Restriction Enzymes

Question 1

What is a plasmid?

Answer 1

Extra-chromosomal DNA that replicates independent of a chromosome.
Plasmids are smaller than a chromosome and can be present in multiple copies.

Question 2

What are plasmids used for?

Answer 2

Plasmids are used in molecular biology research to move genes of interest into an organism so the genes can be studied.

Question 3

What is a recombinant plasmid?

Answer 3

Recombinant plasmids are the result of genes being ligated into a plasmid, combining DNA from two different sources.

Question 4

What is DNA ligase?

Answer 4

DNA ligase is an enzyme involved in DNA replication, gluing two pieces of DNA together.
Ligase makes phosphodiester bonds connect to the BACKBONE of the two pieces of DNA. ATP (ribose, not deoxy) is provided as an energy source.

Question 5

What do restricted enzyme digested pieces do?

Answer 5

These have base sequences on their ends that are complementary to one another. Hydrogen bonding can occur before the phosphodiester bond is made.

Question 6

How is a recombinant plasmid made?

Answer 6

A vector/plasmid without an insert has an insert ligated into it.

Question 7

What is pUC18?

Answer 7

Commonly used plasmid in molecular biology.

Question 8

What do most plasmids contain?
What does pUC18 contain?

Answer 8

Most plasmids contain an antibiotic resistance gene.
pUC18 contains an ampicillin resistance gene (ampR).
Other plasmids may barry a gene that makes the bacterium resistant to another antibiotic.

Question 9

What happens when ampicillin is added to media with a plasmid and Escherichia coli?

Answer 9

When a plasmid is moved into E. coli, the ampicillin will force the bacterium to keep the plasmid inside the cell. Now, the gene of interest can be studied in E. coli.

Question 10

What is an origin of replication (ori)?

Answer 10

The origin of replication is where replication begins.
Plasmids have this; it is needed for the plasmid to be replicated in the bacterium.

Question 11

What are polylinker or polycloning sites?

Answer 11

Polylinker/polycloning sites are located on plasmids. They contain several restriction enzyme sites that are very close together.
When a gene is glued into a plasmid, that is where it will be glued.

Question 12

What do bacteria containing pUC18 have?

Answer 12

Bacteria containing pUC18 have hundreds of copies per cell.
They are also forced to maintain the plasmid by being grown with ampicillin in the media.

Question 13

How can plasmids be moved into a bacterium?

Answer 13

Transformation.

Question 14

Example of transformation?

Question 15

What is a restriction endonuclease?

Answer 15

An enzyme that cuts the DNA at a specific DNA sequence (the restriction enzyme site) within the DNA strands, rather than at the very end. They can leave a sticky end (or cohesive end or overhang) or blunt end on a double-stranded DNA.

Question 16

What is EcoR1?

Answer 16

A restriction enzyme that cuts at 5’GAATTC3’, between the G and the A.

This sequence is considered palindromic.

Question 17

What is HindIII?

Answer 17

A restriction enzyme that cuts the backbone between the AA sequence between 5’AAGCTT3’.

Question 18

What does it mean when EcoR1 generates a 5’ overhang?

Answer 18

It means that there will be a strand that is sticking out with its 5’ end.

Question 19

Does EcoRV make an overhang like EcoR1?

Answer 19

No, EcoRV makes a blunt end. There will be no overhang.

Question 20

When ligase is added along with ATP, it can make the phosphodiester bonds between the insert and the plasmid. How many phosphodiester bonds are needed?

Question 21

How many pieces of insert is needed for a vector/plasmid?

Question 22

How many EcoR1 sites will be in the recombinant plasmid?

Question 23

Why do bacteria make restriction endonucleases?

Answer 23

Bacteria produce restriction endonucleases to chop up DNA from a bacteriophage that may have infected the bacterium.

“Restriction” is used because it limits bacteriophage infection.

Question 24

What is the restriction modification system?

Answer 24

1. Restriction enzyme/endonuclease: used to cut up bacteriophage DNA
2. Methylase: adds methyl group (CH3) to some bases in restriction enzyme sites on bacterial chromosome

Question 25

Where is DNA methylated?

Answer 25

DNA is methylated on restriction enzyme sites and cannot be digested by the restriction endonuclease by a certain bacterium.

Question 26

Restriction endonuclease nomenclature

Answer 26

The first letter in the restriction enzyme name indicates the genus and the second and third letters indicate the species from which the enzyme was isolated.
The three letters are italicized.

Question 27

Why are numbers used in restriction endonucleases?

Answer 27

This is because bacteria can make more than one restriction enzyme. Numbers are used to indicate the order in which the enzymes were discovered in the species.

Question 28

How do molecular biologists use restriction endonucleases?

Question 29

Using restriction endonucleases to make a recombinant plasmid: Step 1

Answer 29

The gene of interest is cut with a restriction endonuclease that cuts on both sides of the gene, meaning each restriction endonuclease site flanks the gene.
This will be the insert in the recombinant plasmid.

Question 30

Using restriction endonucleases to make a recombinant plasmid: Step 2

Answer 30

The gene of interest is ligated to a plasmid vector digested with the same restriction endonuclease. There needs to be four times as many pieces of the insert as the vector when this is done. The most favored reaction is for the vector to close up without taking up an insert.

Note that the recombinant plasmid now has two EcoR1 sites.

Question 31

Using restriction endonucleases to make a recombinant plasmid: Step 3

Answer 31

The recombinant plasmid is moved into a bacterium, which reproduces up to a billion times and each bacterium makes multiple copies of the plasmid.
The researcher can then study the gene of interest.

Question 32

What is pHUG21?

Answer 32

A plasmid that contains the Plesiomonas shigelloides heme transport genes.

Question 33

What happens when a restriction enzyme map is made?

Answer 33

The restriction enzyme sites are denoted by the name of the restriction enzyme.

Question 34

How was pHUG21 made?

Answer 34

It was made by using a vector similar to pUC18, which had a similar polylinker.

Question 35

What information is shown on restriction enzyme maps?

Question 36

What can recombinant plasmids be used for?

Answer 36

They can be used by researchers to overexpress a gene so a protein can be purified and studied more closely.

Question 37

What are expression vectors?

Answer 37

Vectors that are sued to overexpress a gene.