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Restriction endonucleases and their applications

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Restriction endonucleases est ct o e do uc easesand their applications

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’

Only Compatible, base-pairable ends

li tcan ligate

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

Selection of restrictionSelection of restriction enzymes

Of Over 250 commercially available and over 2,000 total, how do I know what to use?

• Cutting frequency• Easy to work with • Economical