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Restriction Endonuclease as

Molecular ScissorsPresented By:-

Avni JethvaM.Sc Microbiology

(Sem2)

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Content

Introduction

History

Restriction enzymes Nomenclature

Different recognition sequences

Classification Applications

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Introduction

Bacteria possess restriction systems as

protection mechanism against incoming

DNA.

This system employs restriction enzymes

like endo- & exo- nucleases.

Nucleases cleave phosphodiester bonds

between nucleotides.

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History

1962-Arber & Dussoix :E.coli can restrict lambdaphage DNA

1965-Arber: E.colimethylate their DNA

1968-1st restrictionendonuclease in E.colidiscovered

1970-Smith, Wilcox &Kelly: Discovered 1st DNA-specific restrictionendonuclease inH.influenzae

http://www.pnas.org/content/vol102/issue17/images/large/zpq0150578910004.jpeg

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Restriction enzymes

Specific endonucleases

Recognize specific short sequences of DNA andcleave the DNA at or near the recognitionsequence

Recognition sequences: usually 4 or 6 bases butthere are some that are 5, 8, or longer

Recognition sequences are palindromes

Palindrome: sequence of DNA that is the samewhen one strand is read from left to right or theother strand is read from right to left consists ofadjacent inverted repeats

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Blunt & Sticky ends

http://en.wikipedia.org/wiki/Image:Restriction_enzyme.jpghttp://en.wikipedia.org/wiki/Image:Sma1.jpg

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Nomenclature

Restriction enzymes are named based on the bacteriain which they are isolated in the following manner:

Derive names from the bacteria

Genus- first letter capitalizedSpecies- second and third letters (small case)

Additional letters from strains

Roman numeral designates different enzymes

from the same bacterial strain, in numerical order

of discoveryExample: EcoRI

E Escherichia

co coli

R R strain

I first enzyme discovered fromEscherichia

coliR

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Different Recognition Sequences

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Classification

3 Basic types:-1)TypeI - cleavage occurs at random

site, so not used for gene manipulation.

2)TypeII recognize a particular

target in duplex DNA, so give fragments of

defined length.

eg: SmaI (blunt ends)

BamHI (sticky ends)

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3)TypeIII(rare) - asymmetric recognition

sites, nicks at measured distances from

recognition sequences. Not used for gene

manipulation.

eg: EcoRI

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The EcoRV-DNA complex

PDB code: 1RVC

Only the DNA from the structure isshown.

The backbone of the segmentsseparated by cleavage are shown indifferent shades of blue.

The orange atoms are magnesium ionsbound to the DNA that play a role in thecleavage reaction.

These ions are located at the cleavagesite; blunt ends are created.

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The EcoRV-DNA complex

The EcoRVendonucleasehas two sub-units (shown in

blue and gray). The DNA is

highlydistorted

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Applications

rDNA Technology

Plasmid Insertion

Southern Blotting DNA Sequencing

PCR

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DNA Sequencing.

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PCR