↑GCG-5' 3'-CC↑GG-5'
Indicate the fragments obtained from the given DNA sequence after cleavage with these enzymes.
[Arrows or boxes work well to mark the sequences to indicate the cuts and fragments
obtained by each enzyme]
a)
Bam HI fragments [2 pts]
5'-GAATTCCCGGCGCTTTG
GATCCATGGCCATGGCCATT-3'
3'-CTTAAGGGCCGCGAAACCTAG
GTACCGGTACCGGTAA-5'
b)
Hha I fragments [2 pts]
5'-GAATTCCCGGCG
CTTTGGATCCATGGCCATGGCCATT-3'
3'-CTTAAGGGCC
GCGAAACCTAGGTACCGGTACCGGTAA-5'
c)
Hae III fragments [4 pts]
5'-GAATTCCCGGCGCTTTGGATCCATGG
3'-CTTAAGGGCCGCGAAACCTAGGTACC
CCATGG
GGTACC
CCATT-3'
GGTAA-5'
DNA fragments can be visualized in polyacrylamide gel electrophoresis by staining with ethidium
bromide , which binds to double-stranded DNA and fluoresces an intense orange when irradiated
by UV light.
Restriction endonucleases cleave DNA at sites with inverted repeat sequences referred to as
palendromic sequences.
The biological role of restriction enzymes in bacteria is to
A) repair DNA.
D) All of the above.
B) induce DNA crossover.
E) None of the above.
C) cleave foreign DNA.
Which of the following DNA sequences contains an 8 base palindromic site? (Note: Only one strand
is shown.)
A) CAGTCC
D) GAGAGAGA
B) GCATCC
E) GCATATGC
C) CGATTAGC
The first three bases of the 6-base recognition cleavage site of HindIII are AAG. What is the
complete sequence of this 6 bp site?
A) AAGAAG
D) AAGCUU
B) AAGCTT
E) None of the above.
C) AAGGAA
Design a potential DNA-restriction enzyme site. Show both strands.
Answer: Any palindromic site of four or more base pairs would be appropriate.
How can DNA fragments of various sizes be separated?
Answer: Electrophoresis is used to separate DNA. Fragments migrate according to
size, with larger fragments migrating more slowly than smaller fragments. Agarose gels
are used for larger fragments. Polyacrylamide is used for shorter fragments, and can
resolve fragments differing by as little as one base. Several techniques for visualization
can be used, including ethidium bromide staining, or autoradiography.
What is a DNA probe and how is it used to identify a specific DNA restriction fragment?
Answer: A DNA probe is used to identify specific DNA fragments. The probe sequence
is complementary to the DNA sequence of interest, thus it can base pair, or hybridize,
with the DNA strand. Probes can be small oligomers or long fragments. They are
labeled for detection using radioactivity or another tag, such as fluorescent probes.
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BIOCHEMISTRY 461
Dr. Bourque
Chapter 5 Study Questions Key
Fall 2009
[8 pts] Name 4 necessary and different features which make DNA sequencing possible by the chain
termination of DNA synthesis method.
Answer: two points each for any four of the following - Controlled random chain terminations for each base
- one reaction for each base
- dideoxyribonucleotides
- DNA polymerase
- gel electrophoresis which can resolve DNA fragments differing by one base
- ability to label so that products of reaction are detectable
- equimolar mixture of reaction products
- other unique answers may also qualify for credit
[2 pts] Show the expected full-length sequence of the growing DNA primer in this template-primer
complex:
(template)
3' - (base paired)-GGTCAATGACAC -5'
(primer) 5' - 32P-(base paired)-CCAGTTACTGTGOH - 3'
[8 pts] The above primer is extended by synthesis in DNA sequencing reactions in a typical chain
termination sequencing experiment. The following illustration shows a DNA gel electrophoresis
pattern of DNA fragments from which the 12-base sequence of the extended primer can be deduced.
Label each lane correctly with the name of the dideoxynucleotide base (A,T,G, or C) that must be
used to produce this data.
[2 pts each for naming the lanes correctly]
(Name the base for each gel lane below)
_T_ _G_ _C_ _A_
(Larger)
(Smaller)
[2 points] Starting with 1 copy of a double-stranded DNA, how many copies of that DNA will there
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be after 4 repetitive cycles of PCR (polymerase chain reaction)
16 copies
[1 point] Current DNA sequencing methods use fluorescence
labeling of dNTPs to
permit automated detection of chain termination synthesis reaction products for all four DNA bases.
[1 point] What absolutely necessary component of a chain termination DNA sequencing reaction
mixture is missing from the following list:
Primer
dNTPs
template DNA
a dideoxy-NTP
DNA polymerase
a “label” to detect the reaction products
[1 point] Chemical synthesis of small DNA molecules proceeds from the 3'
[10 pts]
to the 5'
end.
