Restriction Enzymes/Restriction Analysis Flashcards
Restriction Endonucleases (5)
- 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
Type I Restriction Enzymes
- 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
Type II Restriction Enzymes
- endonuclease activity only (methylase is another enzyme)
- Cut sites within recognition site
- ATP-independent
- palindromic
Different Restriction Enzymes can generate different types of ends
- sticky or cohesive ends (3’/5’ overhang)
- blunt or flush ends
Recognition Sites
-palindromes w/ 2 fold symmetry about a central axis
Isoschizomers
-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
Electrophoresis
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
Gel Electrophoresis
-widely used method for separating DNA (or RNA) based on differences in their lengths
Gel Electrophoresis is very useful for analyzing DNA because (3):
- DNA has 1 unit charge/residue, its charge is proportional to its length
- 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
- can be used to separate DNA fragments on the basis of size alone
Gel electrophoresis is used for (4):
- size analysis of restriction fragments
- restriction mapping
- purification of DNA fragments to be cloned/sequenced
- DNA sequencing
2 types of gels
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
Restriction Enzyme Mapping
- 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
DNA strider
-software for DNA sequence analysis
ORF maps
- 6-frame translation
- start codons=short ticks
- stop codons=long ticks
- useful for scanning a new DNA sequence for potential coding capacity