Restriction Enzymes

Question 1

what are restriction enzymes?

Answer 1

these are enzymes that cut dsDNA at very specific locations

Question 2

How does bacteria protect its own DNA from restriction enzymes?

Answer 2

by having restriction modification systems on their genomes

Question 3

How are restriction enzymes named?

Answer 3

genus, species, somtimes strain, and order of discovery

Question 4

what order was HaeII discovered?

Answer 4

2nd identified

Question 5

how long are cut sites?

Answer 5

4 to 8 bases long typically

Question 6

what type of ends can be resulted in cut sites?

Answer 6

blunt or sticky ends (2-3 types)

Question 7

what are isoschizomers?

Answer 7

recognizes the same recognition sites

Question 8

what are neoschizomers?

Answer 8

recognizes the same recognition sites but cleaves at different positions

Question 9

describe restriction enzyme buffers.

Answer 9

• pH and ionic strength requirements vary
  -comes with buffers optimized for their functions

Question 10

what is the best incubation temperature for restriction enzymes

Answer 10

37 degrees celcius

Question 11

what triggers enzymes to cleave the wrong sequences?

Answer 11

• too much enzymes
• wrong buffers
• long reaction times
• high glycerol
• organic solvents

Question 12

how are two DNA fragments cut by the same restriction enzyme joined together?

Answer 12

using sticky ends (with ligase and ATP)

Question 13

how are two fragments joined together when cut by different restriction nucleases?

Answer 13

blunt ends using ATP and ligase

Question 14

you can characterize DNA by the location of:

Answer 14

restriction enzyme sites

Question 15

describe the process of restriction mapping:

Answer 15

1. add up sizes within digestion
2. analyze single RE digestion with the smallest number of DNA fragments
3. decide whether it is better for A to be close to B or far from B
4. determine the sizes of the DNA fragments resulting from the digestion of A+B
5. compare predicted DNA fragment sizes from double RE digestions from the two maps and determine which would give the same sizes as actual digestion

Question 16

how many fragments are produced when cut with two restriction enzymes?

Answer 16

three

Question 17

in southern DNA what is blotted?

Answer 17

DNA - DNA

Question 18

in Northern DNA what is blotted?

Answer 18

DNA - RNA

Question 19

in western DNA what is blotted?

Answer 19

antibody - protein

Question 20

what is RNA interference?

Answer 20

• this is mRNA translation and stability by making RNA into single strands again (after hairpinning)
• defense mechnism

Question 21

How is RNA silenced during RNA interference?

Answer 21

destroyed or silenced through heterochromatin formation

Question 22

What is CRISPR?

Answer 22

bacterial defence

Question 23

what are the steps of CRISPR?

Answer 23

1. short viral DNA sequence is integrated into CRISPR locus
2. RNA is transcribed from CRISPR locus, processed, and bound to Cas proteins
3. small crRNA in complex with Cas seeks out and destroys viral sequences