14265_Restriction Enzymes (1).ppt

WHAT ARE RESTRICTION WHAT ARE RESTRICTION ENZYMES?ENZYMES?

Restriction Enyzmes Restriction Enyzmes – molecular scissors – molecular scissors able to cut DNAable to cut DNA

HOW DO RESTRICTION HOW DO RESTRICTION ENZYMES WORK?ENZYMES WORK?

Usually cut DNA at a “palindrome” such as Usually cut DNA at a “palindrome” such as GAATTC.GAATTC.

Palindrome – word or phrase when spelled Palindrome – word or phrase when spelled backwords, spells the same word or phrasebackwords, spells the same word or phrase

Ex. Ex. BOBBOBMADAM I’M ADAMMADAM I’M ADAMA Toyota! Race fast, safe car. A Toyota A Toyota! Race fast, safe car. A Toyota

HOW DO RESTRICTION HOW DO RESTRICTION ENZYMES WORK?ENZYMES WORK?

GAATTCCTTAAG

5’ 3’

3’ 5’

| | | | | | “Restriction site”or

“Recognition Sequence”

HOW DO RESTRICTION HOW DO RESTRICTION ENZYMES WORK?ENZYMES WORK?

RE’s cut DNA’s RE’s cut DNA’s phosphodiester bonds phosphodiester bonds and hydrogen bonds.and hydrogen bonds.

HOW DO RESTRICTION HOW DO RESTRICTION ENZYMES WORK?ENZYMES WORK?

- RE’s generate two different types of “cuts”

- Sticky ends

- Blunt cuts.

WHERE DO RE’S COME FROM?WHERE DO RE’S COME FROM?

BacteriaBacteria

““Immune system” to Immune system” to protect against protect against bacteriophages bacteriophages (bacteria-infecting (bacteria-infecting viruses like Lambda).viruses like Lambda).

HOW ARE RE’S NAMED?HOW ARE RE’S NAMED?

After bacteria which produces them.After bacteria which produces them.

GenusGenus

SpeciesSpecies

StrainStrain

Order IsolatedOrder Isolated

Escherichia

coli

R

I

EcoRI

Haemophilus

influenzae

d

III

HindIII

Bacillus

amylo.

H

I

BamHI

Recognition Site G^AATTC A^AGCTT G^GATCC

Types of Restriction EnzymesTypes of Restriction Enzymes

Cleavage

site

Location of methylase

Examples

Type I Random

Around 1000bp away from recognition site

Endonuclease and methylase located on a single protein molecule

EcoK I

EcoA I

CfrA I

Type II Specific

Within the recognition site

Endonuclease and methylase are separate entities

EcoR I

BamH I

Hind III

Type III Random

24-26 bp away from recognition site

Endonuclease and methylase located on a single protein molecule

EcoP I

Hinf III

EcoP15 I

Restriction enzymes that have the same recognition sequence as Restriction enzymes that have the same recognition sequence as well as the same cleavage site are Isoschizomers.well as the same cleavage site are Isoschizomers.

SphI (CGTAC/G) and BbuI (CGTAC/G)SphI (CGTAC/G) and BbuI (CGTAC/G)

Restriction enzymes that have the same recognition sequence but Restriction enzymes that have the same recognition sequence but cleave the DNA at a different site within that sequence are cleave the DNA at a different site within that sequence are Neoschizomers. Eg:SmaI and XmaINeoschizomers. Eg:SmaI and XmaI

C C C G G G C C C G G G C C C G G GC C C G G G

G G G C C C G G G C C CG G G C C C G G G C C C

Xma I Sma IXma I Sma I

Isoschizomers and NeoschizomersIsoschizomers and Neoschizomers

HOW DO RESTRICTION HOW DO RESTRICTION ENZYMES WORK?ENZYMES WORK?

Must provide correct temperature and Must provide correct temperature and buffer (salt, pH) for enzyme to work.buffer (salt, pH) for enzyme to work.

Mimics cellular conditions of bacteria they Mimics cellular conditions of bacteria they come from.come from.

WHAT ARE RE’S USED FOR?WHAT ARE RE’S USED FOR?

Genetic engineering – Genetic engineering – pasting together DNA pasting together DNA from two different from two different organisms. organisms.

HOW DO RESTRICTION HOW DO RESTRICTION ENZYMES WORK?ENZYMES WORK?

Which are more useful in genetic Which are more useful in genetic engineering? RE’s that generate sticky ends engineering? RE’s that generate sticky ends or ones that produce blunt cuts?or ones that produce blunt cuts?

STICKY ENDS

HOW IS DNA PASTED HOW IS DNA PASTED TOGETHER?TOGETHER?

Ligase – another enzyme which Ligase – another enzyme which reconnects phosphodiester bonds.reconnects phosphodiester bonds.

WHAT ELSE ARE RE’S USED WHAT ELSE ARE RE’S USED FOR?FOR?

Forensics – DNA Fingerprinting for crime Forensics – DNA Fingerprinting for crime scene investigation and paternity testing.scene investigation and paternity testing.

Everyone’s DNA has a different sequence Everyone’s DNA has a different sequence – even though only 0.1% different.– even though only 0.1% different.

How frequently would EcoRI cut DNA?How frequently would EcoRI cut DNA?4

6= once every 4096 bp

Lambda (48,514 bp) would expect about 12 EcoRI sites

THOUGHT QUESTIONTHOUGHT QUESTION

Bacteria are prokaryotes.Bacteria are prokaryotes.

Prokaryotes do not have a nucleus.Prokaryotes do not have a nucleus.

Both DNA and RE’s are in cytoplasm.Both DNA and RE’s are in cytoplasm.

Why isn’t bacterial DNA cut by RE’s?Why isn’t bacterial DNA cut by RE’s?

Biological RoleBiological Role

Most bacteria use Restriction Enzymes as a Most bacteria use Restriction Enzymes as a defence against bacteriophages.defence against bacteriophages.

Restriction enzymes prevent the replication of Restriction enzymes prevent the replication of the phage by cleaving its DNA at specific sites.the phage by cleaving its DNA at specific sites.

The host DNA is protected by Methylases which The host DNA is protected by Methylases which add methyl groups to adenine or cytosine bases add methyl groups to adenine or cytosine bases within the recognition site thereby modifying the within the recognition site thereby modifying the site and protecting the DNA.site and protecting the DNA.

MethylationMethylation

In humans, methyl groups are used to tag In humans, methyl groups are used to tag genes to turn them on or off. genes to turn them on or off.