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PCR lab
Week 1
LE 16-8
Sugar
Adenine (A)
Sugar
Thymine (T)
Sugar
Sugar
Guanine (G)
Cytosine (C)
DNA structure
LE 16-7
5 end
Hydrogen bond
3 end
1 nm
3.4 nm
3 end
0.34 nm
Key features of DNA structure
5 end
Partial chemical structure
Space-filling model
LE 16-5
Sugar–phosphate
backbone
Nitrogenous
bases
5 end
Thymine (T)
Adenine (A)
Cytosine (C)
Phosphate
Sugar (deoxyribose)
3 end
DNA nucleotide
Guanine (G)
DNA structure is antiparallel
• There is a 3’ end and a
5’ end
• Each strand is
unidirectional
• Many enzymes that
replicate DNA are
unidirectional also
Hydrogen bonding between DNA
bases
• A with T, C with G
• CG pairs have 3 bonds,
AT have two
LE 16-9_1
The parent molecule has
two complementary
strands of DNA. Each base is
paired by hydrogen bonding
with its specific partner, A with
T and G with C.
LE 16-9_2
The parent molecule has
two complementary
strands of DNA. Each base is
paired by hydrogen bonding
with its specific partner, A with
T and G with C.
The first step in replication is
separation of the two DNA
strands.
LE 16-9_3
The parent molecule has
two complementary
strands of DNA. Each base is
paired by hydrogen bonding
with its specific partner, A with
T and G with C.
The first step in replication is
separation of the two DNA
strands.
Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
LE 16-9_4
The parent molecule has
two complementary
strands of DNA. Each base is
paired by hydrogen bonding
with its specific partner, A
with T and G with C.
The first step in replication is
separation of the two DNA
strands.
Each parental strand now
serves as a template that
determines the order of
nucleotides along a new,
complementary strand.
The nucleotides are connected
to form the sugar-phosphate
backbones of the new strands. Each
“daughter” DNA
molecule consists of one
parental strand and one new
strand.
Cast and Mold- each can copy the
other
How is DNA replicated?
• It was expected, but not proven, that DNA was
replicated semiconservatively
• Competing models were the conservative
model and the dispersive model
LE 16-10
Parent cell
Conservative
model. The two
parental strands
reassociate after
acting as templates
for new strands,
thus restoring the
parental double
helix.
Semiconservative
model. The two
strands of the
parental
molecule
separate, and each
functions as a
template for
synthesis of a new,
comple-mentary
strand.
Dispersive model.
Each strand of both
daughter molecules
contains
a mixture of
old and newly
synthesized
DNA.
First
replication
Second
replication
Meselson-Stahl experiment
• They labeled the
nucleotides of the old
strands with a heavy
isotope of nitrogen
• The first replication
produced a band of hybrid
DNA, eliminating the
conservative model
• A second replication
produced both light and
hybrid DNA, eliminating the
dispersive model and
supporting the
semiconservative model
LE 16-11
Bacteria
cultured in
medium
containing
15N
DNA sample
centrifuged
after 20 min
(after first
replication)
Bacteria
transferred to
medium
containing
14N
DNA sample
centrifuged
after 40 min
(after second
replication)
First replication
Conservative
model
Semiconservative
model
Dispersive
model
Less
dense
More
dense
Second replication
DNA Polymerase
• Copies DNA
• Requires primers (primase)
• Requires unwound DNA (helicase)
– These are DNA binding proteins
• Works in a unidirectional manner (5’-3’)
• PCR uses Taq polymerase
PCR
•
•
•
•
Polymerase Chain Reaction
Uses Taq polymerase
Taq= Thermophilus aquaticus
PCR amplifies DNA samples
PCR
• Step 1- Melting
– DNA denatures
• Step 2- Annealing
– Primers bind to
complementary sequences
• Step 3- Elongation
– Taq DNA polymerase adds
free nucleotides to strands
• Cycle is complete, DNA
has doubled
• Process can begin again
Ingredients for PCR
1. dNTPs
2. Mg++ containing Buffer
3. Taq polymerase
4. Primers for your gene
of interest
5. Thermal cycler
6. A gene (piece of DNA)
you are interested in
All together = DNA xerox
machine!
dNTPs
• Individual DNA
nucleotides
• Four kinds- A, C, G,
and T
• They match up
with template
DNA
Taq Polymerase
• DNA polymerase
isolated from
Thermophilus aquaticus
bacteria
• Lives in hot springsheat resistant
• Optimal Taq temp- 72C
Primers
• Single-stranded DNA
sequences of 15-30 bp
specific to gene of
interest
• One at the 5’ start, the
other at the 3’ end of
your gene
Thermal Cycler
• Melting point
of DNA= ~94C
• Annealing
temp = 55C
• Optimal Taq
polymerase
temp= 72C
PCR II
February 1, 2008
DNA, replication, and PCR
DNA Lecture review
• DNA subunits are called _______.
