http://ca.youtube.com/watch?v=7PvnyAEC65A&eurl=http://genomics.xprize.org/
Dr. Kathleen Hill
khill22@uwo.ca
Office Hours: Monday noon to 5pm
Room 333 Western Science Centre
Hill Research and Teaching Website
http://www.uwo.ca/biology/Faculty/hill/index.htm
Study Guide for 2581b: WebCT site
281b Dr. Hill
DNA Extraction
Ever wonder where DNA comes from?
Now you can isolate it yourself using
items found around your house!
Materials:
Cheese cloth
Funnel
Test tube
Glass rod
Beaker
Ziploc bag
½ litre of water
What is the
function of
soap, salt and
alcohol in DNA
extraction?
2-3 salt pouches
Dish soap
2-3 slices of orange
Rubbing alcohol
Procedure:
1. Place a square of cheesecloth in the funnel and put in the test tube.
2. Mix salt, water, and a couple drops of dish soap in the beaker this is your
extraction buffer!
3. Put orange slices and some extraction buffer into a Ziploc bag, smush it up, then
let it sit for a few minutes
4. Pour the orange and extraction buffer mixture through the cheesecloth and collect
it in the test tube
5. Add some ice cold rubbing alcohol and let sit
6. Remove the extracted DNA (the white layer at the top) with a glass rod!
2581b Hill C9
DNA
Extraction
In the
laboratory a common method of DNA
Ever wonder where DNA comes from?
extraction
Now
you can isolate ituses
yourselfSDS
using (soap) to soluabilize membranes
items
around your house!
andfound
inactivate
proteins and Proteinase K to digest
proteins and then extractions with Phenol:
Chloroform to physically remove proteins from DNA.
Salt and alcohol permit precipitation of DNA
Materials:
Cheese cloth
Funnel
Test tube
Glass rod
Beaker
Ziploc bag
½ litre of water
2-3 salt pouches
Dish soap
2-3 slices of orange
Rubbing alcohol
Procedure:
1. Place a square of cheesecloth in the funnel and put in the test tube.
2. Mix salt, water, and a couple drops of dish soap in the beaker this is your
extraction buffer!
3. Put orange slices and some extraction buffer into a Ziploc bag, smush it up, then
let it sit for a few minutes
4. Pour the orange and extraction buffer mixture through the cheesecloth and collect
it in the test tube
5. Add some ice cold rubbing alcohol and let sit
6. Remove the extracted DNA (the white layer at the top) with a glass rod!
2581b Hill C9
Recombinant DNA Technology
Key Concepts
Two key properties of nucleic acids
ACGT
TGCA
Complementary
5’
3’
ACGT Antiparallel
TGCA
3’
5’
2581b Hill C9
Recombinant DNA Technology
Key Concepts
A key property of
Protein:nucleic acid interactions
Sequence specific binding
2581b Hill C9
Restriction Endonucleases Bind to
Specific Palindromic DNA Sequences
• Palindrome (Rotational symmetry)
• Cuts: through covalent bonds in the sugar
phosphate backbone of DNA
– Blunt/flush -Blunt ends
– Staggered
-Single stranded “sticky ends”
• 3’ overhang
• 5’ overhang
2581b Hill C9
Fig. 9.2
Certain restriction endonucleases cleave
the covalent bonds of the sugar
phosphate backbone at sites in the
palindromic sequence that create
Blunt or flush ends
2581b Hill C9
Fig. 9.2.b
Certain restriction endonucleases cleave
the covalent bonds of the sugar phosphate
backbone at sites in the palindromic
sequence that create
5’ sticky or cohesive ends
Hydrogen bonds possible
Hybridization possible with complementary sequence
2581b Hill C9
Fig. 9.2.c
Certain restriction endonucleases cleave
the covalent bonds of the sugar phosphate
backbone at sites in the palindromic
sequence that create
3’ sticky or cohesive ends
Hydrogen bonds possible
Hybridization possible with complementary sequence
2581b Hill C9
Restriction
Endonucleases
•derived from
prokaryotes
•have the Genus
and species
name of their
prokaryote
origin
•numbered since
there may be
many of these
enzymes in a
single species
2581b Hill C9
Restriction Endonucleases are derived
from prokaryotes
•Restriction Endonucleases cut double stranded DNA
at specific palindromic sequences
•Bacteria use restriction enzymes to protect from
invading viral nucleic acids
•Bacteria methylate their DNA to protect it from
digestion by their own restriction enzymes
2581b Hill C9
Fig. 9.3
Determining the frequency that a
palindrome is present in a DNA sequence
***Note Above: The
assumption was made that
A, C, T, G occur with equal
frequencies in the DNA
target sequence***
2581b Hill C9
Fig. 9.3
***Note: you may be
asked to determine the
average restriction
fragment size in cases
where there are
nonequivalent frequencies
of the four nucleotides***
2581b Hill C9
Fig. 9.3
Restriction Endonucleases with shorter
recognition sequences tend to have more
frequent cut sites and produce shorter
fragments
Frequent cutter
Less frequent cutter
2581b Hill C9
Fig. 9.3.a
1.The average fragment length for a four base
cutter is 256 bp
2.The average fragment length for a six base
cutter is about 4kb
2581b Hill C9
Restriction Endonuclease
Digestion
EcoRI
Cutting DNA
Circular DNA with one cut = one fragment
Linear DNA with one cut = two fragments
2581b Hill C9
Fig. 9.4.a
The concept of a
complete vs. partial digest
Complete Digest:
All possible sites
cut in all templates
in the reaction
Partial Digest: The
reaction is
incomplete and not
all sites and
templates are cut
2581b Hill C9
A linear DNA molecule has two target sites
for restriction enzyme AvaI. What is the
maximum number of DNA lengths that can
be produced if the sample is only partly
digested?
A. 3
B. 4
C. 5
D. 6
E. 9
2581b Hill C9
A linear DNA molecule has two target sites
for restriction enzyme AvaI. What is the
maximum number of DNA lengths that can
be produced if the sample is only partly
digested?
A. 3
B. 4
C. 5
D. 6
E. 9
2581b Hill C9
A linear DNA molecule has two
target sites for restriction enzyme
AvaI. What is the maximum number
of DNA lengths that can be produced
if the sample is only partly digested?
Question variables to be aware of:
•Linear or circular
•Number of target sites
•Type of digest: partial or complete
2581b Hill C9
Restriction Fragment Analysis
Gel electrophoresis separates DNA
fragments primarily on the basis of
size/length
Restriction Enzyme digest
of plasmid DNA
2581b Hill C9
Fig. 9.5.a
DNA is electrophoresed
through a polymer
Agarose vs. Acrylamide Gels
Agarose: larger migration space for DNA
Polyacrylamide: smaller migration space for DNA
2581b Hill C9
Fig. 9.5.a
•DNA is loaded (pipetted) into the wells of
the gel
•Sucrose or glycerol provide density so the
DNA sample sinks into the wells of the
submerged gel
•A dye helps to see the sample fall into the
well
DNA is negatively charged and migrates
at a rate relative to its size/length
2581b Hill C9
Fig. 9.5.b.2
Anatomy of a DNA Gel
-
Ethidium Bromide
is a dye that
intercalates with
DNA and fluoresces
upon UV exposure
DNA Migration
Size markers assist in
determining fragment
length
UV Light
Ethidium
Bromide
Migration
+
2581b Hill C9
Fig. 9.5.b.2
Genomic DNA
after Digestion
Cutting a complex
DNA sample with a
frequent cutter
results in a smear
Size markers assist
in determining
fragment length
2581b Hill C9