Gel Electrophoresis & Restriction Enzymes

Question 1

Pre 1970s

Answer 1

• few tools available to study nucleic acids
• those that were available either provided minimal information and/or were very tedious

-ultracentrifugation in CsCl gradients to study bulk properties of DNA sequence (buoyant density)

Question 2

Restriction endonucleases

Answer 2

• first reported in 1970
• thousands now known, many commercially available
• recognized specific DNA sequences and cleave DNA
• restrict entry of foreign DNA into bacterial cells
• Type II REs cleave DNA within the recognition site
• Type II most important for molecular biology
• DNA restriction site is a palindrome
• DNA fragments cut by REs can be reassembled with ligand enzyme

Question 3

Restriction enzyme facts

Answer 3

1st three letters of the name refer to species of bacteria the enzyme was isolated from

• other letters refer to particular bacterial strain
• Roman numeral referees to order in which enzyme was discovered
• most REs recognize palindromic DNA sequences
• most RE recognition sequences are 4-6 bp long
• shorter RE sites tend to occur more frequently in DNA

Question 4

How often to restriction enzymes cut DNA sequences

Answer 4

• if we assume all 4 bases are equally present in DNA and nucleotide order is essentially random
  
  • probability an RE will cut DNA is (0.25)^n where n is the number of bases in the RE recognition sequence
• eg. For an RE with a 6 base recognition sequence, P=0.25^6=0.000244
  
  • thus the average size of DNA fragments produced by a 6-cutter is 1/0.000244= 4096 bp
• caveats:
  
  1. Assumption that equal frequency of the 4 bases is violated by real DNA sequences
  2. DNA sequenecs not always random order, but effect of the departure from randomness on the frequency of RE cut sites is small

Question 5

Gel electrophoresis

Answer 5

• a method for sorting DNA and RNA fragments by size
• at neutral pH, DNA molecules are negatively charges because of the phosphate groups
• in an electrical field, DNA will tend to move toward the positive electrode
• cannot be done in a liquid - needs a gel
• usually use agarose gel - an uncharged polysaccharide purified from agar of the seaweed: agar agar

Question 6

Ethidium bromide

Answer 6

-EtBr is an intercalating dye between the bases

Question 7

Migration of DNA molecules

Answer 7

• linear DNA molecules migrate at a rathe that is inversely proportional to the log of their molecular mass (or number of base pairs)
• smaller sized DNA molecules migrate more rapidly than larger ones
  
  • the pores in the agarose gel matrix impede the larger DNA molecules
• need some fragments of known size to calibrate it

Question 8

Factors that affect mobility of DNA fragments

Answer 8

1. Agarose concentration in gel
2. Topology (physical conformation) of DNA molecule
3. Voltage

Question 9

Concentration of agarose

Answer 9

• higher conc of agarose, the smaller the average pore size in the gel matrix
• smaller pores creates more resistance to DNA movement, favouring small DNA fragments and giving better resolution of size differences of small fragments

Question 10

Voltage applied to DNA affects rate of migration

Answer 10

-higher voltage will cause fragments to migrate faster

Question 11

Factors that DONT influence migration rate

Answer 11

• the %GC sequence of DNA molecule

- one exception: AT-rich sequences, called bent DNA

Question 12

Purpose of restriction mapping

Answer 12

• often DNA fragments are too large to just sequence in one pass
• restriction site mapping provides a view of DNA landmarks that give a broader view of DNA organization
• useful as a preliminary step to cloning or sub cloning particular DNA sequences of interest