Chapter9 (and Section 8-4): Genetic Engineering

deadstructureBiotechnology

Dec 14, 2012 (4 years and 8 months ago)

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Chapter 9:

Genetic Engineering

Section 9
-
2:

Manipulating DNA

Tools of DNA Manipulation


Biologists have tools to cut, separate, and
read DNA sequences, and splice together
those sequences in almost any order



Tool #1: Cutting DNA


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


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



This is an example using
SmaI

EcoRI

Other Restriction Enzymes

** Note that the restriction enzyme recognition sequences are always palindromes.

Tool #2: Separating DNA


Electrophoresis
is a technique used to
separate DNA fragments cut by restriction
enzymes.



Fragments move through a special gel made
of
agarose


Electrophoresis


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


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


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


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


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


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


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


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