Restriction enzymes

Where are restriction enzymes found?

In bacteria and archaea

Role of restriction enzymes

Defence mechanisms in organisms against viruses or plasmids

Where are restriction enzymes found?

In bacteria and archaea

Role of restriction enzymes

Defence mechanisms in organisms against viruses or plasmids

What can restriction enzymes recognise?

Specific DNA sequences

What can restriction enzymes cleave?

Phosphodiester bonds within the DNA backbone at or near recognition sites

Classification of restriction enzymes

Based on recognition sites and cutting patterns

Recognition sites for restriction enzymes

Specific DNA sequences

Typically palindromic

Cutting patterns

Specific position within the recognition site where the enzyme cleaves the DNA

Step 1 of DNA cleavage and restriction sites

Binds to DNA and creates a double-stranded break at or near the recognition site

Step 2 of DNA cleavage and restriction sites

Cleavage results in formation of DNA fragments with either blunt ends or sticky ends

Blunt ends

Straight cut

Sticky ends

Overhanging ends

Type 1 restriction enzymes

Multifunctional enzymes that recognise specific DNA sequences

Cleavage sites are variable and non-specific

3 subunits

What do the subunits of Type 1 Restriction enzymes do?

1=recognise DNA sequence

2=DNA cleavage

Where do type 1 restriction enzymes cleave DNA?

At significant distance from their recognition sites

What do type 1 restriction enzymes require for their activity?

both ATP and S-adenosyl methionine (SAM)

Type II restriction enzyme

Most common

Recognise specific palindromic DNA sequences

Where do type II restriction enzymes cleave DNA?

At or near their recognition site

What do type II restriction enzymes need for activity?

Do not require ATP

Uses of type II restriction enzymes

-DNA cloning

-Recombinant DNA technology

-Gene editing techniques eg CRISPR-Cas9

-DNA fingerprinting

Type III restriction enzymes

Recognise specific DNA sequences

Similar to Type II in terms of recognition and cleavage patterns

Where do type III restriction enzymes cleave DNA?

Short distance from their recognition sites

What do type III restriction enzymes require for activity?

ATP

Palindromic sequences

DNA sequence can be read the same on both strands when read in the same direction

What does EcoRI recognise and produce?

Recognise=GAATTC

Produces=DNA fragments with sticky ends (5' overhang)

What does HindIII recognise and produce?

Recognise=AAGCTT

Produces=DNA fragments with sticky ends (5' overhang)

What does BamHI recognise and produce?

Recognise=CTCGAG

Produces=DNA fragments with sticky ends (3' overhangs)

What does Hae III recognise and cleave?

Recognise=GGCC

Cleave=DNA at specific recognition site

What can restriction enzyme digestion and ligation be used for?

Cloning

Gene editing

DNA mapping

Function of DNA ligase

Catalyses the formation of phosphodiester bonds between DNA fragments

Gel electrophoresis

Separates DNA fragments based on their size

Process of Gel electrophoresis

-Electrci field is applied to gel matrix

-DNA fragments migrate through gel

-Smaller DNA fragments move more quickly through. gel matrix

-Larger fragments move more slowly

Restriction mapping

Determination of the location of restriction sites on DNA molecule

How can restriction mapping be achieved?

Through digestion of DNA:

-Restriction enzymes

-Gel electrophoresis

What can restriction enzymes identify in terms of medical and diagnostic ways?

-Detect single nucleotide polymorphisms (SNPs)

-Restriction fragment length polymorphism (RFLP) analysis

-Genetic mutations

eg CF, Sickle cell anaemia, Huntington's disease

Techniques used to analyse patients DNA sample

-Polymerase chain reaction-RFLP

-Allele-specific PCR (AS-PCR)

-Restriction enzymes

What is the purpose of looking at patients DNA?

-Genetic testing

-Disease diagnosis

-Genetic predisposition

-Early detection of inherited disorders

Detection of BRCA1/BRCA2 mutations

Restriction enzymes in identifying genes

Analyse sample using RFLP analysis

Diagnosis of Thalassemia

Restrcition enzymes

RFLP analysis and PCR-RFLP=look for specific globulin genes

Thalassemia

Abnormal haemoglobin production

Detection of Cystic Fibrosis

Mutation in cystic fibrosis transmembrane conductance regulator (CFTR) gene

PCR-RFLP and AS-PCR techniques

Future developments in restriction enzymes

-Nanopore sequencing

-Single-cell genomics

-High-throughout screening methods