1. Biotechnology
Restriction Enzymes
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2. Introductory Question
You want to make recombinant DNA molecule from DNA isolated from humans and bacteria. What would you do first to make the recombinant DNA molecule?
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3. Essential Questions
What are restriction enzymes and where are they found in nature?
What role to they play in recombinant DNA Technology?
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4. Restriction Enzymes Cut at specific sites along the DNA
Each enzyme recognizes a unique cut site
Reflect: How are restriction enzymes like the scissors in the introductory activity?
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5. Where are restriction enzymes found in nature?
Proteins found in bacteria
Protect bacteria against DNA from invading organisms including viruses or other bacteria
Protect the bacteria by cutting the foreign DNA
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6. Naming Restriction Enzymes
Restriction enzyme name reflects bacterium where the enzyme originated
Letters represent the genus and species of the bacterium
Roman numeral indicate the order in which the enzymes were discovered
Ex. EcoRI – first enzyme isolated from Escherichia coli RY13
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7. Restriction Site
A short sequence of DNA that the restriction enzyme recognizes
Each restriction enzyme recognizes distinct restriction sites
Both strands are cut
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8. Characteristics of the Restriction Site
Four to six nucleotides in length
Symmetrical meaning the sequence for both strands are the same when read in 5’ to 3’ direction
Symmetrical restriction site known as the palindromic sequences 5’ 3’ 3’ 5’
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9. Two Types of Cut Sites
Blunt End – restriction enzyme cuts the two strands of DNA such that the ends are even across both strands
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10. Two types of Cut Sites
Sticky ends- restriction enzyme cuts the two strands in a staggered way.
This results in one end of DNA being single- stranded and overhangs the other strand
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11. Restriction Fragment DNA after cut with restriction enzymes
Fragments created differ in size depending on where the restriction site is located
The size of the fragment is defined by the number of paired nucleotides along the fragment
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12. Determination of Fragment Size
Can be determined by conducting agarose gel electrophoresis
Compare the DNA Fragments to known standards after they migrate in the gel under an electrical current and stained.
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13. Questions Answer the following questions on a separate piece of paper
In what organism is restriction enzymes found in nature and what role do they play in this organism?
Explain what the name for the restriction enzyme BamHI represents.
Explain what scientists are referring to when they say a restriction site has a “palindromic sequence”.
Explain the difference between staggered end and blunt end cut sites.
Explain how the size of the DNA fragment is determined. Provide a detailed answer.
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