Restriction endonucleases est ct o e do uc eases and their - - PowerPoint PPT Presentation

Restriction endonucleases est ct o e do uc eases and their applications

Hi t f t i ti History of restriction endonucleases and its role e do uc eases a d ts o e in establishing molecular bi l biology

Restriction enzymes

• Over 10,000 bacteria species have been

screened for restriction enzymes O 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 iti discovered 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 d from 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 ) p DNA from cleavage.

• These enzymes recognize the same DNA

y g sequence as the restriction enzyme they accompany, but instead of cleaving the sequence, they disguise it by methylating one of the bases in h DNA t d each DNA strand.

Classification of Restriction enzymes enzymes

Class I Class II (93%) Class III Restriction-methylase

• n the same subunit

Homo-dimers, methylase on a separate subunit Restriction-methylase

• n the same subunit

p ATP-dependent Mg++ dependent ATP-dependent Binds to DNA recognition site and t DNA d l recognize symmetric DNA sequences and l ithi th Cut the DNA at the recognition site and th di i t f cuts DNA randomly - any DNA as long as it comes in contact cleave within the sequences then dissociate from the DNA

Type II Restriction enzymes are endonucleases that cut DNA at specific sites, and are most useful for molecular biology research

Type II Restriction enzymes Recognition sites Recognition sites are P li d i Palindroimes: 121 IFFI ABA IFFI, ABA AAGCTT TTCGAA

How do I know what sequence How do I know what sequence each enzyme cut?

• Test by cutting DNA of known sequence

y 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
• Invitrogen

Invitrogen

• 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 enzyme

What 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 be Recognition sequence may be interrupted or ambiguous

Acc I: GT(at/gc)AC ( g ) Bgl I: GCCNNNNNGGC g Afl III: ACPuPyGT Afl III: ACPuPyGT

Three types of ends produced Three types of ends produced by type II restriction enzymes

• 3’-overhang (protruding)
• 5’-overhang

5 overhang

• Blunt end

5’-overhang

EcoR I

5’-----------------------gaattc---------------------------3’ 5 -----------------------gaattc---------------------------3 3’-----------------------cttaag--------------------------5’ X EcoR1 5’-----------------------g + aattc---------------------------3’ g 3’-----------------------cttaa + aattc 3 g---------------------------5’

3’-overhang

Pst I:

5’-----------------------ctgcag---------------------------3’ 5 -----------------------ctgcag---------------------------3 3’-----------------------gacgtc--------------------------5’ X PstI 5’-----------------------ctgca-3’ + 5’-g---------------------------3’ g 3’-----------------------g-5’ + 5 g 3 3’- actgc---------------------------5’

Blunt end

EcoR V

5’-----------------------gatatc---------------------------3’ 5 -----------------------gatatc---------------------------3 3’-----------------------ctatag--------------------------5’ X EcoR V 5’-----------------------gat + atc---------------------------3’ g 3’-----------------------cta + atc 3 tag---------------------------5’

Only Compatible, base-pairable ends li t can ligate

Odds of cutting at a segment of DNA

• 4 bp cutter: 44 = 256 bp
• 6 bp cutter: 46 = 4 kb

p

• 8 bp cutter: 48 = 64 kb
• ??? How many do you predict Eco R1 to

cut catfish genome of 8 x 109 bp g p

What do you expect if What do you expect if you digest with your you digest with your plasmid DNA with 4- p bp cutters

What do you expect if What do you expect if you digest with your you digest with your plasmid DNA with 6- p bp cutters

What do you expect if What do you expect if you digest with your you digest with your plasmid DNA with 8- p bp cutters

Restriction mapping

EcoR1 EcoR1 EcoR1 Pst I Pst I E E E P P

10 kb

0.6 kb 2.4 kb 6.0 kb 1.0 kb 1.5 kb 0.8 kb 7.7 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 if What do you expect if you digest with your you digest with your genomic DNA with 4- g bp cutters

What do you expect if What do you expect if you digest with your you digest with your genomic DNA with 6- g bp cutters

What do you expect if What do you expect if you digest with your you digest with your genomic DNA with 8- g bp cutters

Selection of restriction Selection of restriction enzymes

Of Over 250 commercially available and

• ver 2,000 total, how do I know what to

use?

• Cutting frequency
• Easy to work with
• Economical