Micro chapter 9
Compare selection and mutation.
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If you put a gene in a virus, the next step in genetic modification would be a.insertion of a plasmid. b.transformation. c.transduction. d.PCR. e.Southern blotting.
C
If you put the gene for Bt-toxin from Bacillus thuringiensis into a tomato plant, the resulting plants will
Be toxic to the insects that eats the plants
The use of microorganisms, cells, or cell components to make products such as hormones, antibiotics, food, or vaccines is known as
Biotechnology
To express a human gene in a bacterium, cDNA must be made because bacteria
Cannont remove introns
DNA fingerprints are actually
DNA fragment
Recombinant DNA can be introduced into a host cell by any of the following methods
Polymerase chain reaction
NAME IT This virus family, normally associated with AIDS, may be useful for gene therapy.
Retroviridae
The shotgun sequencing technique is used to
Sequence entire genomes
The following steps are necessary to clone eukaryotic genes in bacteria. What is the third step?
Splice exons together
Sequencing a genome provides
the order of nucleotides in a genome
Which of the following statements correctly differentiates a genomic library from a cDNA library?
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All of the following are true of the polymerase chain reaction (PCR)
A heat-stable DNA polymerase is used in the reaction process. Billions of copies of a DNA sequence are made in a few hours. An automated thermocycler is used to heat and cool the reaction samples. Short pieces of DNA called primers are added to the reaction mixtures.
The process of making multiple copies of a DNA molecule is referred to as
Amplification
Restriction enzymes were first discovered with the observation that a.DNA is restricted to the nucleus. b.phage DNA is destroyed in a host cell. c.foreign DNA is kept out of a cell. d.foreign DNA is restricted to the cytoplasm. e.all of the above
B
The following enzymes are used to make cDNA. What is the second enzyme used to make cDNA? a.reverse transcriptase b.ribozyme c.RNA polymerase d.DNA polymerase
B
Which of the following is the fourth basic step to genetically modify a cell? a.transformation b.ligation c.plasmid cleavage d.restriction-enzyme digestion of gene e.isolation of gene
B
The following sequential steps are used to make a recombinant cell. Which of these steps occurs LAST? Insert gene into a vector. Vector is taken up by cell. Grow cells containing vector with gene. Isolate gene of interest.
Grow cells containing vector with gene.
Describe a recombinant DNA experiment in two or three sentences. Use the following terms: intron, exon, DNA, mRNA, cDNA, RNA polymerase, reverse transcriptase.
In a eukaryotic cell, RNA polymerase copies DNA; RNA processing removes the introns, leaving the exons in the mRNA. cDNA can be made from the mRNA by reverse transcriptase.
Differentiate the following terms. Which one is "hit and miss"—that is, does not add a specific gene to a cell? a.protoplast fusion b.gene gun c.microinjection d.electroporation
In protoplast fusion, two wall-less cells fuse together to combine their DNA. A variety of genotypes can result from this process. In b, c, and d, specific genes are inserted directly into the cell.
Pieces of human DNA stored in yeast cells.
Library
Which of the following statements is true regarding the polymerase chain reaction (PCR)?
PCR is used to make copies of DNA in vitro.
Which of the following can be used as vectors to genetically modify cells?
Plasmids, shuttle vectors, and viruses
In the blue-white screening procedure, bacteria that are transformed with recombinant plasmid and cultured in media containing ampicillin and X-gal will
Produce white colonies
Which of the following might specifically be used as part of a reverse genetics approach to studying a gene?
RNA interference
Restriction enzymes
Recognize palindromic double standed sequences of DNA and cut the DNA in a specific location Restriction enzymes are useful in genetic engineering when they make staggered cuts in DNA.
Which of these answers is true for restriction enzymes
Restriction enzymes are useful in genetic engineering when they make staggered cuts in DNA.
What are the functions of reverse transcriptase in making cDNA
Reverse transcriptase copies mRNA into single-stranded DNA, as well as Reverse transcriptase destroys the original RNA of the mRNA-DNA hybrid.
If a recombinant plasmid is put in solution with E. coli, the bacteria may pick up the plasmid by
Transformation
The basic steps to genetically modify a cell are listed below. Which step would come LAST?
