BIOL 261: CH. 12 - BIOTECHNOLOGY AND SYNTHETIC BIOLOGY

Why is the use of thermostable DNA polymerase critical in PCR?

The use of thermostable DNA polymerase is critical in PCR because the reaction involves high temperatures that would denature regular DNA polymerases.

What are examples of thermostable DNA polymerases used in PCR?

Examples of thermostable DNA polymerases used in PCR include Taq polymerase and Pfu polymerase.

Why is Pfu polymerase useful in PCR when high accuracy is crucial?

Pfu polymerase is useful in PCR when high accuracy is crucial because it has proofreading activity, allowing it to correct errors during DNA synthesis.

How have genes for thermostable DNA polymerases been used for commercial production?

Genes for thermostable DNA polymerases have been cloned into Escherichia coli for commercial production, allowing for large-scale enzyme production.

From what organism is Taq polymerase isolated?

Taq polymerase is isolated from Thermus aquaticus, a thermophilic bacterium.

What does gel electrophoresis employ to separate nucleic acids by size and charge?

Gel electrophoresis employs an agarose gel to separate nucleic acids by size and charge.

What is gel electrophoresis used to separate?

Gel electrophoresis is used to separate nucleic acids based on their size and charge.

What is the first step in cloning a gene of interest into E. coli?

The first step is to isolate the gene.

What is an example of a gene that can be cloned?

An example is the gene for insulin.

Where is the insulin gene found?

It is found in pancreatic cells.

How is DNA isolated from pancreatic cells?

DNA is isolated by cutting it with restriction enzymes.

What do restriction enzymes do?

They cut DNA at specific restriction sites.

What are the two types of ends produced by restriction enzymes?

They can produce sticky ends or blunt ends.

What is an example of a restriction enzyme?

An example is EcoRI.

What technique is used to separate DNA fragments after cutting?

Electrophoresis is used to separate DNA fragments into bands.

What stain is used to visualize DNA in gel electrophoresis?

Ethidium bromide is used to stain the gel.

How is stained DNA visualized?

A transilluminator (UV light) is used to make ethidium bromide fluoresce.

What happens after DNA bands are visualized?

The DNA bands are cut off from the gel.

What technique is used to make more copies of the DNA?

PCR (polymerase chain reaction) is used.

What machine is used in PCR?

A thermocycler is used, which heats up and cools down the reaction.

What is a key enzyme used in PCR?

Taq polymerase is used in PCR.

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What experiment is set up to introduce the cloned gene into E. coli?

A transformation experiment is set up.

Is E. coli naturally competent for transformation?

No, E. coli is not naturally competent.

How is E. coli made competent?

It is treated with cold Ca²⁺.

What is used to carry the gene into E. coli?

A plasmid is used.

What is an example of a cloning vector plasmid?

An example is pUC19, which is a double-stranded DNA plasmid.

What does the LacZ gene code for?

LacZ codes for beta-galactosidase, an enzyme that breaks lactose into glucose and galactose.

What color indicates that beta-galactosidase is being produced?

Blue indicates that beta-galactosidase is being produced.

What color indicates that beta-galactosidase is not being produced, and what does this mean?

White indicates that beta-galactosidase is not being produced, meaning these are mutant colonies that contain the gene of interest.

What enzyme is used to cut the pUC19 plasmid and the foreign DNA?

EcoRI is used to cut the pUC19 plasmid and the foreign DNA.

What is mixed with the cut plasmid to facilitate DNA insertion?

The cut plasmid is mixed with the foreign DNA and ligase.

What is the next step after mixing the plasmid/DNA with ligase?

The plasmid/DNA mixture is transformed into competent E. coli.

How is E. coli prepared for transformation?

E. coli is shocked with cold Ca2+ before adding the plasmid/DNA mixture.

What happens after E. coli is incubated with the plasmid/DNA mixture?

The cells are plated on X-gal and ampicillin media.

What is done after plating the transformed E. coli?

The plates are incubated overnight.

What type of colonies should be looked for after incubation?

White colonies should be looked for after incubation.

What is done with the white colonies?

The white colonies are transferred to fresh media to obtain a pure culture of mutant cells.

What is the next step after obtaining a pure culture of mutant cells?

The next step is to determine what the inserted DNA codes for.

What is Agrobacterium tumefaciens?

Agrobacterium tumefaciens is a plant pathogen that contains the Ti (tumor-inducing) plasmid.

What is the function of the Ti plasmid in Agrobacterium tumefaciens?

The Ti plasmid can be used to transfer DNA directly into certain plants.

What is Bt toxin, and what organisms does it affect?

Bt toxin from Bacillus thuringiensis, a gram-positive rod-shaped bacterium that produces endospores, is toxic to moth and butterfly larvae.

How does PCR work and when would you use it?  What is Taq?  Where did it come from and why is that beneficial?

PCR (Polymerase Chain Reaction) works by amplifying specific DNA sequences through repeated cycles of denaturation, annealing, and extension.

• It is used when there is a need to replicate small amounts of DNA to analyze or study.

• Taq polymerase is an enzyme that synthesizes DNA strands.

• It comes from Thermus aquaticus, a heat-loving bacterium, which is beneficial because it can withstand the high temperatures required for the denaturation step in PCR.

What is the purpose of molecular cloning?

The purpose of molecular cloning is to isolate and replicate a specific gene or DNA fragment in a host organism, allowing for detailed study or production of a protein.

Why is PUC19 a great cloning vector?

