Download Chapter9 (and Section 8-4): Genetic Engineering

Chapter 9:
Genetic Engineering
Section 9-2:
Manipulating DNA
Tools of DNA Manipulation
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Biologists have tools to cut, separate, and
read DNA sequences, and splice together
those sequences in almost any order
Tool #1: Cutting DNA
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Restriction enzymes are proteins that cut
DNA at specific sequences
Each RE recognizes a different sequence –
there are more than 100
DNA can be cut into smaller, precisely sized
fragments allowing scientists to work with a
few hundred nucleotides at a time
Restriction Enzymes
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When RE cut DNA they can leave blunt
ends or sticky ends
Sticky ends are single-stranded regions on
either side of the cut
This is an example using EcoRI
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This is an example using SmaI
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EcoRI
Other Restriction Enzymes
** Note that the restriction enzyme recognition sequences are always palindromes.
Tool #2: Separating DNA
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Electrophoresis is a technique used to
separate DNA fragments cut by restriction
enzymes.
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Fragments move through a special gel made
of agarose
Electrophoresis
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Step 1: Cut DNA using RE
Step 2: Place fragments at one of the gel in
wells
Step 3: Apply electric current. (Set gel into
buffer solution that conducts current –
Negative electrode at the end with the DNA
fragments)
Electrophoresis
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DNA has a negative charge, so the current
will pull the DNA fragments toward the
positive electrode
Smaller fragments move through the gel
faster than larger fragments
Tool #3: Reading DNA
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Once REs have cut a sample of DNA,
fragments can be placed in a test tube with
DNA polymerase and nucleotides to “read”
the fragments
Complementary DNA strand produced using
chemically modified nucleotides that stop
assembly at certain spots – fragments then
separated by electrophoresis
Reading DNA
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After electrophoresis, gel has a pattern of
bands that reveals the DNA sequence
Done by computers
Used in Human Genome Project (handout)
Tool #4: Splicing DNA
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Sticky ends left by some REs
If two samples of DNA are cut with the same
RE, their sticky ends can be matched up and
enzymes can be used to permanently join
the fragments
Newly joined pieces of DNA are called
recombinant DNA
Recombinant DNA
http://www.eng.auburn.edu/~yylee/che595/Reading%20Assignments/Recombinant%20DNA.htm
Cell Transformation
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Involves inserting new genes into a cell,
changing the cell’s genetic makeup
Uses recombinant DNA
Can be done in prokaryotes and eukaryotes
Transforming Bacteria
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Some bacteria have their regular DNA plus a small,
circular, extra piece of DNA called a plasmid
Plasmid can be made recombinant using REs – new
genes are spliced in
Recombinant plasmids are mixed into bacterial
cultures - under the right conditions they will be
picked up by some bacteria
These bacteria will then reproduce more bacteria
containing the recombinant plasmid
Transforming Eukaryotes
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More difficult to get a eukaryote to accept
foreign DNA because they are more complex
Yeasts (eukaryote) contain plasmids like
bacteria, therefore are commonly used for
transformation
Animal and plant cells without plasmids have
been transformed by injecting new DNA