Transformation
Self-replicating DNA for transmitting a gene from one organism to another.
Vector
Design an experiment using vaccinia virus to make a vaccine against the AIDS virus (HIV).
You could engineer a vaccinia virus to express HIV proteins, then use the engineered virus as a vaccine. The engineered vaccinia might express the HIV spike protein and induce neutralizing antibodies, or it might express some non-structural protein and induce a CMI response.
You are attempting to insert a gene for saltwater tolerance into a plant by using the Ti plasmid. In addition to the desired gene, you add a gene for tetracycline resistance (tetR) to the plasmid. What is the purpose of the tetR gene?
You probably used a few plant cells in a Petri plate for your experiment. You can grow these cells on plant-cell culture media with tetracycline. Only the cells with the new plasmid will grow.
Some commonly used restriction enzymes are listed in Table 9.1 on page 248. a.Indicate which enzymes produce sticky ends. b.Of what value are sticky ends in making recombinant DNA?
a.BamHI, EcoRI, and HindIII make sticky ends. b.Fragments of DNA produced with the same restriction enzyme will spontaneously anneal to each other at their sticky ends.
Assume you insert a specific gene into a plasmid and use blue-white screening. You plate the transformed E. coli cells on an ampicillin X-gal medium. Cells that produce blue colonies are
ampicillin resistant but do not contain the new gene
Recombinant DNA technology is used for all of the following
amplification of DNA for microbe identification insertion of genes from humans or plants into bacteria or viruses hepatitis B vaccine production using yeast cells human insulin production by bacterial cells
When two DNA pieces cut with the same restriction enzyme are combined, sticky ends will
associate by complementary base pairing and hydrogen bonds
If DNA ligase was not used in the creation of a recombinant plasmid,
base-pairing would occur but the sugar phosphate backbone would not be connected
For the introduction of a genetically modified plasmid into E. coli,
calcium chloride and heat shock can be used
Which of the following can be used as vectors to genetically modify cells?
calcium chloride and heat shock can be used
In nature, the function of restriction enzymes is to
destroy bacteriophage DNA
All of the following are benefits and improvements made possible by recombinant DNA technology
development of genetic screening procedures for early detection of genetic diseases development of new, safer vaccines Improed weed and pest control
Which of the following is an advantage of obtaining the protein product human growth hormone by recombinant DNA technology rather than extraction from cadavers?
eliminates the need to extract the protein from tissues that might harbor pathogens purity speed cost-effectiveness
cDNA is made from a
mRNA templates
In genetic engineering, antibiotic resistance is often cloned into a vector to
make direct selection of a clone possible
List the four properties of vectors.
most important property is self-replication; serve as vehicles for the replication of desired DNA sequences Preservation
Which of the following is an example of a cloning vector
plasmid
step in Southern blotting?
transfer of DNA fragments to filters addition of radioactive probe made from the gene of interest digestion of sample DNA with restriction enzyme separation of DNA fragments by gel electrophoresis
A good cloning vector
should have a gene or genes that allows for selection of transformed host cells
Which of the following statements correctly differentiates biotechnology and rDNA technology
Biotechnology involves any use of microorganisms or cells to make products, regardless of the means used. rDNA technology involves the specific use of molecular modifications in microorganisms or cells, in which a gene from one cell is inserted into another cell, altering the recipient cell to make some desired product.
the following purpose of genetic modification
Create multiple copies of a gene of interest. Create hormones such as insulin or human growth hormone. Create proteins used in vaccines (e.g., hepatitis B vaccine). Modify the characteristics of an organism.
An ampicillin-sensitive culture of E. coli is transformed with a plasmid that contains the gene of interest plus an ampicillin-resistant gene. If it is then plated on an ampicillin-containing growth medium:
only the bacteria with the plasmid will grow
Why did the use of DNA polymerase from the bacterium Thermus aquaticus allow researchers to add the necessary reagents to tubes in a preprogrammed heating block?
