Genetics: Lecture 7

Question 1

Molecular Cloning?

Answer 1

When you have a gene of interest in an organism that encodes a protein which could be useful.

We do this when we want to harness that gene and use it for other applications

Question 2

Goal of molecular cloning?

Answer 2

Isolate the gene of interest (make us able to make changes if we want to in the gene sequence).

Make more protein

Question 3

What is molecular cloning interested in?

Answer 3

We are interested in plasmids (circles of DNA in bacteria)

• We take our DNA that we have an incorporate it into a plasmid, then being able to shuttle the new DNA around using a plasmid vector.

Question 4

Cloning a gene involves ______?

Answer 4

Copying just a piece of DNA rather than the entire organism.

Question 5

What is a plasmid?

Answer 5

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

Question 6

Recombinant DNA?

Answer 6

Molecules of DNA from two different sources that are combined together in the lab.

Question 7

Recombinant Bacterium?

Answer 7

A bacterium host with a recombinant DNA plasmid.

Question 8

Process of cloning a gene?

Answer 8

1. Gene inserted into plasmid
2. Plasmid put into bacterial cell
3. Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest.
4. Basic research and various applications (copies of gene and protein is harvested)

Question 9

Use of gene cloning?

Answer 9

• The gene for pest resistance is inserted into plants to make them resistant to the pests.
• A gene is inserted into bacteria to alter it and allow it to clean up toxic waste.
• Human growth hormone treats stunted growth
• Protein dissolves blood clots in heart attack therapy
• Insulin is made by pharmaceuticals to be able to treat/regulate patients

Question 10

Where is gene cloning used?

Answer 10

• Cleaning toxic waste
• Plant farmers
• Pharmaceuticals

Question 11

We can make recombinant DNA with?

Answer 11

Restriction enzymes.

Question 12

Restriction enzymes?

Answer 12

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

Question 13

Sticky ends?

Answer 13

Single-stranded ends left hanging after the DNA is cut.

Question 14

Palindromic sequence of DNA?

Answer 14

DNA that is read the same but backwards between the different strands.

Question 15

Example of palindromic sequence?

Answer 15

5’ G-A-A-T-T-C 3’
3’ C-T-T-A-A-G 5’

Question 16

Steps of making recombinant DNA with restriction enzymes?

Answer 16

1. Restriction enzyme cuts the sugar-phosphate backbones at each arrow.
2. DNA fragment (cut by same enzyme) from another source is added. Base pairing of sticky ends produces various combinations.
3. DNA ligase seals the strands together

Question 17

What can the sticky ends do?

Answer 17

The free base pairs can hydrogen bond with complementary sticky ends. This occurs when another DNA fragment is introduced.

Question 18

DNA ligase?

Answer 18

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

Question 19

Contents of Plasmid Map?

Answer 19

• AmpR (ampicillin resistor)
• Lac Operon
• Multicloning site

Question 20

AmpR?

Answer 20

Ampicillin Resistor kills off things that are unnecessary for the plasmid to keep the necessary items in the plasmid.

Question 21

LacZ in Plasmid Map?

Answer 21

• Encodes enzyme beta-galactosidase which cleaves glycosidic bonds to create a glucose and galactose.
• When something is added to the Multi-cloning site, the LacZ will stop functioning.

Question 22

Multicloning site?

Answer 22

Composed of many different restriction sites that are put there by people. This is engineered to have the MCS be as flexible as possible with as many restriction sites as needed for that cell.

Question 23

Adding DNA to bacteria is?

Answer 23

Transforming the bacteria.

The use of the word “transform” is from Griffith’s idea!

Question 24

DNA Gel Electrophoresis is?

Answer 24

used to visualize and purify the DNA, separating segments out by size.

Question 25

Where is the DNA placed in Gel Elecrophoresis?

Answer 25

It is placed in wells on the negative side of the power source.

Question 26

Why is it on the negative side?

Answer 26

DNA is negatively charged so it wants to move to the positive side. Having it on the negative side lets it go as far as it needs to to the positive.

Question 27

What does Electrophoresis tell us?

Answer 27

It tells us the size of the DNA segments.

Question 28

Which segments go farther?

Answer 28

The shorter ones

Question 29

Which segments go less far?

Answer 29

The longer ones

Question 30

What is PCR?

Answer 30

PCR stands for Polymerase Chain Reaction.

Question 31

What does a PCR do?

Answer 31

Its job is to copy or amplify a DNA sequence.

• Instead of natural DNA replication, a PCR creates two new sequences at the end rather than 1

Question 32

What happens in PCR?

Answer 32

There are three steps to PCR:

1. Denaturation
2. Annealing
3. Extension

Question 33

PCR denaturation?

Answer 33

• strands are heated to 95 degrees celsius and separated in half.

Question 34

PCR Annealing?

Answer 34

• strands are cooled to 65 degrees and DNA primers will attach, forming a place for DNA polymerase to latch on and begin amplifying the strands.

Question 35

PCR Extension?

Answer 35

• strands are heated back up to 72 degrees and new nucleotides are able to be added.

Question 36

Cycle 1 of PCR yields?

Answer 36

2 molecules!

Question 37

Taq polymerase?

Answer 37

An unusual heat-stable DNA
polymerase from Thermus aquaticus

• It is isolated and used in our reaction to work with the heat change/ withstand it!

Question 38

What goes into the tube for PCR?

Answer 38

1. The DNA that is to be replicated
2. Primers
3. Nucleotides
4. Taq Polymerase
5. Buffer (water and salt)