Download Document

Biotechnology Chapter 20
Homework 5-1 – Basics of DNA Recombination and Gene Cloning
Powerpoint: Gene cloning 2010.
Reading: Campbell pp. 396- 403.
1) What does the term Recombinant DNA mean?
2A) List three potential benefits of inserting a gene from one organism into another.
2B) List three potential risks of inserting a gene from one organism into another.
3A) What is gene cloning?
3B) What is a plasmid?
3C) What is a restriction enzyme?
3D) What kind of sequences do restriction enzymes typically recognize?
3E) Why are restriction enzymes useful in biotechnology?
4) List the 5 steps necessary to clone a gene.
5) Inserting a Eurkaryotic gene in a bacterial host:
A) Why can’t a eukaryotic gene simply be inserted directly into a bacterial host?
B) What does the enzyme reverse transcriptase do? What organism is the source
of reverse transcriptase?
C) Explain the process for preparing eukaryotic genes for insertion into a bacterial
host.
6) What enzyme is needed to seal the nicks after sticky ends are combined?
7) What is meant by the term transformation? How can transformation be
accomplished in the lab?
8) List 4 different methods for screening for cell colonies that contained the desired
DNA insert.
9) What is a DNA library?
10) Campbell p. 405, Concept check 20.1, #1 and 2.
5-1 Multiple Choice Practice:
1) Petroleum-lysing bacteria are being engineered for the removal of oil spills. What is
the most realistic danger of these bacteria to the environment?
A) Mutations leading to the production of a strain pathogenic to humans
B) Extinction of natural microbes due to the competitive advantage of the “petrobacterium”
C) Destruction of natural oil deposits
D) Poisoning of the food chain
2) The role of restriction enzymes in DNA technology is to
A) Provide a vector for the transfer of recombinant DNA.
B) Produce cDNA from mRNA
C) Produce a cut (usually staggered) at specific recognition sequences on DNA.
D) Reseal “sticky ends” after base-pairing of complementary bases
E) Digest DNA into single strands that can hybridize with complementary sequences.
3) Which of the following DNA sequences would be most likely to be a restriction site?
(Hint: Look for palindromic sequence in choices)
a) AACCGG
TTGGCC
b) AAGG
T T CC
c) CTGCAG
GACGTC
4) Which of the following is a difficulty in getting prokaryotic cells to express
eukaryotic genes?
A) The genetic code differs between the two because prokaryotes substitute the base
uracil for thymine
B) The ribosomes of prokaryotes substitute the base uracil for thymine.
C) The signals that control gene expression are different and prokaryotic promoter
regions must be added to the vector.
D) Prokaryotic cells cannot splice out introns.
E) C + D are both true.
5) Yeast has become important in genetic engineering because it
A) Has RNA splicing machinery.
B) Has plasmids that can be genetically engineered
C) Allows the study of eukaryotic gene regulation and expression
D) Grows steadily and rapidly in the laboratory.
E) Does all of the above.
6) A plasmid has two-antibiotic-resistance genes, one for ampicillin and one for
tetracycline. It is treated with a restriction enzyme that cuts in the middle of the
ampicillin gene. DNA fragments containing a human globin gene were cut with the
same enzyme. The plasmids and fragments are mixed, ligase, and used transform
bacterial cells. Clones that have taken up the recombinant DNA are the ones that
A) Are blue and can grow on plates with both antibiotics
B) Can grow on plates with ampicillin but not with tetracycline
C) Can grow on plates with tetracycline but not with ampicillin
D) Cannot grow with any antibiotics.
E) Can grow on plates on tetracycline and are not blue.
MC Answers on last page of packet.
5-2 Biotechnology Techniques
Powerpoint: Biotechnology Techniques 2012
Reading: Campbell, p. 403-411, especially studying insert Research method boxes and
419 to 420.
1) DNA microarray
A) What is the purpose of a DNA microarray?
B) Give three examples of applications of microarray experiments.
C) Campbell p. 411, Concept Check, #3 (What If?)
2) Gel Electrophoresis
A) Describe a gel electrophoresis experiment. What is the purpose of gel electrophoresis?
B) Why does DNA move toward the positive electrode rather than the negative electrode
in gel electrophoresis?
C) In a particular gel, 3 bands of DNA are visible. Band A has moved 3 cm from
the starting point, band B has moved 5 cm, and band C has moved 6.5 cm.
Which band contains the smallest molecular weight DNA? How do you know?
D) Since DNA is invisible to the naked eye, how do we visualize the bands of DNA?
Analyzing Fragments formed by Digestion of DNA by Restriction Enzymes
3) Linear vs. Circular DNA - If you start with linear DNA, how many cut sites do there
have to be to produce 2 fragments that will run separately on a gel? If you start with a
circular plasmid of DNA, how many cut sites do there have to be to produce 2 fragments
that will run separately on a gel?
4)This segment of DNA has restriction sites I and II which create restriction
fragments a,b, and c. Which of the following gel(s) produced by
electrophoresis would represent the separation and identity of these
fragments?
5) Given the following restriction map
A
B
C
D
Draw a sketch of the pattern that would be obtained if the 4 fragments were separated
by gel electrophoresis.
