Transcript Using Gel Electrophoresis to analyze DNA, RNA and proteins

Using Gel Electrophoresis to
analyze DNA, RNA and proteins
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A little background
• Did you know that acids and bases
neutralize each other?
• An acid is anything that will dissociate a
hydrogen ion (the acid is an electron
acceptor)
• A base is anything that will bind to a
hydrogen ion (the base is an electron
donor)
• Carboxyl groups and phosphate groups
are good acids
• Amine groups are good bases!
• If you put an acid in a basic buffer, the
acid will become negatively charged.
Draw it!
• If you use an acidic buffer, a basic group
will become positively charged. Draw it!
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What is DNA?
• DNA stands for
deoxyribonucleic acid
• 99% of it is found in the
nucleus (nDNA). 1% is
found in mitochondria
(mDNA)
• It is a polymer: that
means individual units
were linked together to
create the long strand
seen here
• The individual units
(monomers) are
nucleotides
Pictures from:http://publications.nigms.nih.gov/thenewgenetics/chapter1.html
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What are nucleotides?
• The DNA code of a
gene—the sequence
of its individual DNA
building blocks,
labeled A (adenine), T
(thymine), C
(cytosine), and G
(guanine) and
collectively called
nucleotides
Pictures from:http://publications.nigms.nih.gov/thenewgenetics/chapter1.html
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Let’s see them up-close
• Notice their sugar
component
(deoxyribose)
• Notice their base (a
purine or a pyrimidine)
• Notice their phosphate
groups! When put into a
basic buffer, the
hydrogen atoms
dissociate and the
phosphate groups
become large,
negatively charged
groups! They act like an
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Picture
from:http://fig.cox.miami.edu/~cmallery/150/gene/c5x29nucleotides.jpg
acid!
So the DNA is now negatively charged, now what?
• In 1973, Dr. Edwin
Southern isolated
genomic DNA
• Used a restriction
enzyme to cut the DNA
into smaller fragments of
varying sizes
• Poured an agarose gel
• Loaded the sample in the
gel
• Applied an electrical
current
• DNA separates according
to size (smallest “runs”
the fastest).
Image from:http://www.scifair.org/+images/Electrophoresis.gif
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OK, but gels are slippery and can break, plus it’s
difficult to store for long term, what else can we
do? A Southern Blot!
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Image from:http://www.gbiosciences.com/Image/BE315.jpg
Now we have a piece of membrane with our
DNA stuck to it. But I can’t see anything,
now what?
• We have to hybridize our
membrane with a probe.
• A probe is basically a
piece of a gene that is
labeled (fluorescence or
radioisotope).
• Anywhere there is a gene
on the blot that matches
(basepairs) with our
probe, they will hybridize
• They remain hybridized
even after washing to
remove unbound probe.
This is our probe!
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Let’s review how we can manipulate DNA
Image from:http://openlearn.open.ac.uk/file.php/2645/S377_1_007i.jpg
Short term
Quick viewing
of DNA
fragments
Long term storage
Blot can be reused
(re-hybridized to
other probes) and
stored easily
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http://www.sumanasinc.com/webcontent/animations/content/gelelectrophoresis.html
But that’s not all! RNA molecules
can be manipulated, too!
• Did you know that RNA is
also a polymer?
• It is single stranded
• Uracil not thymine!
• And, RNA can be
manipulated on a gel just
like DNA.
• This technique is called a
“northern blot”
• Not capitalized, just a
“joke” taken from Dr.
Southern’s technique.
Negative charges when placed in a basic buffer!
http://www.steve.gb.com/science/prokaryotes_and_eukaryotes.html
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Northern blotting
• This technique is used to
find out which genes are
transcribed!
• You don’t need restriction
enzymes because mRNA
segments are short.
• Ex: Different genes
transcribed in different
tissues
• Ex: Different genes
transcribed at different
times of cell cycle within
the same cell
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Just because you see an RNA...
• Doesn’t mean the gene was
fully “expressed!”
• Remember, ultimately the
protein is the end product of
gene expression.
• Don’t worry! Proteins are
polymers too!
• Their monomers are amino
acids!
• When placed in a basic buffer,
they have negative charges
too!
• Guess what? Western blots
are used for protein analysis.