• They are comprised of a sugar, a ____, and a
_______.
• There are 4 kinds of DNA bases: __, __, __
and _______.
• Adenine always binds with ______ and
guanine with_______- this is “______’s rules”.
• DNA bases cling together by _____ bonds.
More DNA facts
• DNA is the universal code to make ________.
• The sides of the DNA ladder run 5’-3’ down
one side, and 5’-3’ up the other. This is called
_____ structure.
• DNA is copied with the enzyme __________.
• DNA’s melting point is___________.
• People have about ______DNA base pairs per
haploid cell.
DNA Polymerase
• Copies DNA
• Requires primers (primase)
• Requires unwound DNA (helicase)
– These are DNA binding proteins
• Works in a unidirectional manner (5’-3’)
• PCR uses Taq polymerase
PCR
•
•
•
•
Polymerase Chain Reaction
Uses Taq polymerase
Taq= Thermophilus aquaticus
PCR amplifies DNA samples
PCR
• Step 1- Melting
– DNA denatures
• Step 2- Annealing
– Primers bind to
complementary sequences
• Step 3- Elongation
– Taq DNA polymerase adds
free nucleotides to strands
• Cycle is complete, DNA
has doubled
• Process can begin again
Gel Electrophoresis
• Phoresis- “carrying” (G)
• Moves (carries) DNA
through a gel using
electricity
• Speed depends on DNA
length
• Used for isolation,
purification, and
measurement of DNA
fragments
DNA is Negatively Charged
• Phosphates each carry a
single negative charge
• m/Z ratio for all DNA
segments is ~equal
• DNA will move to (+)
electrode (“Run to the
red”)
Agarose Gel
•
•
•
•
•
Purified from seaweed
Porous at molecular level
DNA moves through pores
Buffer conducts electricity
Large DNA molecules move
slower than small ones
Density can be varied
Loading a Gel
• DNA is mixed with
loading dye
• Dye-DNA mixture is
placed into gel wells
Loading a gel
• Put pipette tip in well
below buffer level
• Depress plunger to 1st
stop- avoid bubbles
• Remove pipette tip
BEFORE releasing
plunger
• Change tips before
loading next well
A Jar of Marbles
• Space in between the
marbles would allow
sand to fall
• Large grains would fall
slower
Detection- DNA Staining in Gel
• Ethidium bromide is
used
• Intercalates DNA
• Fluorescent under UV
light
• Intercalates DNA
DNA Intercalation
• Ethidium bromide sticks
between the rungs of
the DNA ladder
• Can impair proper DNA
replication
• Wear gloves, please
Sorting DNA by size
• Which lane(s) have the
largest DNA fragments?
The smallest?
• What do you think is in
lane M?
• M is a marker
• Also called a “ladder”
• 4th Band from top in
lane M=500 bp
• 5th Band is 400 bp
• How big are the bands
Purifying DNA
• Desired DNA fragments
can be cut directly from
gel, purified, and used
What will we find in our DNA?
• In order to tell students
apart, we must have DNA of
different length
• We are looking for the “Alu
repeat” at one place in the
genome (the PV92 locus of
chromosome 16)
• Some folks got it, some folks
don’t
• Some folks got it half the
time…
The Eukaryotic genome
• Human DNA is >99%
identical
• The PV92 locus of
chromosome 16 is
dimorphic
• Some people have an
Alu repeat
Eukaryotic Genomes Contain introns
Introns are spliced out during translation
5 Exon
Pre-mRNA
Intron
Exon
Intron
Exon
3
5 Cap
Poly-A tail
1
30
31
Coding
segment
104
105
146
Introns cut out and
exons spliced together
Poly-A tail
5 Cap
5 UTR
1
146
3 UTR
The Eukaryotic Genome
• Contains introns
• Introns are spliced out
(Retro-)transposons move around the
genome across many generations
19.16
Mammals
25-50%
Primates
Alu
10%
Much of the Eukaryotic genome is
“Junk DNA”
• 500,000 Alu sites in the
human genome
• PV92 on chromosome
16 is just one place
were the Alu sequence
can be found
(sometimes….)
Gene frequencies
• If we know how
common a gene is, we
can predict its
distribution in the
population
• If a coin is flipped twice,
what are the odds of
getting
• 2 heads?
• 2 tails?
• One of each?
Hardy-Weinberg Equilibrium
• Coin flip is based on a
“gene frequency” of
50%
• Genes do not always
have 50% frequency
• What if the frequency
is 40%?
• We can use algebra…
• The Hardy-weinberg
equation!