11.1 Restriction Endonuclease and Gel Electrophoresis

Question 1

Type I restriction endonucleases

Answer 1

Recognise specific specific DNA sequences the cleave somewhere else. “Restrict” entry of foreign DNA into bacterial cells.

Question 2

Type II restriction endonucleases (restriction enzymes)

Answer 2

Cleave DNA within the recognition site.

Question 3

DNA restriction site

Answer 3

A palindromic sequence that reads the same on the top strand as the bottom strand but only in 5’ to 3’ of both.

Question 4

Restriction endonucleases EcoRI

Answer 4

GAATTC - G AATTC with sticky ends

Question 5

Nomenclature of REs

Answer 5

Derived from species strain and the order in which they were isolated.

Question 6

Bacterial REs do not attack their own host DNA

Answer 6

host DNA methylates a base in every copy of the RE site in its own genome so as to not be targeted

Question 7

Gel electrophoresis

Answer 7

Sorts DNA and RNA fragments by size, must be done in agar gel with a buffer to provide ions for current flow ( and increase pH).

Question 8

DNA-binding fluorescent dye

Answer 8

EtBr (ethidium bromide) is an intercalating DYE, binds in minor grooves.

Question 9

Size fractionation of DNA

Answer 9

Migration rate of linear DNA = 1/(log[bp]) (by is # of base pairs/molecular mass)

Question 10

Agarose concentration

Answer 10

Increase in concentration decreases gel pore size. Smaller pores are more resistant to DNA movement and favours smaller particles for better resolution of small fragments.

Question 11

DNA topology

Answer 11

Supercoiled (linear/circular) - fastest
Linear - medium
relaxed circular - slowest

Question 12

Voltage on migration speed

Answer 12

Greater voltage speeds up the rate of migration of DNA fragments

Question 13

DNA migration is not affected by:

Answer 13

The GC content of the DNA molecules