.
7) Polymerase Chain Reaction (PCR)
A) What is PCR and how is it used?
B) What is the difference between PCR and Gene Cloning? What are the advantages
and disadvantages of each technique?
C) List the 3 steps in one PCR cycle.
D) Why is the polymerase used in automated PCR systems taken from a bacteria
that lives in a hot springs?
E) What happens to the number copies of the target DNA after each cycle?
8) Southern Blot
A) What is the purpose of a Southern Blot?
B) Briefly describe how a Southern Blot experiment is conducted.
9) DNA Fingerprinting
A) What is a Restriction Fragment Length Polymorphorism (RFLP)?
B) What regions of our DNA are most useful in identifying individuals? Why aren’t
coding regions of our DNA (genes) very useful?
C) Draw a diagram and also explain verbally why regions that contain no differences in
the individual repeat DNA sequences (Short Tandem Repeats or STR’s) are so useful in
distinguishing individuals. (Specifically, the sequence of an individual repeat is identical
between individuals but what is different between individuals?)
D) What is a DNA fingerprint? How is a DNA fingerprint made?
E) Give two examples of how DNA fingerprints are used.
F) Who is the father of the baby in the DNA fingerprint below?
10) One application of DNA fingerprinting technology has been to identify stolen children and
return them to their parents. Bobby Larson was taken from a supermarket parking lot in New
Jersey in 1978,when he was 4 years old. In 1990, a 16-year-old boy called Ronald Scott was
found in California, living with a couple named Susan and James Scott, who claimed to be his
parents. Authorities suspected that Susan and James Scott might be the kidnappers and that
Ronald Scott might be Bobby Larson. DNA samples were obtained from Mr. And Mrs. Larson
and from Ronald, Susan, and James Scott. PCR of several VNTR loci was used to generate a
DNA fingerprint of each individual with the results shown in the figure below.
From the information in the figure, what can you say about the parentage of
Ronald Scott? Explain.
5-3 Applications of Biotechnology
– Reading Campbells, pp. 412 -420; 426-432.
- Powerpoint: DNA Sequencing and the Human Genome Project Powerpoint
- 1) Sanger Sequencing Method
- A) What is does the Sanger Sequencing method accomplish?
- B) What is a dideoxyribonucleotide? What happens to the elongation of a DNA
strand when a dideoxyribonucleotide is incorporated in place of a normal
deoxyribonucleotide?
- C) Explain how is the sequence of nucleotides determined.
Human Genome Project
2A) What is the Human Genome Project?
2B) (IB Question): Outline 3 outcomes of the Human Genome project.
2C) Discuss potential benefits of knowing the sequence of a person’s genes.
2D) Discuss potential concerns about knowing the sequence of person’s genes.
5-4 Gene Therapy,Cloning, Stem Cells
Gene Therapy, Cloning Organims, Stem Cells Powerpoint
1A) What is gene therapy?
1B) What kind of diseases are currently targets of gene therapy?
1C) What vector is commonly used to insert DNA into new host cells?
1D) What are the risks of gene therapy?
Cloning of an organism from differentiated cells
2A) Who is Dolly the Sheep? What is different about her?
2B) List the steps necessary to do cloning using differentiated cells.
2C) How efficiently does this technique work?
2D) What are the potential benefits of cloning organisms?
2E) What are some of the potential concerns of cloning organisms?
Production of differentiated tissue cells from stem cells
3A) What are stem cells? What are differentiated cells?
3B) State two potential medical benefits of stem cell research.
3C) Why is the harvesting of stem cells from embryos so controversial in the US?
3D) Breakthroughs: What important breakthroughs took place in 2007 and in 2010?
5-5 Unit Review
PART 1: Multiple Choice (Answers at end).
Use the following choices to answer questions #1-3.
A) Restriction enzyme
B) Reverse transcriptase
C) Ligase
D) DNA polymerase
E) RNA nuclease
1) Which is the first enzyme used for the production of cDNA
2) Which enzyme is used in the polymerase chain reaction?
3) Which is the first enzyme used in the production of DNA fragments for DNA
fingerprinting?
Part 2: Campbell’s Self-Quiz Problems:
p. 424, #1,5,6,8
PART 3: PRACTICE AP ESSAY:
2002 AP EXAM
1) The human genome illustrates both continuity and change.
A) Describe the essential features of two of the procedures/techniques below. For each of the
procedures/techniques you describe, explain how its application contributes to understanding
genetics.
• The use of bacterial plasmids to clone and sequence a human gene.
• Polymerase chain reaction (PCR)
• Restriction fragment length polymorphism (RFLP) analysis
B) 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.
PART 1 ANSWERS:
1) B 2) D 3) A
Answers to MC questions after 5-1:
1) B
2) C
3) C
Answer to MC for 5-2: C
Answer to 5-2 Restriction Map:
4) E
5) E
6) C
Answer to First Paternity Problem: Sample 4
Answer to Paternity Kidnapping Case: The Larsons are likely to be Ronald
Scott’s parents. Almost all of his bands on the gel are
found in either Mr or Mrs Larson. There is not a significant match with the
Scotts.