Restriction enzymes

Question 1

What is the difference between endonucleases and restriction endonucleases?

Answer 1

Endonucleases cut randomly rather than at specific points.

Question 2

What is the natural function of restriction endonucleases?

Answer 2

They occur naturally in bacteria to protect the bacteria from bacteriophage.

Question 3

Why doesn’t the bacterial genome get cleaved by the restriction enzymes?

Answer 3

Bacterial DNA is methylated at the restriction sites and is therefore protected.

Question 4

What are Type II restriction endonucleases?

Answer 4

They recognise a specific DNA sequence that is often 4 or 6 nucleotides in length. They cut precisely at or near this site.

Question 5

What does palindromic mean and what relevance does this have to type II restriction endonucleases?

Answer 5

It means the sequence will be read the same in each direction. Type II restriction endonuclease restriction sites are often palindromic.

Question 6

What is the recognition site of EcoRI and does it create blunt or sticky ends?

Answer 6

GAATTC, sticky.

Question 7

What is the recognition site of BamHI and does it cut blunt or sticky?

Answer 7

GGATCC, sticky.

Question 8

What is the recognition site of AluI and does it cut blunt or sticky?

Answer 8

AGCT, blunt.

Question 9

What is the restriction site of Sau3A and does it cut blunt or sticky?

Answer 9

GATC, sticky.

Question 10

What is the benefit of sticky ends?

Answer 10

The ends can base pair together via hydrogen bonds which is helpful if you want to rejoin ends.

Question 11

What is the benefit of the same sticky ends being produced by different restriction enzymes?

Answer 11

Different fragments can be joined together even if they have been cut by different restriction enzymes.

Question 12

What is the frequency of restriction sites in a tetranucleotide?

Answer 12

Once every 245 nucleotides.

Question 13

What is the frequence of a hexanucleotide restriction site?

Answer 13

Once every 4096 nucleotides.

Question 14

What is the process for using restriction enzymes?

Answer 14

Reaction is carried out in a microfuge tube using sterile components. It requires an appropriate buffer for the enzyme and is usually incubated at 37 degrees for an hour for the digestion to complete.

Question 15

How can the restriction enzyme be inactivated?

Answer 15

EDTA, heat or phenol.

Question 16

How can agarose gel electrophoresis be used together and provide use?

Answer 16

Used for analysing, mapping and purifying DNA molecules.

Question 17

What is agarose?

Answer 17

A highly pure form of agar, extracted from seaweed.

Question 18

How can an agarose gel be prepared?

Answer 18

Various plastic shit, buffer, agarose, ethidium bromide, pour into a casting tray and wait for it to set.

Question 19

How can you prepare DNA for electrophoresis?

Answer 19

Mix the digested DNA with a loading buffer that contains a dye so you can see what you’re doing. The buffer will also contain sucrose/glycerol to make the sample heavier than the elctrophoresis buffer. The sample will sink to the bottom of the well.

Question 20

Which direction does DNA move in electrophoresis?

Answer 20

From the cathode towards the anode.

Question 21

Do smaller or larger fragments move further throughout the electrophorese?

Answer 21

Smaller fragments move further.

Question 22

How can the DNA fragments be seen after the electrophorese?

Answer 22

Stain with ethidium bromide during or after the electrophorese and place the gel on a UV transilluminator.

Question 23

How can the size of the unknown fragments be determined?

Answer 23

Using size markers, usually a HindIII digest of lambda bacteriophage DNA run next to the unknown sample.

Question 24

What is restriction mapping?

Answer 24

Working out the relative positions of the restriction sites for particular enzymes.

Question 25

How do you perform restriction mapping?

Answer 25

Do a single and double restriction enzyme digest. Electrophorese them in parallel, accurately measure the sizes and work out the relative positions on the DNA molecule.

Question 26

What can restriction maps be useful for?

Answer 26

Cloning particular genes or comparing molecules from different sources.

Question 27

What ligase can be used to insert a gene into a cloning vector and where does this ligase originate from?

Answer 27

T4 DNA ligase and from bacteriophage T4 where the function is the repair DNA.

Question 28

How does ligation of blunt and sticky ends differ?

Answer 28

Sticky ends have to be complementary and base pair by hydrogen bonding. Blunt ends are less efficient but any fragments can be joined.

Question 29

What is the difference between inter and intra-molecular association?

Answer 29

Inter is ligating two separate fragments from different sources whereas intra is making a loop with the same fragment. There is no control over which sticky ends ligate.

Question 30

What is the problem with inter and intra-molecular association?

Answer 30

There is no control over which molecules join and only a few ligation products will be the desired recombinants.

Question 31

What can be used to remove the problem of inter and intra-molecular association and how does it work?

Answer 31

Alkaline phosphatase. It removes the 5’ terminal phosphates. It will be used on vector or fragments so no intra-molecular association can occur.