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The 20 Amino Acids
Glycine
Serine
Alanine
Threonine
Valine
Cysteine
Leucine
Isoleucine
Methionine Glutamic acid
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Aspartic acid
Phenylalanine
Lysine
Arginine
Asparagine
Glutamine
Tyrosine
Histidine
Tryptophan
Proline
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Zwitterions and pI
• The pH at which an amino acid exists in solution as a
neutral molecule, a zwitterion.
• Since zwitterions are not charged, they will not move
• Isoelectric points (pI) are for proteins and reflect when
their overall amino acid charges are balanced
(electrically neutral). As such, they will not migrate to
either side of an electrophoresis chamber.
• As a zwitterion the amino acid is electrically neutral
and will not migrate to either side of the
electrophoresis chamber.
• the pH at which an amino acid exists determines its
net electrical charge and the type of charge (+ or -).
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If you increase the pH of a solution of an amino acid
by adding hydroxide ions, the hydrogen ion is
removed from the -NH3+ group. During
electrophoresis, this amino acid would move toward
the anode (the positive electrode)
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If you decrease the pH by adding an acid to a solution of an
amino acid, the -COO- part of the zwitterion picks up a
hydrogen ion.
This time, during electrophoresis, the amino acid would move
towards the cathode (the negative electrode).
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Cellulose serum protein
electrophoresis (SPE)
• Proteins separate
according to their
electrical charges
• Small amount of
serum or urine
• Current passed
through strip
• Proteins stained
http://www.labtestsonline.org/understanding/analytes/electrophoresis/test.html
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Bands of SPE
• Albumin
– Most obvious band. Single
protein
• α1- globulins
– Almost entirely a1-antitrypsin
• α2 -globulins
– Mainly α2 -macroglobulin and
haptoglobin
• β-globulins
– β 1-transferrin
– β 2-C3 complement
• γ-globulins
– immunoglobulin
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Example: normal and abnormal
serum protein electrophoresis
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In contrast to cellulose strips, SDS gels allow
separation of proteins according to their size
because of the negative detergent binding to the
amino acids in the same ratio.
http://employees.csbsju.edu/hjakubowski/classes/ch331/Techniques/TechElectrophoresis.htm
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For a western, we use antibodies
as a probe.....Why?
A DNA probe would have nothing to bind to on a protein. We need to use antibodies.
http://employees.csbsju.edu/hjakubowski/classes/ch331/Techniques/TechElectrophoresis.htm
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http://www.biology.arizona.edu/IMMUNOLOGY/activities/western_blot/west1.html
Let’s play
• We have a family of 5: Dad, mom, Son 1,
Son 2, and Son 3.
• Who has the blue toenail gene? What test
would you perform?
• Who expresses the transcript? What test
would you perform?
• Who actually has blue toenails (no
cheating by asking to remove shoes and
socks!) What tests could you perform?
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Here’s the Southern blot:
+C
-C
D
M
S1
S2
S3
Questions:
•Why have a positive
control?
•Why have a negative
control?
•What happened to
mom’s BT gene?
•What can you say about
the sons’ BT genes?
•Which parent did they
inherit them from?
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Here’s the northern blot:
+C
-C
D
M
S1
S2
S3
Questions:
•Who expresses the BT
transcript?
•Does this necessarily
mean they have BTs?
•What blot will provide
you with proof?
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Here’s the western blot
+C
-C
D
M
S1
S2
S3
Who has
?
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What if you ran out of a basic
buffer?
• Do you have to cancel your experiments?
• What if you had an acidic buffer available?
• What charges would your molecules now
have?
• How would you set up your
electrophoresis equipment?
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Enzyme-linked Immunosorbant
Assay (ELISA)
• Quicker than a Western
• Not as specific as a western:
antibodies do go “bad” and
could bind aberrantly to an
antigen they should not bind.
• False positives: in addition to
an antibody “going bad”
antibodies tend to stick to the
positive charges on the plastic
dish. Blocking and washing
are VERY important!
• Pregnancy tests (confirmed
with blood test and
ultrasound).
• HIV diagnosis (must be
confirmed with western gel)
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http://www.biology.arizona.edu/IMMUNOLOGY/activities/elisa/technique.html
Step 1
Step 2
Step 3
Step 4
Step 5
Step 6
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