This question refers to the roles of the following enzymes/chemicals in construction of a
cDNA library. Write the number of the description on the right hand column in the
blank space to which it refers in the left hand column.
__3___ DNA ligase
(1)
synthesizes the first strand of cDNA as
a complement to the mRNA template
(2)
hydrolyzes the mRNA after synthesis of
the first cDNA strand
(3)
links the cDNA to the vector DNA
(4)
Synthesizes the second strand of the
cDNA
(5)
primes first cDNA strand synthesis
__5___ oligo dT
__2___ NaOH
___1__ Reverse transcriptase
___4__ DNA polymerase
[1 pt each ] ANSWER TRUE OR FALSE
F
If a gene is inserted into a tetracycline antibiotic resistance gene in a plasmid, host cells
containing the resulting recombinant DNA plasmid will grow in the presence of tetracycline.
T
An elephant hemoglobin cDNA can be expressed as a translatable mRNA in E.coli cells.
Fill in the Blank Questions [12 pts - 1 pt for each answer]
The enzyme that catalyzes the formation of a phosphodiester linkage at a break in a DNA strand is
DNA ligase .
Restriction endonucleases cleave DNA at sites with inverted repeat sequences referred to as
palindromic sequences.
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Complementary, single-strand overhangs that are produced by some restriction endonucleases are
referred to as sticky ends or cohesive ends .
The Sanger technique for sequencing DNA involves the use of 2',3'-dideoxy
that terminate chain elongation.
nucleotide analogs
In solid-phase synthesis of oligonucleotides, the 2'deoxyribonucleotide-3'-phosphoramidite is added
to the _5’_ end of the growing oligonucleotide.
PCR is the abbreviation for Polymerase Chain Reaction which is an in-vitro DNA replication
technique used to make multiple copies of a DNA molecule.
Bacterial plasmid DNA and bacteriophage DNA are commonly used ___vectors___________ to
introduce foreign DNA into a bacterium.
The enzyme terminal transferase
can be used to add nucleotides to the 3' end of DNA.
Complimentary DNA (cDNA) is formed by the action of reverse transcriptase on __mRNA__.
DNA fragments can be visualized in polyacrylamide gel electrophoresis by staining with ethidium
bromide which binds to double-stranded DNA by intercalation (insertion) between base pairs and
which fluoresces an intense orange color when irradiated by UV light.
Multiple Choice Questions [2 pts each]
1. The biological role of restriction enzymes in bacteria is to
A) repair DNA.
D) All of the above.
B) induce DNA crossover.
E) None of the above.
C) cleave foreign DNA.
Ans: C
2. What do Southern, Northern, and Western blots detect, respectively?
A) DNA, RNA, and protein D) protein, DNA, and RNA
B) DNA, protein, and RNA E) RNA, protein, and DNA
C) RNA, DNA, and protein
Ans: A
3. Reagents necessary for sequencing by chain termination include
A) template DNA, deoxyribonucleoside triphosphates (dNTPs), primer,
dideoxy nucleotide analogs, DNA polymerase, and radioactive probe.
B) template DNA, deoxyribonucleoside triphosphates (dNTPs), primer,
dideoxy nucleotide analogs, and DNA polymerase.
C) template DNA, deoxyribonucleoside triphosphates (dNTPs), primer,
dideoxy nucleotide analogs, RNA polymerase.
D) All of the above.
E) None of the above
Ans: B
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4. Plasmids used in recombinant DNA technology typically
A) possess a gene for antibiotic resistance.
B) replicate independently of the host genome.
C) are circular double stranded molecules.
D) all of the above.
E) a and b.
Ans: D
5. A polylinker site contains
A) many closely spaced restriction enzyme sites.
B) links for antibiotic resistance.
C) sequences allowing linkage to mRNA.
D) All of the above.
E) None of the above.
Ans: A
6. Why are met and trp often used to design DNA probes from amino acid
sequences?
A) They have ony one possible codon sequence each..
B) Met is the first amino acid in the protein chain.
C) Both are used often in proteins.
D) All of the above.
E) None of the above.
Ans: A
7. For identification of a gene, to which strand of DNA must the probe be complementary?
A) either strand
D) only the template strand
B) both strands
E) None of the above.
C) only the coding strand
Ans: A
8. Reverse transcriptase is normally found in
A) plants.
D) All of the above.
B) retrovirus.
E) None of the above.
C) mitochondria.
Ans: B
9. The probe used to isolate a gene from a genomic library is often
A) the ligand that binds to the protein.
D) All of the above.
B) its promoter region.
E) None of the above.
C) cDNA constructed from an mRNA of the gene.
Ans: C
10. Genes can be inserted into eukaryotic cells by
A) viruses.
D) All of the above.
