restriction enzyme
TRANSCRIPT
Hi t f t i tiHistory of restriction endonucleases and its role e do uc eases a d ts o ein establishing molecular
bi lbiology
Restriction enzymes
• Over 10,000 bacteria species have been screened for restriction enzymesO 2 500 t i ti h b f d• Over 2,500 restriction enzymes have been found
• Over 250 distinct specificities• Occasionally enzymes with novel DNA sequence• Occasionally enzymes with novel DNA sequence
specificities are still found while most now prove to be duplicates (isoschizomers) of already di d ifi itidiscovered specificities.
Restriction Enzyme Function
• It is generally believed that the biological function of restriction enzymes is to protect cells from foreign DNA.
• Infecting DNA is cleaved (restricted) by the restriction enzyme(s) preventing it f f ll li ti dfrom successfully replicating and parasitizing the cell.
Why the bacteria does not kill itself? The Restriction Enzyme Modification Systems
if everything gets cleaved, how come the bacteria does not kill itself?
• Usually, organisms that make restriction enzymes also make a companion modification enzyme (DNA methyltransferase) that protects their own ( y ) pDNA from cleavage.
• These enzymes recognize the same DNA y gsequence as the restriction enzyme theyaccompany, but instead of cleaving the sequence, they disguise it by methylating one of the bases in
h DNA t deach DNA strand.
Classification of Restriction enzymesenzymes
Class I Class II (93%) Class III
Restriction-methylase on the same subunit
Homo-dimers, methylase on a separate subunit
Restriction-methylase on the same subunit
p
ATP-dependent Mg++ dependent ATP-dependent
Binds to DNA recognition site and
t DNA d l
recognize symmetric DNA sequences andl ithi th
Cut the DNA at the recognition site and th di i t fcuts DNA randomly -
any DNA as long as it comes in contact
cleave within the sequences
then dissociate from the DNA
Type II Restriction enzymesare endonucleases
that cut DNA at specific sites, and are most useful for molecular biology research
Type II Restriction enzymesRecognition sitesRecognition sites
are P li d iPalindroimes:
121IFFI ABAIFFI, ABAAAGCTTTTCGAA
How do I know what sequenceHow do I know what sequence each enzyme cut?
• Test by cutting DNA of known sequencey g q
• Commercial sources are tested already, y,and you find a catalog
Some popular Biotechnology Companies
• Life Technologies (BRL/GIBCO)• New England Biolabs• Amersham Pharmacia Biotech• Qiagen
P• Promega• Clonetech• InvitrogenInvitrogen• Stratagene• ...
Nomenclature of restriction enzyme
• Eco R1: E coli• Pst I: Providencia stuartii• Hind III: Haemophilus influenza• Not I: Norcardia otitidis-caviarum
• What do you name a restriction enzymeWhat do you name a restriction enzyme isolated from Xanthomonas graminis?
How long is the recognition sequence
• 4 bp: e.g., Taq 1, HpaII, MspI
• 6 bp: e.g., EcoR1, HindIII, BamH1, PstI, salI
• 8 bp: Not I, Sfi I
Recognition sequence may beRecognition sequence may be interrupted or ambiguous
Acc I: GT(at/gc)AC( g )
Bgl I: GCCNNNNNGGCg
Afl III: ACPuPyGTAfl III: ACPuPyGT
Three types of ends producedThree types of ends produced by type II restriction enzymes
• 3’-overhang (protruding)• 5’-overhang5 overhang• Blunt end
5’-overhang
EcoR I
5’-----------------------gaattc---------------------------3’5 -----------------------gaattc---------------------------33’-----------------------cttaag--------------------------5’
X EcoR1
5’-----------------------g+ aattc---------------------------3’g
3’-----------------------cttaa +aattc 3
g---------------------------5’
3’-overhang
Pst I:
5’-----------------------ctgcag---------------------------3’5 -----------------------ctgcag---------------------------33’-----------------------gacgtc--------------------------5’
X PstI
5’-----------------------ctgca-3’+ 5’-g---------------------------3’g
3’-----------------------g-5’ + 5 g 33’- actgc---------------------------5’
Blunt end
EcoR V
5’-----------------------gatatc---------------------------3’5 -----------------------gatatc---------------------------33’-----------------------ctatag--------------------------5’
X EcoR V
5’-----------------------gat+ atc---------------------------3’g
3’-----------------------cta + atc 3tag---------------------------5’
Odds of cutting at a segment of DNA
• 4 bp cutter: 44 = 256 bp• 6 bp cutter: 46 = 4 kbp• 8 bp cutter: 48 = 64 kb
• ??? How many do you predict Eco R1 to cut catfish genome of 8 x 109 bpg p
What do you expect ifWhat do you expect if you digest with youryou digest with your plasmid DNA with 4-p
bp cutters
What do you expect ifWhat do you expect if you digest with youryou digest with your plasmid DNA with 6-p
bp cutters
What do you expect ifWhat do you expect if you digest with youryou digest with your plasmid DNA with 8-p
bp cutters
Restriction mappingEc
oR1
EcoR
1
EcoR
1
Pst I
Pst I
E EEP P
10 kb
0.6 kb 2.4 kb 6.0 kb 1.0 kb
1.5 kb 0.8 kb7.7 kb
10 kb
What do you expect to see with double digest, if reaction is complete?
0.6 kb, 0.9 kb*, 1.5 kb, 6.0 kb, 0.2 kb, 0.8 kb.
What do you expect ifWhat do you expect if you digest with youryou digest with your genomic DNA with 4-g
bp cutters
What do you expect ifWhat do you expect if you digest with youryou digest with your genomic DNA with 6-g
bp cutters
What do you expect ifWhat do you expect if you digest with youryou digest with your genomic DNA with 8-g
bp cutters