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Alex's Genetics > Lecture 6a > Flashcards

Lecture 6a Flashcards

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Biology 101 flashcards
1
Q

Who invented PCR?

A

Kary Mullis

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2
Q

What did Kary Mullis win the Nobel Prize for in 1993?

A

For inventing PCR

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3
Q

What is meant when we say PCR is error-prone?

A

Mistakes can occur early on when using PCR. When this happens, the mistakes can be amplified.

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4
Q

What is PCR great for?

A

Diagnostics in which we visualize the presence or absence of mutations

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5
Q

When we are cloning genes using PCR amplified genes, what do we need to determine?

A

Their DNA sequences

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6
Q

How do we visualize PCR products?

A

We run the PCR products on an agarose gel.

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7
Q

Where is the DNA added when running PCR products?

A

DNA is added into the sample wells.

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8
Q

What happens after we have loaded the DNA into the sample wells?

A

A current is applied.

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9
Q

What happens with DNA when the current is applied? Why?

A

The current makes DNA migrate to the anode (+ electrode). This occurs because DNA is negatively charged.

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10
Q

How thick is the agarose gel?

A

Typically 5-10 mm (0.25 inch) thick

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11
Q

How are DNA fragments separated in the agarose gel?

A

DNA fragments are separated by size.

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12
Q

Describe the steps of visualizing PCR products with agarose gel.

A

1) Load the DNA samples into the wells.
2) Apply the electric current
3) DNA separation by size

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13
Q

How can we picture the agarose gel for the DNA?

A

The agarose gel is like an obstacle course for the DNA.

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14
Q

How is DNA fragment size related to speed through agarose gel?

A

The larger the DNA fragment, the slower it makes its way through the gel to the anode (+ electrode).

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15
Q

What is the chemical in the agarose gel called?

A

Ethidium bromide

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16
Q

What does Ethidium bromide do?

A

As the DNA migrates through the gel, the Ethidium bromide intercalates between the bases of DNA and stays there. When we shine UV light on the gel, the Ethidium bromide makes the DNA fluorsce under the UV light.

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17
Q

What does the UV light do?

A

UV light makes the DNA fluoresces orange-white when the ethidium bromide is intercalated with it.

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18
Q

What is a polylinker?

A

It consists of several cleavage sites for many different restriction enzymes. We can cleave at different sites to insert DNA.

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19
Q

Do any of the restriction enzymes for the polylinker cleave elsewhere in the vector DNA? If so, which?

A

No! None of these restriction enzymes in the polylinker cleave anywhere else in the vector DNA.

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20
Q

What are the 3 most popular antibiotic resistance genes?

A

AmpR, CamR and KanR.

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21
Q

What is the AmpR gene?

A

Resistance to ampicillin

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22
Q

What is the CamR gene?

A

Resistance to chloramphenicol

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23
Q

What is the KanR gene?

A

Resistance to Kanamycin

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24
Q

Do all sticky ends have 5’ overhangs?

A

No! They can also have 3’ overhangs.

25
Q

What restriction enzymes produce 5’ overhangs?

A

EcoR and BamH

26
Q

In relation to the cDNA, how does the polylinker get cleaved?

A

The polylinker gets cleaved to match the cDNA sticky ends so that the cDNA can fit into the plasmid.

27
Q

What can computer programs tell you about PCR DNA fragments?

A

They can tell you the size of the restriction fragments.

28
Q

After we’ve cloned a gene into a plasmid, describe what do we do to check DNA fragment sizes.

A

1) Cleave the DNA with restriction enzymes in 5 separate reactions, each with different restriction enzymes.
2) Separate the DNA fragments by gel electrophoresis
3) Compare the DNA fragment sizes seen on the gel to the computer-generated restriction map of the plasmid.

29
Q

What happens if we try to express human proteins in bacteria?

A

The human protein will be rapidly degraded.

30
Q

What does a fusion gene produce?

A

A fusion protein, which is part bacterial and part human.

31
Q

What happens when we introduce a fusion protein into bacteria?

