Genetic Lecture 7

Question 1

DNA Technology The Genetic Revolution

Answer 1

How humans can interfere and make changes to DNA

Question 2

Plasmid (a.k.a. Vector)

Answer 2

A small, circular piece of DNA that is replicated in a
bacteria.

Question 3

Gene of interest

Answer 3

A gene that encodes a protein that could be useful.

Question 4

Foreign DNA

Answer 4

The DNA we want to isolate and make changes if we need to, and to make more protein.

Question 5

Recombinant DNA

Answer 5

Molecules of DNA from two different sources that are
combined together in the lab. It’s called this because you are recombining two things that very often aren’t possible in nature.

Question 6

Recombinant Bacterium

Answer 6

A bacterium host with a recombinant DNA plasmid.E. coli is the bacteria that is used most often in these experiments.

Question 7

When did cloning a gene first happen?

Answer 7

In 1975, seemed unnatural and a lot people talked about the ethics of it.

Question 8

Steps of cloning a gene?

Answer 8

Gene inserted into a plasmid. Plasmid put into a bacterial cell. Host cells grown in culture to form a clone of cells that contain the cloned gene of interest. Basic research and various applications.

Question 9

Where is the bacteria typically grown in?

Answer 9

A flask that has a medium good for the bacteria. These bacteria have a new generation every 20 minutes.

Question 10

Two uses of cloning a gene?

Answer 10

Isolating the placid and taking out the genes of interest. This can be used for gene for pest resistance inserted into plants, or gene used to alter bacteria for cleaning up toxic waste. Another way is to harvest the protein expressed from the gene of interest. This can be used as human growth hormone treats stunted growth or protein dissolves blood clots in heart attack therapy (mostly in pharmaceutical ways).

Question 11

Restriction Enzymes

Answer 11

Cut DNA into fragments based upon recognizing a
specific sequence of nucleotides.

Question 12

Restriction site

Answer 12

Read same in both directions (antiparallel). Where the divide occurs. It can be recognized by some enzymes that will be able to cut right at the specific spot.

Question 13

Sticky Ends

Answer 13

Single-stranded ends left hanging after the DNA is cut. The free base pairs can hydrogen bond with
complementary sticky ends.

Question 14

Steps for Making Recombinant DNA with Restriction
Enzymes?

Answer 14

Restriction enzyme cuts the sugar-phosphate backbones at each arrow. DNA fragment from another source is added. The fragment from different DNA molecule cut by the same restriction enzyme. Base pairing of sticky ends produces various combinations. DNA ligase seals the strands.

Question 15

DNA ligase

Answer 15

Catalyzes the formation of the covalent bonds
that close up the sugar- phosphate backbones.

Question 16

Plasmid maps

Answer 16

Are graphical representation of plasmids, that show the locations of major identifiable landmarks on DNA like restriction enzyme sites, gene of interest, plasmid name and length etc. Circle of DNA.

Question 17

Amplicillain resistance (antibiotic)

Answer 17

So when you introduce the bacteria with this plasmid in it, it will make it resistant to ampicillin. By adding ampicillin in your medium to kill off everything else that doesn’t have your plasmid.

Question 18

Multi cloning site

Answer 18

Different restriction sites that have been put there by people. To give flexibility in the lab to put your piece of DNA in. The number next to the enzyme is its position.

Question 19

The first restriction enzyme discovered?

Answer 19

EcoR1

Question 20

LacZ arrow

Answer 20

If they added a piece of DNA in the multi cloning sites, it will stop LacZ from working. Gives a chemical that turns blue when lacZ is working. Want it to not be blue, because it means the bonds are broken.

Question 21

DNA Gel Electrophoresis

Answer 21

To visualize and purify the DNA. Separates DNA of different sizes.

Question 22

Gel electrophoresis steps?

Answer 22

In the tube a bit of restriction enzyme, plasmid, and DNA are put in the DNA that you want to insert into the plasmid, close that up and put into the incubator for a few hours. Then mix with your bacteria, then go to the plate and see if you get blue or white colonies.

Question 23

What is the jelly in gel electrophoresis made of?

Answer 23

Agarose gel

Question 24

What are the wells?

Answer 24

Where the DNA is placed in, it’s made by the comb.

Question 25

What is in the tube?

Answer 25

A mixture of DNA molecules of different sizes.

Question 26

The charge of DNA?

Answer 26

Negatively because of the phosphate groups. The DNA is placed on the negative side and then moves to the positive electrodes.

Question 27

Cathode

Answer 27

Negative end

Question 28

Anode

Answer 28

Positive end

Question 29

What molecules move faster through the gel?

Answer 29

The shorter molecules.

Question 30

What does higher density gel do?

Answer 30

Used to separate smaller molecules. All the molecules will move slower because there is more molecules stoping them (that they have to move through).

Question 31

What happens if the gel isn’t dense enough?

Answer 31

The shorter ones will just go right through it.

Question 32

How can people take just one piece of the DNA out of the gel?

Answer 32

By cutting it out with a blade.

Question 33

PCR (Polymerase Chain Reaction)

Answer 33

To copy (amplify) a DNA sequence. It’s very sensitive and uses enzymes from nature to our advantage to amplify DNA. Works with a target sequence. A lot of ideas came from DNA’s own replication.

Question 34

Taq polymerase

Answer 34

An unusual heat- stable DNA polymerase from Thermus aquaticus. With this they could isolate the protein with DNA polymerase from the organism to use in the PCR reactions.

Question 35

Steps of PCR?

Answer 35

Denaturation (95 degrees C) separates the DNA strands. Annealing (60 degrees C) the DNA primers are added (use them instead of RNA because they are more robust and won’t get degraded). Extension (72 degrees C) where new nucleotides can be added on the 3’ end.

Question 36

How many does each cycle produce in PCR?

Answer 36

Cycle 1 is 2 molecules
Cycle 2 is 4 molecules
Cycle 3 is 2 of the 8 molecules match the target sequence and are the right length.
The DNA amount doubles at each cycle.

Question 37

Results of PCR?

Answer 37

After 3 cycles, two molecules match the target sequence exactly. After 30 more cycles, over 1 billion molecules match the target sequence.