B) chemical treatment.
E) None of the above.
C) microinjection.
Ans: D
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11. Animals that harbor a foreign gene as a result of recombinant gene manipulation
are called
A) transgenic.
D) All of the above.
B) mutants.
E) None of the above.
C) aliens.
Ans: A
Short-Answer Questions
1. A number of tools are critical to gene exploration. Name at least four.
Answer: Tools include restriction enzyme analysis, blotting techniques (Southern
and Northern), DNA sequencing, solid-phase synthesis of nucleic acid, PCR, computer
analysis, other answers may be correct.
2. How can DNA fragments of various sizes be separated?
Answer: Electrophoresis is used to separate DNA. Fragments migrate according to
size, with larger fragments migrating more slowly than smaller fragments.
Agarose gels are used for larger fragments. Polyacrylamide is used for shorter
fragments, and can resolve fragments differing by as little as one base. Several
techniques for visualization can be used, including ethidium bromide staining, or
autoradiography.
3. What is a DNA probe?
Answer: A DNA probe is used to identify specific DNA fragments. The probe
sequence is complementary to the DNA sequence of interest, thus it can base
pair, or hybridize, with the DNA strand. Probes can be small oligomers or long
fragments. They are labeled for detection using radioactivity or another tag, such
as fluorescent probes.
4. What are some important aspects that are the basis of the Sanger DNA sequencing method?
Answer: Sanger sequencing is based upon replication of DNA under conditions in
which the chain is terminated by controlled interruption, resulting in various sizes
of DNA fragments. By including limited amounts of radioactive bases, strands
differing by one base in length can be observed. By reading the bands, the
sequence can be determined. (Fluorescent or dye tags can also be used in an
automated process.)
5. Explain the basis of the polymerase chain reaction.
Answer: Using primers that flank the sequence of interest and carrying out
repetitive replication reactions can make copies of a particular DNA fragment. Template
DNA, DNA polymerase, dNTPs, and other necessary reagents are added to the
reaction mixture. The DNA is heated to separate the strands, cooled to allow the
primers to anneal, and then replicated. The process is repeated many times,
each time doubling the number of DNA copies present. The enzyme used is
thermostable, and can withstand the repetitive heating and cooling steps.
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6. Describe two ways PCR can be used in medical diagnosis.
Answer: PCR can be used to identify the presence of infecting bacteria or viruses;
can detect if certain mutations, such as those found in cancer cells, are present;
and can be used as a diagnostic tool to investigate known gene mutations. [etc...]
7. Briefly outline the steps necessary to create a recombinant DNA molecule.
Answer: Both the fragment of interest and the vector DNA are cut with restriction
enzyme(s), which create complementary sticky ends. The pieces of DNA are
allowed to anneal and DNA ligase is added to join the ends.
8. How is a single gene of interest identified on a plate containing many different gene
library clones?
Answer: By using a probe specific for the DNA of interest, the clone can be
identified. The probe is designed to hybridize to the DNA of the clone that has
been transferred to a membrane. The probe is labeled with radioactivity or
another tag so that it can be easily detected and the proper clone identified and
selected from the original plate.
10. A gene is inserted into an ampicillin resistance gene in a plasmid. Will cells containing the
resulting recombinant plasmid be sensitive or resistant to ampicillin?
Answer: When inserted into a gene, the new DNA interrupts the previous gene.
Thus, the antibiotic gene is unlikely to be functional, the clone will be sensitive to
the antibiotic (, and the clone will not grow.
11. Briefly outline how a cDNA library is made.
Answer: Reverse transcriptase is used to convert mRNA molecules into DNA, and
the RNA is then digested away by alkali. Terminal transferase is used to add
several residues to the end of the fragment, such as G. Then, a complementary
primer, such as polyC, can be used to synthesize the opposite strand. Linkers
can be added so the fragment can be inserted into a vector, and then into a
host, such as bacteria.
12. Why are foreign genes often inserted with a different promoter, such as using the
metallothionein gene promoter 5' to a growth hormone gene?
Answer: Promoters are selected that are easy to control, and can be turned on or
off as desired. Thus, metallothionein, which is induced by the presence of heavy
metals, can be mediated by altering metals concentrations.
13. How is gene disruption used to determine the function of a gene?
Answer: If a gene is completely disrupted (also called gene knockout), a functioning
protein is not made. By examining the knockout organism, clues to the protein's
function can be made by observation and testing.
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14. How is a gene gun used?
Answer: DNA is coated onto tungsten pellets, and the microprojectiles are shot into
the tissue at very high velocity.
15. What advantage can be gained by splicing together portions of two different
genes?
Answer: This technique allows the creation of novel bifunctional genes. Distinct
functional domains are paired and aligned into one gene, often resulting in
proteins with unique characteristics and activities.
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