Restriction Enzymes/Restriction Analysis

Question 1

Restriction Endonucleases (5)

Answer 1

• enzymes that catalyze the ds cleavage of DNA at specific or non-specific base sequences
• name comes from “restriction modification
• many bacteria use site specific methylation to mark their own DNA and DNA cleavage to inactivate foreign DNA (e.g viral DNA)
• strain specific restriction enzymes and methylases work in pairs
• can be used to create recombinant molecules

Question 2

Type I Restriction Enzymes

Answer 2

• methylase and endonuclease activity in 1 enzyme
• cleavage sites outside of recognition site in non specific sequence (up to 10 kb away from recognition site)
• cleavage requires ATP

Question 3

Type II Restriction Enzymes

Answer 3

• endonuclease activity only (methylase is another enzyme)
• Cut sites within recognition site
• ATP-independent
• palindromic

Question 4

Different Restriction Enzymes can generate different types of ends

Answer 4

• sticky or cohesive ends (3’/5’ overhang)

- blunt or flush ends

Question 5

Recognition Sites

Answer 5

-palindromes w/ 2 fold symmetry about a central axis

Question 6

Isoschizomers

Answer 6

-restriction enzymes that recognize the same DNA sequence (may or may not cleave at the same position)
e.g. SacI and SstI recognize and cleave in the same position
HhaI and HinP1I recognize same sequence but cleave at different positions

Question 7

Electrophoresis

Answer 7

The movement of charged molecules in an electric field w/ negatively charged molecules moving toward the positive electrode and positively charged ones toward the negative electrode

Question 8

Gel Electrophoresis

Answer 8

-widely used method for separating DNA (or RNA) based on differences in their lengths

Question 9

Gel Electrophoresis is very useful for analyzing DNA because (3):

Answer 9

1. DNA has 1 unit charge/residue, its charge is proportional to its length
2. The molecular sieving effect determines the relative mobility of DNA molecules at a given gel concentration, effect is proportional to the length of the molecule
3. can be used to separate DNA fragments on the basis of size alone

Question 10

Gel electrophoresis is used for (4):

Answer 10

1. size analysis of restriction fragments
2. restriction mapping
3. purification of DNA fragments to be cloned/sequenced
4. DNA sequencing

Question 11

2 types of gels

Answer 11

Agarose
-forms gels w/ pores 100-300 nm, depending on conc of agarose used
-useful for resolving fragments b/w 100 and 50 000 nucleotides
Polyacrylamide
-cross linked polymer, used for electrophoretic analysis of proteins and small nucleic acids
-can be used to resolve fragments that differ by as little was one nucleotide in length

Question 12

Restriction Enzyme Mapping

Answer 12

• digest a DNA molecule w/ 2 different restriction enzymes w/ different target sequences and compare the fragment size
• restriction maps are easy to generate if there are few cut sites for the enzymes used
• restriction mapping is generally only feasible for small molecules (although rare base cutters (e.g. NotI) make it possible to generate maps for larger fragments

Question 13

DNA strider

Answer 13

-software for DNA sequence analysis

Question 14

ORF maps

Answer 14

• 6-frame translation
• start codons=short ticks
• stop codons=long ticks
• useful for scanning a new DNA sequence for potential coding capacity