Restriction Enzymes/Determination of Fragment Size

Question 1

Restriction Enzymes [What is their function, What do they recognize, What do they do to the DNA, What are the products

Answer 1

-RE cleave DNA
-recognizes specific sequences of 4 to 8 bp
-hydrolizes a phosphodiester bond in each strand

Products
-Cleavage within the recognized
sequence occur on both strands
in the center → Blunt end
-Cleavage at staggered positions
on each strand, generating ends
with one strand or the other having
an overhang → sticky ends

Question 2

What is Restriction Mapping?

Answer 2

Process of determining the sites of restriction cleavage within a
piece of DNA

Question 3

What are Restriction fragments?

Answer 3

Resulting DNA fragments after cleavage with restriction enzyme

Question 4

Mobility of double-stranded plasmid DNA on agarose gels (in order of least to most travel)

Answer 4

a) Nicked circular DNA (travels the least)
b) linear DNA
c) supercoiled DNA (travels the most)

Question 5

How are DNA fragments detected? [What assay is required? What dye do you need to use? How does the dye work?]

Answer 5

-separated by agarose gel electrophoresis
-visualized by staining with Ethidium Bromide
-dye that intercalates between bases of nucleic acids and is highly fluorescent upon UV light exposure

Question 6

Reagents for restriction enzyme experiment

Answer 6

-Plasmid
-BanH1 (restriction enzyme)
-Pst1 (restriction enzyme)
-10x BSA (protein to prevent DNA from sticking to eppendorf tube and pipettes)
-10x Buffer 3 (optimal pH for enzymes)
-deionized water