Lecture 5: restriction enzymes

Question 1

what do restriction enzymes do

Answer 1

cut double stranded DNA at specific sequences

Question 2

how does a bacterium protect its own DNA

Answer 2

Some restrictiion enzymes cleave only at DNA recognition sites that are not methylated

Question 3

how are restriction enzymes named

Answer 3

• They are named based on their origin
• They have the first letter of the bacteria’s genus, two letters of the species, the strain (if applicable), and the order of discovery

Question 4

based on the rules of naming, what would this be Escherichia coli RY13, 5th identified

Answer 4

EcoRV

Question 5

based on the rules of naming, what would this be Haemophilus aegypticus, 2nd identified

Answer 5

HaeII

Question 6

how big are the cut sites made by restriction enzymes

Answer 6

They are usually 4 to 8 bases long, however they can be longer

Question 7

true/false RE cut sites are usually palindromic

Answer 7

true

Question 8

what are the types of ends that RE cut sites can produce

Answer 8

• blunt
• sticky

Question 9

what are variable bases

Answer 9

• represent potential bases at one spot
• P= purine
• Y= pyrimidine
• W= weak
• S= strong

Question 10

Which of the following DNA sequences are potential RE recognition sites?

a) 5’ GCACCACG 3’
b) 5’ GCACAGTGC 3’
c) 5’ GCACGTGC 3’
d) 5’ GCAACG 3’

Answer 10

c) 5’ GCACGTGC 3’

Question 11

what is an isoschizomer

Answer 11

RE that cuts in a similar manner to another RE. They recognize the same recognition site

Question 12

what is a neoschizome

Answer 12

RE that recognize the same cut but at different positions

Question 13

what is the probabillity of occurrence of an RE

Answer 13

(1/4)^n

Question 14

true/false Buffers for RE activity are provided by the company you order them from

Answer 14

true

Question 15

what is the buffer composition for RE activity

Answer 15

pH and ionic strength requirements vary

Question 16

what is the incubation temp for RE activity

Answer 16

Most work best at 37 degrees celsius but can be at higher temperatures.

Question 17

what is heat inactivation in RE

Answer 17

the way to stop RE activity

Question 18

true/false Restriction enzymes always only cleave their canonical sequences

Answer 18

• false
• under certain conditions, some restriction enzymes can cleave non-canonical sequences which are similar but different from usual recognition sequence

Question 19

what triggers star activity

Answer 19

• Too much enzyme
• Wrong buffer
• Long reaction time
• High glycerol
• Organic solvents

Question 20

true/false when using RE, the sticky ends need to be compatible to properly reform the DNA

Answer 20

true

Question 21

joining 2 fragments cut by diff restriction nucleases is how much less efficient

Answer 21

10-100 times less

Question 22

what is star activity

Answer 22

when RE cleave similar, but not identical, sequences to their canonical ones under non-standard conditions

Question 23

what does linear restriction mapping do

Answer 23

characterize DNA by location of restriction enzyme sites

Question 24

descrbe the steps in linear restriction mapping

Answer 24

1. add up the sizes of the DNA fragments within each digestion
2. analyze the single RE digestion w the smallest # of fragments first
3. see the two potential cut spots … they will be identical
4. overlay the other enzyme on top, flipping once again (we know its 3 and 7, but which side is which)
5. then count how the pieces would divide, and see which map matches the predicted outcome
6. idk look at slide 14+15 of her slides if you’re confused

Question 25

describe the steps in circular restriction mapping

Answer 25

1. add up fragments within each digest 2. analyze the single digest w the largest number of DNA fragments *always place one RE site at 12 o clock position* 3. place the location of the next RE so that the resultation DNA fragments will match those obtained by double RE digest 4. *idk look at slide 16 of her slides if you're confused*