PUC19 is a great cloning vector because it has a high replication rate, a small size, and features like an antibiotic resistance gene and a multiple cloning site, which make it easy to insert and select for recombinant DNA.

What is EcoRI? What is it used for?

EcoRI is a restriction enzyme that cuts DNA at specific sequences.

• It is used to cleave DNA into smaller fragments for cloning or analysis, typically recognizing the sequence GAATTC.

Explain how you could clone a gene for insulin production into E. coli.

To clone a gene for insulin production into E. coli, the insulin gene would first be inserted into a plasmid vector, which is then introduced into E. coli through transformation.

• The bacteria would then express the insulin gene, producing insulin protein, which could be harvested.

What is a reporter gene? The product of which reporter gene yields a green color?

A reporter gene is a gene used to monitor the presence or activity of a gene of interest.

• The product of the GFP (Green Fluorescent Protein) reporter gene yields a green color under UV light.

What is the advantage of using genetic engineering to make insulin?

The advantage of using genetic engineering to make insulin is that it allows for the production of human insulin in large quantities, which is safer and more effective than using insulin from animal sources.

What is a transgenic plant?

A transgenic plant is a plant that has been genetically modified to contain DNA from another species, typically to enhance traits like pest resistance or improved nutritional content.

What is rBST and how is it made?  What is it used for?

rBST (recombinant bovine somatotropin) is a synthetic hormone produced by genetically engineered bacteria.

• It is used to increase milk production in dairy cows.

Explain why recombinant vaccines might be safer than some vaccines produced by tradition methods?

Recombinant vaccines might be safer because they use only specific proteins or antigens from the pathogen, reducing the risk of contamination or adverse reactions associated with live or whole-pathogen vaccines.

What are the important differences among a recombinant live attenuated vaccine, a vector vaccine and a subunit vaccine?

A recombinant live attenuated vaccine uses a weakened form of the live pathogen to stimulate an immune response.

• A vector vaccine uses a harmless virus to deliver genes from the pathogen to stimulate immunity.

• A subunit vaccine contains only specific pieces (antigens) of the pathogen rather than the whole organism, minimizing the risk of side effects.

After digesting a DNA sequence, a restriction endonuclease can generate

sticky ends.

overhangs.

blunt ends.

blunt ends, overhangs, or sticky ends.

blunt ends, overhangs, or sticky ends.

What is the temperature used for the extension step?

94 °C

60 °C

72 °C

72 °C

How do the strands separate during PCR?

The high heat of the denaturation step breaks the hydrogen bonds between the two strands.

The cycling of the temperatures breaks the hydrogen bonds between the two strands.

The primers separate the strands during the annealing step.

The DNA polymerase breaks the hydrogen bonds between the two strands.

The high heat of the denaturation step breaks the hydrogen bonds between the two strands.

What is a thermocycler?

The machine that controls the heat of the reaction, cycling between the different temperatures of the different steps during PCR

The process of cycling through the different temperatures of a PCR reaction 30 times

The special DNA polymerase, used in a PCR reaction, that can tolerate the high temperatures

The name for the DNA primers used in a PCR reaction

The machine that controls the heat of the reaction, cycling between the different temperatures of the different steps during PCR

What is the sequence of the temperatures of a typical PCR reaction?

60 °C, 72 °C, 94 °C

94 °C, 60 °C, 72 °C

94 °C, 72 °C, 60 °C

72 °C, 94 °C, 60 °C

72 °C, 60 °C, 94 °C

94 °C, 60 °C, 72 °C

True or False?
The key steps in cloning a foreign gene into a vector, regardless of the application, involve isolating the insert fragment, ligating the insert into a vector, and transforming it into a host.

True

Cells that have "insertional inactivation" of the lacZ gene are

white.

blue.

fluorescent green.

yellow.

white.

The genes encoding green fluorescent protein (GFP) and β-galactosidase are typically used in cloning as

reporter genes.

global control genes.

transcription regulators.

promoter sequences.

reporter genes.

A(n) ________ gene is a gene that encodes a protein that is easy to detect and assay.

encoder

translational

recorder

reporter

reporter

The enzyme that covalently links both strands of a vector and inserted DNA in molecular cloning is

DNA phosphatase.

DNA ligase.

DNA transferase.

DNA hydrolase.

DNA ligase.

The Ti plasmid is best suited for genetically manipulating

plants.

viruses.

fish.

Agrobacterium spp.

plants.

What is the end goal of PCR?

To quickly increase the number of copies of a specific DNA sequence

To increase the pool of different DNA sequences

To allow cells to make DNA faster, thereby growing faster

To quickly increase the number of copies of a specific DNA sequence

PCR stands for

polymerase copy reaction.

polymerase chain reaction.

polymerization copying rapidly.

polymerase chain reaction.

Which of the following is an application that uses PCR?

Sequencing a gene, diagnosing a disease, and providing enough DNA for cloning into another organism

Providing enough DNA for cloning into another organism

Sequencing a gene

Diagnosing a disease

Sequencing a gene, diagnosing a disease, and providing enough DNA for cloning into another organism

Which of the following is NOT a common step in molecular cloning using plasmids?

Hybridize DNA sequences with slightly mismatched oligonucleotides.

Insert the vectors into a host.

Fragment DNA into small segments.

Ligate DNA into vectors.

Hybridize DNA sequences with slightly mismatched oligonucleotides.

True or False?
One fundamental technique of genetic engineering includes the ability to cut DNA into random fragments.

True

True or False?
An effective way to introduce DNA into plant cells is using the Ti plasmid, which comes from a plant pathogen.

True