Download Restriction Enzyme Digestion

Restriction Enzyme Digestion
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Definition
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http://www.esemag.com/0105/dna2.jpg
 Restriction enzyme:
A protein that recognizes a particular sequence of DNA and cuts
the DNA at that site (the restriction site)
 Digestion:
the act of breaking down into pieces
Restriction enzymes
 Recognize short
specific sequences,
often palindromes
 Isolated from bacteria
as endogenous
“restrictors” of bacterial
pathogens
http://arbl.cvmbs.colostate.edu/
Different enzymes…different
sites…different cuts
http://www.phschool.com/science/
Eco RI
http://www.phschool.com/science/
Pst I
http://www.phschool.com/science/
Sma I
Animations
Cutting and pasting
3D animation
Quick Quiz
A restriction enzyme cuts:
A.
B.
C.
D.
Single-stranded DNA
RNA
Proteins
Double-stranded DNA
Quick Quiz
A restriction enzyme works by identifying a
specific DNA sequence and cleaving:
A. The sugar-phosphate backbone of one
strand
B. The sugar-phosphate backbone of both
strands
C. The nitrogenous bases from one strand
D. The nitrogenous bases from both strands
Examples
 Restriction enzyme digestion
is used for…
 Identifying individuals
(DNA fingerprinting)
 Identifying species
(e.g. Mytilus)
 Cloning
(moving genes in and out of
plasmids)
http://www.nottingham.ac.uk/biology
http://www.bio.davidson.edu/courses/genomics/method/inducepromoter.gif
Quick Quiz
ITS PCR products must be cut by restriction
digestion to identify the different Mytilus
species because:
A. Both mussel species have an ITS PCR
product of the same size
B. Both mussel species have the same
restriction sites
C. ITS PCR products can vary in size
D. Neither mussel species gives good ITS
PCR bands
Non-Examples
Restriction enzyme
digestion is NOT
used for…
Cutting RNA
Sequencing DNA
Digestion Procedure
Prepare Master Mix
Add buffer and enzyme in correct proportions.
Add restriction digest master mix to DNA
Mix thoroughly by flicking tube.
Incubate
Temperature and time depend on enzyme.
View result by gel electrophoresis
Downstream Applications
 Ligation and…..
http://www.kochi-u.ac.jp/~tatataa/tech2/gene/ligation.jpg
 Bacterial transformation
http://hthbiotech.sandiegostc.org/tr-2a-b.html
END
Resources
http://www.phschool.com/science/biology_place/biocoach/red/intro.html
http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/cuteffects.html
http://web.indstate.edu/thcme/mwking/molecular-medicine.html#restriction
http://www.fermentas.com
California State Chemistry Standards
Grades 9-12
2. Properties of matter result from the ability of atoms to form
bonds…
6. Solutions are homogenous mixtures of two or more
substances
10b. Students know the bonding characteristics of carbon…
California State Biology Standards
Grades 9-12
5d. Students know the general structure and function of DNA
California State
Investigation and Experimentation
Standards
Grade 7
a. Select and use appropriate tools and technology to perform
tests, collect data, analyze relationships, and display data
Grades 9-12
c. Identify possible reasons for inconsistent results, such as
sources of error or uncontrolled conditions
d. Formulate explanations by using logic and evidence
k. Recognize the cumulative nature of scientific evidence
l. Analyze situations and solve problems that require combining
and applying concepts from more than one area of science
National Standards
Grades 6-12
Content Standard A: Science as Inquiry
Content Standard C: Life Science
Content Standard E: Science and Technology