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Restriction Enzymes
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Restriction Enzymes
• Enzyme that cuts DNA at specific nucleotide sequences known as restriction sites.
• Found in bacteria and have evolved to provide a defense mechanism against invading viruses.
• In bacteria they selectively cut up foreign DNA in a process called restriction
• To cut the DNA, restriction enzyme makes two incisions, each strand of the DNA double helix.
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Picking a palindromeWords that read the same forwards as
backwards
Hannah
Level
Madam
hannaH
leveL
madaM
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Restriction Enzymes
• Over 3000 have been identified• More than 600 available commercially• Routinely used for DNA modification and
manipulation in laboratories.• http://en.wikipedia.org/wiki/Restriction_enzyme
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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.
5’
5’
3’
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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Each of the double strands of the DNA
molecule is complimentary to the other; thus adenine pairs with thymine, and guanine with
cytosine.
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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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Restriction enzymes recognize and make a cut within specific palindromic sequences, known as restriction sites, in the genetic code. This is usually a 4- or 6 base pair
sequence.
Example?
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Hae IIIHaeIII 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 was found HaeIII could go to work cutting
(cleaving) the DNA
5’ TGACGGGTTCGAGGCCAG 3’3’ ACTGCCCAAGGTCCGGTC 5’
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These cuts produce what scientists call“blunt ends”
5’ TGACGGGTTCGAGG CCAG 3’3’ ACTGCCCAAGGTCC GGTC 5’
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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 exampleR 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”Remember how HaeIII produced a “blunt end”? EcoRI, for instance, 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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Some more examples of restriction sites of restriction enzymes with their cut sites:
HindIII: 5’ AAGCTT 3’ 3’ TTCGAA 5’
BamHI: 5’ GGATCC 3’ 3’ CCTAGG 5’
AluI: 5’ AGCT 3’ 3’ TCGA 5’
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Separating Restriction Fragments, I
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Separating Restriction Fragments, II