Taq Polymerase stable at high temps - 72 degrees • Added for extension of DNA synthesis • Can read template strand and match to complementary nucleosides rapidly. Application: PCR copies DNA - allows for AIDS to be detected in early stages
For Agrobacterium tumefaciens to be used to introduce foreign DNA into a plant cell, that DNA must first be
inserted into the T-DNA region of the Ti plasmid of A. tumefaciens
Compare and contrast the following terms: a. cDNA and gene restriction fragment and gene c.DNA probe and gene d.DNA polymerase and DNA ligase e.rDNA and cDNA f.genome and proteome
. a.Both are DNA. cDNA is a segment of DNA made by RNA-dependent DNA polymerase. It is not necessarily a gene; a gene is a transcribable unit of DNA that codes for protein or RNA. b.Both are DNA. A restriction fragment is a segment of DNA produced when a restriction endonuclease hydrolyzes DNA. It is not usually a gene; a gene is a transcribable unit of DNA that codes for protein or RNA. c.Both are DNA. A DNA probe is a short, single-stranded piece of DNA. It is not a gene; a gene is a transcribable unit of DNA that codes for protein or RNA. d.Both are enzymes. DNA polymerase synthesizes DNA one nucleotide at a time using a DNA template; DNA ligase joins pieces (strands of nucleotides) together. e.Both are DNA. Recombinant DNA results from joining DNA from two different sources; cDNA results from copying a strand of RNA. f.The proteome is the expression of the genome. An organism's genome is one complete copy of its genetic information. The proteins encoded by this genetic material comprise the proteome.
Match the following choices to the statements in questions 7 through 10. a.antisense b.clone c.library d.Southern blot e.vector
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The following picture shows bacterial colonies growing on X-gal plus ampicillin in a blue-white screening test. Which colonies have the recombinant plasmid? The small satellite colonies do not have the plasmid. Why did they start growing on the medium 48 hours after the larger colonies?
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A population of cells carrying a desired plasmid.
Clone
A gene that hybridizes with mRNA.
Antisense
The DNA probe, 3' -GGCTTA, will hybridize with which of the following? a.5' -CCGUUA b.5' -CCGAAT c.5' -GGCTTA d.3' -CCGAAT e.3' -GGCAAU
B
You have a small gene that you want replicated by PCR. You add radioactively labeled nucleotides to the PCR thermal cycler. After three replication cycles, what percentage of the DNA single strands are radioactively labeled? a.0% b.12.5% c.50% d.87.5% e.100%
D
A plasmid that has been cleaved with EcoRI can recombine with another plasmid that has been
Digested with EcoRI
Special considerations must be taken when using bacteria to produce a eukaryotic protein. What is the cause for this additional difficulty?
Eukaryotic genes contain introns, which prokaryotic cells cannot remove.
Southern blotting is used to
Identify particuoar seuqencez of DNA
How does RNAi "silence" a gene?
In RNAi, siRNA binds mRNA creating double-stranded RNA, which is enzymatically destroyed.
What type of test can determine whether these patients have contracted HIV from Dr. B.?
Reverse-transcription PCR using a primer for an HIV gene can be used to amplify DNA for analysis.
Suppose you want multiple copies of a gene you have synthesized. How would you obtain the necessary copies by cloning? By PCR?
The gene can be spliced into a plasmid and inserted into a bacterial cell. As the cell grows, the number of plasmids will increase. The polymerase chain reaction can make copies of a gene using DNA polymerase and a primer for the gene.
During the Southern blotting technique, what is the purpose of transferring the DNA fragments from the gel to a nitrocellulose filter?
This step attaches the DNA fragments to a permanent substrate, which then can be probed.
Which of the following is a safety issue related to the use of recombinant DNA?
allergic reactions to components in genetically modified foods, reduction in populations of "desirable" insects due to the use of Bt insecticide, and spread of bioengineered traits, such as herbicide resistance, into related weed species
n genetic engineering, antibiotic-resistance genes are usually cloned into vectors to
allow selection for bacteria containing the vector
Genetic technology has enabled screening for a variety of genetic conditions, and use of this technology is becoming more widely available. Which of the following is likely to become an important issue that will need to be addressed?
the need for legislation to protect the privacy of individuals' genetic information