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Restriction

Enzymes

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  • 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.

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  • The specific site called: RESTRICTION SITE .

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  • They are biological scissors.

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  • 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.

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Restriction Enzymes

Learning intention

  • Describe the process of cutting DNA using restriction enzymes

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Success criteria

  • recall that enzymes are proteins 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

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DNA technologies are used in a range of industries; your body and your food are made of cells with DNA and protein.

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  • 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.

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Restriction Enzymes - purposes

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

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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

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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

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https://www.sciencelearn.org.nz/resources/2035-restriction-enzymes

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Specific restriction enzymes cut at specific DNA sequences.

For example:

EcoRI is an enzyme that cuts at the following sequence: GAATTC

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EcoRI was discovered in E. coli bacteria.

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The resulting pieces of DNA are called “restriction fragments.”

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Many restriction enzymes

  • HindIII was discovered in H. influenza

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  • PstI was discovered in P. stuartii

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  • EcoRV was discovered in E. coli

Protruding ends are also called “sticky” ends. Why might these be useful?

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Restriction fragments

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

When you cut two separate molecules of DNA with the same restriction enzyme, the fragments will have matching sticky ends.

This is how recombinant DNA is created.

Purpose: making recombinant DNA

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Booklet 3 Part A

Example sequence. Cut with EcoRI.

T C C A G C T G G A C G A A T T C T T C A G A T G A A T T C A A A

A G G T C G A C C T G C T T A A G A A G T C T A C T T A A G T T T

  • Number of fragments: 3
  • Size of each fragment: 12bp, 9bp, 4bp

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  • EcoRI:

🡺🡺is isolated from E.coli strain RY13.

🡺🡺 I indicates it was the first enzyme of that type isolated from E. coli RY13.

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Blunt end

Sticky end

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RE

name

Origin

Restriction site

EcoRI

Escherichia coli

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BamHI

Bacillus amyloliquefaciens H

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HindIII

Haemophilus influenza RD

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HaeIII

Haemophilus aegyptius

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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.

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  • 🡺🡺 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).

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  • (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).

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  • This technique able to differentiate minor nucleotide sequence variations in

homologous fragments of DNA

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DNA

Extraction

PCR for the region that you want to do the

study on

Incubation of the DNA with RE

Agarose gel to visualized your results