Restriction Endonuclease

MCB5706:BIOMOLECULAR ENGINEERING

Prof Tan Wen Siang

By: Gol Mohammad Dorrazehi GS 31726

19 March 2012

Restriction

enzymes

History and Definition

Discovery Definition

Mechanism

Naming

Types

Type I

Type II

Type III

Artificial RE

Application

Recombinant DNA Cloning

SNPs genotype DNA

RFLP

DNA sequencing

DNA storage – libraries

Discovery 1952-53: Luria and Human

discovered the phenomenon of

restriction and modification

Over 10,000 bacteria species have been screened for restriction enzymes From them Over 3000 restriction enzymes have

been studied in detail More than 600 are available commercially and are routinely used for DNA modification and manipulation in laboratories

DefinitionRestriction enzymes are Molecular Scissors

Restriction Endonuclease is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences (mostly palindromic or symmetry) known as restriction sites

Palindromic sequence?

eEnglish palindromes:- Mom- Dad- Able was I ere I saw ElbaDNA Palindromes:The EcoRI cutting site:

5'-GAATTC-3'3'-CTTAAG-5'

The HindIII cutting site:5'-AAGCTT-3'3'-TTCGAA-5'

palindromic sequence

Restriction site?

The mirror-like palindrome in which the same forward and backwards are on a single strand of DNA strand, as in GTAATG

The inverted repeat palindrome is

also a sequence that reads the same forward and backwards, but the forward and backward sequences are found in complementary DNA strands (GTATAC being complementary to CATATG)

Inverted repeat palindromes are more common and have greater biological importance than mirror-like palindromes.

DNA Palindromes

Restriction site

Also called cleavage site or restriction

endonuclease site The specific nucleotide site recognized

by restriction endonuclease

Are generally palindromic sequences

Back

Mechanism of function Function:restriction enzymes protect cells from foreign DNA(they restrict the function of infecting DNA) Mechanism:Restriction enzymes recognize a specific sequence of nucleotides, and produce two types of double-stranded cut in the DNA:

BLUNT ENDS

STICKY ENDS

Blunt End cut:

AluI

HaeIII

Sticky End cut:

EcoRI

HindIII

- OH 3’

5’ P -

- P 5’

3’ OH -

Direct fashion

Designed fashion

Self DNA protection

Bacteria protect their own DNA from restriction digestion by methylation of its recognition site

Back

Nomenclature

1. First letter from genus2. Next two letters represent species3. Additional letter or number represent the strain or serotypes

For example. the enzyme HindII was isolated from Haemophilus influenzae serotype d

Eco R1: E coli

Pst I: Providencia

stuartii

Not I: Norcardia

otitidis-caviarum

Back

Derivation of the EcoRI name

Abbreviation Meaning Description

E Escherichia genus

co coli species

R RY13 strain

I First identified order of identificationin the bacterium

Example: EcoR1 Genus: Escherichia Species: coli Strain: R Order discovered: 1

Types of RE

Type I: Cleave at sites remote from recognition site

Type II: Cleave within or at short specific distances

from recognition site

Type III: Cleave outside of their recognition

sequences

Type IV: Target normal DNA

Artificial restriction enzymes:Artificial restriction enzymes can be generated by fusing a natural or engineered DNA binding domain to a nuclease domain

Type I

Capable of both restriction and modification activities

The cofactors S-Adenosyl methionine (AdoMet), ATP, and

Mg2+, are required for their full activity

Contain two R (restriction) subunits,

two M (methylation) subunits and

one S (specificity) subunit

Cleave DNA at random length from recognition

site

Type II

Mostly used for gene analysis and cloning

More than 3500 REs Recognize 4-8 bp sequences

Need Mg 2+ as cofactor

Cut in close proximity of the recognition site

Homodimers ATP hydrolysis is not required

Examples: EcoRI, EcoRII, BamHI, HindIII

Type III

Large enzymes Combination restriction-and-modification Cleave outside of their recognition

sequences Require two recognition sequences in

opposite orientations within the same DNA molecule

No commercial use or availability

Type IV

Cleave only normal and modified DNA (methylated, hydroxymethylated and glucosyl-hydroxymethylated bases).

Recognition sequences have not been well defined

Cleavage takes place ~30 bp away from one of the sites

Artificial RE

generated by fusing a natural or engineered DNA binding domain to a nuclease domain

can target large DNA sites (up to 36 bp) can be engineered to bind to desired DNA

sequences

 Zinc finger nucleases are the most commonly used artificial restriction enzymes Generally used in genetic engineering applicationsBack

Application in cut and paste DNA make genetic engineering possible with coordination of Ligase Enzyme

Rrestriction site could be added to primers (forward&Reverse) that will be used for amplifying gene

Both vector and amplified gene will be cut by RE to produce sticky ends

In ligation mixture the target gene will be inserted to vector with the aid of sticky ends and DNA ligase

RE in Cloning

The Restriction site must be out of the sequence of target gene

RE in Cloningi. Designing of primer with

restriction siteii. Amplifying target gene by

PCR and designed primeriii. Double digestion of vector

and amplified gene by same REs

iv. Ligation of gene and vectorv. Transformation vi. Expression

Back

(SNPs) Single Nucleotide Polymorphisms distinguish gene alleles by specifically recognizing single base changes in DNA

By use of SNP the restriction enzyme can be used to genotype a DNA sample without the need for expensive gene sequencing

RE in SNPs

DNA molecule 1 differs from DNA molecule 2 at a single base-pair location (a C/T polymorphism)

Perform (PCR) to amplify a region surrounding this SNP Fragmenting the sample of DNA by a restriction enzyme ( restriction digest) The resulting DNA fragments are then separated by length through agarose gel electrophoresis Alleles with correct restriction sites will generate two visible bands of DNA on the gel

SNPs

Back

(RFLP) Restriction Fragment Length Polymorphism Molecular biologists have identified

regions of the human genome where restriction fragment lengths are highly variable between individuals

Electrophoresis of these RFLP’s produce different patterns of DNA bands

RE in RFLP

In a similar manner to SNP, restriction enzymes are used to digest genomic DNA for gene analysis by Southern blot This technique identify how many copies (or paralogues) of a gene are present in the genome of one individual, Analysis of RFLP is used in genome mapping, DNA fingerprinting, genetic disease analysis, identifying mutations (polymorphisms) and DNA libraries

RFLP

m

_

+

DNA is negatively charged from the phosphate backbone

Visualize DNA with ethidium bromide – fluoresces ONLY when bound to DNA

Restriction Enzymes for RFLP

Question & Comments?