Restriction Enzymes

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Restriction Enzymes
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• Restriction Enzymes scan the DNA sequence
• Find a very specific set of nucleotides
• Make a specific cut

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Palindromes in DNA sequences

• Genetic palindromes are similar to verbal
  palindromes. A palindromic sequence in DNA is one
  in which the 5 to 3 base pair sequence is
  identical on both strands.

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3
5
3
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• Restriction enzymes recognize and make a cut
  within specific palindromic sequences, known as
  restriction sites, in the DNA. This is usually a
  4- or 6 base pair sequence.

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Restriction Endonuclease Types

• Type I- multi-subunit, both endonuclease and
  methylase activities, cleave at random up to 1000
  bp from recognition sequence
• Type II- most single subunit, cleave DNA within
  recognition sequence
• Type III- multi-subunit, endonuclease and
  methylase about 25 bp from recognition sequence

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Hae III

• HaeIII is a restriction enzyme that searches the
  DNA molecule until it finds this sequence of four
  nitrogen bases.

5 TGACGGGTTCGAGGCCAG 3 3 ACTGCCCAAGGTCCGGTC 5

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Once the recognition site is found Hae III will
cleave the DNA at that site
5 TGACGGGTTCGAGGCCAG 3 3 ACTGCCCAAGGTCCGGTC 5
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These cuts produce blunt ends
5 TGACGGGTTCGAGG CCAG 3 3 ACTGCCCAAGGTCC GGTC
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• The names for restriction enzymes come from
• the type of bacteria in which the enzyme is found
• the order in which the restriction enzyme was
  identified and isolated.

• EcoRI for example
• R strain of E.coli bacteria
• I as it is was the first E. coli restriction
  enzyme to be discovered.

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blunt ends and sticky ends

• Hae III produced a blunt end?
• EcoRI makes a staggered cut and produces a
  sticky end

5 GAATTC 3 3 CTTAAG 5
5 GAATTC 3 3 CTTAAG 5
5 G AATTC 3 3 CTTAA G 5
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• blunt end
• sticky end

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More examples of restriction sites of restriction
enzymes with their cut sites

• Hind III 5 AAGCTT 3
• 3 TTCGAA 5
• Bam HI 5 GGATCC 3
• 3 CCTAGG 5
• Alu I 5 AGCT 3
• 3 TCGA 5

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Separating Restriction Fragments, I
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Separating Restriction Fragments, II
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Gene Cloning

• What is gene cloning? How does it differ from
  cloning an entire organism?
• Why is gene cloning done?
• How is gene cloning accomplished ?
• What is a DNA Library?

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What is DNA cloning?

• When DNA is extracted from an organism, all its
  genes are obtained
• In gene (DNA) cloning a particular gene is copied
  (cloned)

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Why Clone DNA?

• A particular gene can be isolated and its
  nucleotide sequence determined
• Control sequences of DNA can be identified
  analyzed
• Protein/enzyme/RNA function can be investigated
• Mutations can be identified, e.g. gene defects
  related to specific diseases
• Organisms can be engineered for specific
  purposes, e.g. insulin production, insect
  resistance, etc.

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How is DNA cloned?, I
Blood sample

• DNA is extracted- here from blood
• Restriction enzymes, e.g. EcoR I, Hind III, etc.,
  cut the DNA into small pieces
• Different DNA pieces cut with the same enzyme can
  join, or recombine.

DNA
Restriction enzymes
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The action of a restriction enzyme, EcoR I Note
EcoR I gives a sticky end
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DNA Cloning, II

• Bacterial plasmids (small circular DNA additional
  to a bacterias regular DNA) are cut with the
  same restriction enzyme
• A chunk of DNA can thus be inserted into the
  plasmid DNA to form a recombinant

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DNA cloning, III

• The recombinant plasmids are then mixed with
  bacteria which have been treated to make them
  competent, or capable of taking in the plasmids
• This insertion is called transformation

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DNA Cloning, IV

• The plasmids have naturally occurring genes for
  antibiotic resistance
• Bacteria containing plasmids with these genes
  will grow on a medium containing the antibiotic-
  the others die, so only transformed bacteria
  survive

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DNA Cloning, V

• The transformed bacterial cells form colonies on
  the medium
• Each cell in a given colony has the same plasmid
  ( the same DNA)
• Cells in different colonies have different
  plasmids ( different DNA fragments)

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Screening, I

• Screening can involve
• Phenotypic screening- the protein encoded by the
  gene changes the color of the colony
• Using antibodies that recognize the protein
  produced by a particular gene

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Screening, II

• 3. Detecting the DNA sequence of a cloned gene
  with a probe (DNA hybridization)

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Polymerase Chain Reaction

• PCR

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PCR

• invented by Karry B. Mullis (1983, Nobel Prize
  1993)
• patent sold by Cetus corp. to La Roche for 300
  million
• depends on thermo-resistant DNA polymerase (e.g.
  Taq polymerase) and a thermal cycler

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Heat-stable DNA polymerase

• Taq DNA polymerase was isolated from the
  bacterium Thermus aquaticus.
• Taq polymerase is stable at the high temperatures
  (95oC) used for denaturing DNA.

Hot springs at Yellowstone National Park,
Wyoming.
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DNA polymerase requirements

• template
• primer
• nucleotides
• regulated pH, salt concentration, cofactors

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Steps in DNA replication

1. template denatured
2. primers anneal
3. new strand elongation

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Steps in a PCR cycle

• 1) template denatured
• 94 C, 30 sec
• 2) primers anneal
• 45-72 C, depending on primer sequence
• 30 sec 1 min
• 3) new strand elongation
• 72 C depending on the type of polymerase
• 1 min for 1000 nucleotides of amplified sequence

Number of specific DNA molecule copies grows
exponentially with each PCR cycle. Usually run
20-40 cycles to get enough DNA for most
applications (If you start with 2 molecules,
after 30 cycles you will have more than a billion)
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PCR Process

• 25-30 cycles
• 2 minute cycles
• DNA thermal cycler

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Template denatured
Annealing primers
New strand elongation
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Uses for PCR

• Research
• Gene cloning
• Real-time PCR
• DNA sequencing

• Clinical
• DNA fingerprinting
• Crime scene analysis
• Paternity testing
• Archeological finds
• Genetically inherited diseases

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DNA Sequencing
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Chain termination method (Sanger Method),
sequence of single stranded DNA is determined by
enzymatic synthesis of complementary strands
which terminate at specific nucleotide
positions Chemical degradation method
(Maxam-Gilbert Method), sequence of a double
stranded DNA molecule is determined by chemical
treatment that cuts at specific nucleotide
positions
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Dideoxynucleotide (ddNTP)
http//www.ncbi.nlm.nih.gov/books/bv.fcgi?ridhmg.
figgrp.604
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http//www.ncbi.nlm.nih.gov/books/bv.fcgi?ridhmg.
figgrp.605
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http//www.ncbi.nlm.nih.gov/books/bv.fcgi?ridhmg.
figgrp.605
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http//www.ncbi.nlm.nih.gov/books/bv.fcgi?ridgeno
mes.figgrp.6477
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http//www.ncbi.nlm.nih.gov/books/bv.fcgi?ridhmg.
figgrp.607
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Costs and time for sequencing a human genome
(3.2 billion bp)