A

The human protein part won’t be degraded.

32
Q

What was the first commercial production of a human protein?

A

Human Somatostatin protein in the bacterium E. coli

33
Q

Describe the process of using a fusion gene to obtain a human protein.

A

1) The plasmid expresses the fusion gene.
2) mRNA gets translated and produces the fusion protein.
3) CNBr is added, which cleaves the protein to separate the human and bacterial one.

34
Q

Describe the production of human insulin in bacteria.

A

1) Transform into E. coli. This means taking an altered plasmid and inserting it into a bacterial cell.
2) Culture cells
3) Purify b-gal-insulin fusion proteins
4) Treat with CNBr
5) Purify A and B chains
6) Refold and oxidize cysteines

35
Q

What does insulin do?

A

A hormone that promotes glucose uptake.

36
Q

What type of treatment is insulin used for?

A

For diabetic patients

37
Q

What does Factor VIII do?

A

Blood clotting factor

38
Q

What type of treatment is Factor VIII used for?

A

For certain types of hemophilia patients.

39
Q

Where are Insulin and Factor VIII made?

A

Bacteria

40
Q

What is Biological Control?

A

Refers to the use of microorganisms or their products to alleviate disease or damage of plants from environmental conditions.

41
Q

What are two ways to do biological control?

A

1) Nonpathogens
2) Microorganisms that produce helpful toxins

42
Q

How are nonpathogens used for biological control?

A

They are used to compete effectively against pathogens for nutrients or space.

43
Q

How are toxic microorganisms used for biological control?

A

Microorganisms that produce helpful toxins that inhibit other microorganisms or insects, but not the plant.

44
Q

What is a recombinant microorganism?

A

A microorganisms whose DNA is altered in the lab.

45
Q

Are most people for or against the release of recombinant microorganisms?

A

Lots of people oppose it.

46
Q

What is Bioremediation/Biotransformation?

A

The use of microorganisms to reduce environmental pollutants by producing enzymes that transform the structure of the toxic pollutant.

47
Q

What is biotransformation with biodegradation?

A

The toxic pollutant is degraded into nontoxic metabolites.

48
Q

What is biotransformation without biodegradation?

A

The pollutant is rendered less toxic by oxidation/reduction reactions or polymerization reactions.

49
Q

What are Genetically Modified Plants?

A

When we use selective breeding to produce plants with desirable characteristics.

50
Q

What are desirable characteristics we would want in GMP?

A

Larger, disease resistance, and high-quality food.

51
Q

How long have genetically engineered crops been used for?

A

Since the mid-1990s.

52
Q

In 2006, how many crops were transgenic?

A

Roughly 20% of all crops.

53
Q

What was the first way GMP was done?

A

With Agrobacterium tumefaciens

54
Q

Describe the process of cancer from Agrobacterium tumefaciens.

A

This type of bacteria is found within soil and can infect plant cells through wounds on the plant. When the bacterium infects the plant, the T DNA from the Ti plasmid jumps onto the plant cell’s chromosome, promoting cancer.

55
Q

Describe the process of making transgenic plants with A. tumefaciens.

A

1) The gene of interest is inserted into the T DNA of the Ti plasmid.
2) The recombinant Ti plasmid is transferred into A tumefacians.
3) Plant cells are exposed to A. tumefaciens.
4) The T DNA is transferred and incorporated into the plant cell chromosome.
5) Plant cell will now express that gene.

56
Q

What are alternative to making transgenic plants?

A

1) DNA guns
2) Remove the cell wall and get DNA in using conventional methods (like an electric current)

57
Q

What is meant by using DNA guns to make transgenic plants?

A

Shoot pellets at the cell wall to penetrate it and introduce the new DNA.

58
Q

What is resistance to the herbicide glycophosphate an example of?

A

GMP to give a resistance gene

59
Q

What are 3 categories of traits that have been modified in transgenic plants?

A

1) Resistance to viral, bacterial, and fungal pathogens.
2) Resistance to insects.
3) Resistance to herbicides.

Alex's Genetics (38 decks)

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