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Ppt. Restriction Enzymes

Restriction enzymes are biological scissors that recognize specific nucleotide sequences in DNA and cleave the sugar phosphate backbones at those sites. They are used in various applications, including making recombinant DNA, DNA profiling, and analyzing genetic variations. Discovered in bacteria, these enzymes play a critical role in molecular biology and biotechnology.

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52 views

Ppt. Restriction Enzymes

Restriction enzymes are biological scissors that recognize specific nucleotide sequences in DNA and cleave the sugar phosphate backbones at those sites. They are used in various applications, including making recombinant DNA, DNA profiling, and analyzing genetic variations. Discovered in bacteria, these enzymes play a critical role in molecular biology and biotechnology.

Uploaded by

swastikasyangbho
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We take content rights seriously. If you suspect this is your content, claim it here.
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Restriction Enzymes

• Restriction enzymes (RE) are enzymes that have the ability to recognizes a specific, short
nucleotide sequence and cleave the sugar phosphate backbones in double stranded DNA at that
specific site.

• The specific site called: RESTRICTION SITE .

• They are biological scissors.

• RE naturally found in a wide variety of prokaryotes.


➔➔Bacterium is immune to its own restriction enzymes, even if it has the target sequences
ordinarily targeted by them.Why?

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Endonucleases

 Endonucleases are enzymes that cleave the phosphodiester bond within a


polynucleotide chain.
 Some of them have no regard to sequence when cutting DNA, but many others
do so only at specific nucleotide sequences.
 The latter group is often called r`estriction endonucleases or restriction
enzymes.
Restriction Enzymes

Learning intention Success criteria


❑ Describe the process of cutting ❑ recall that enzymes are proteins
DNA using restriction enzymes that catalyze specific chemical
reactions under specific conditions
❑ Recognise how restriction enzymes
are used to make recombinant
DNA.
❑ Identify restriction sites in double-
stranded DNA
DNA technologies are used in a range of industries; your body
and your food are made of cells with DNA and protein.
▪ Enzymes are biological catalysts – found in all cells.
▪ They are molecules (usually proteins) that speed up chemical reactions.
▪ Enzymes have unique chemical structures that mean they only act on
specific substrates
▪ For a particular enzyme to work optimally, it must be under specific
conditions (temperature, pH), otherwise its unique chemical structure
will be disrupted.
▪ Enzymes are critical for a range of cellular processes including digestion,
DNA replication, protein synthesis.
Restriction Enzymes - purposes

Researchers rely on restriction enzymes to assist with many processes in laboratories around
the world:

1. Making recombinant DNA and appraising success


 For research, medicine and agriculture
2. DNA profile analysis
 For disease diagnosis, paternity/family relationship testing, and forensics
Restriction Enzymes

▪ These enzymes were discovered in


bacteria.
▪ They help the bacteria destroy viral DNA.
(bacteria get viruses too!)
▪ They cut between specific bases (letters) of
the double stranded DNA molecule
▪ The DNA is then in multiple pieces
▪ The pieces are separated by gel
electrophoresis for analysis
https://www.sciencelearn.org.n
z/resources/2035-restriction-
enzymes
Specific restriction enzymes cut at
specific DNA sequences.
For example:
EcoRI is an enzyme that cuts at the
following sequence: GAATTC

EcoRI was discovered in E. coli


bacteria.

The resulting pieces of DNA are


called “restriction fragments.”
Many restriction enzymes
❑ HindIII was discovered in H.
influenza

❑ PstI was discovered in P. stuartii

❑ EcoRV was discovered in E. coli

Protruding ends are also


called “sticky” ends. Why
might these be useful?
Restriction fragments

https://www.addgene.org/protocols/dna-ligation/

When you cut two separate molecules


of DNA with the same restriction
Purpose: making recombinant enzyme, the fragments will have
matching sticky ends.
DNA This is how recombinant DNA is
created.
Booklet 3 Part A

Example sequence. Cut with EcoRI.

T C CAG C T G G AC G AAT T C T T CA GAT G AAT T C


AAA
AG G T C GAC C T G C T TAAGAAG T C TAC T TAAG
T
• T T
Number of fragments: 3
• Size of each fragment: 12bp, 9bp, 4bp
• EcoRI:
➔➔siisolated from E.coli strain RY13.
➔➔I indicates it was the first enzyme of that type isolated from E. coli RY13.

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Sticky end

Blunt end

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RE Restriction site
Origin
name

EcoRI Escherichia coli

BamHI Bacillus amyloliquefaciens H

HindIII Haemophilus influenza RD

HaeIII Haemophilus aegyptius

AluI Arthrobacter luteus

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➔
➔Restriction Endonuclease scan the length of the DNA.

 ➔Binds to the DNA molecule when it recognizes a specific sequence.


➔

 ➔➔Makes one cut in each of the sugar phosphate backbones of the double helix –
by hydrolysing the phosphodiester bond (Specifically between the 3’ O atom and
the P atom is broken).

 (Scan ➔➔Recognize ➔➔Cut)

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1. Generation of restriction map.

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2. Recombinant DNA technology (gene cloning).

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3. Restriction Fragment Length Polymorphism (RFLP).

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• Is a tool to study variations among individuals (humans and other species).

• This technique able to differentiate minor nucleotide sequence variations in


homologous fragments of DNA

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PCR for the
region thatyou Incubation of Agarose gel to
DNA the DNA with visualized your
want to dothe
Extraction RE results
study on

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