Download DNA STRUCTURE AND FUNCTION

Laboratory Encounters in Plant
Genomics
Dr. Jan Stephens
Colorado State University
Summer workshop
June 27th & 28th, 2006
5/23/2017
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What is genomics?
What is biotechnology?
What is genetic engineering?
What can genetic engineering do?
What are the basic procedures for
producing a genetically modified
plant product?
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1. Trait Identification
2. Gene Discovery
3. Gene Cloning
4. Gene Verification
5. Gene Implantation
6. Cell Regeneration
7. Testing New Plant
8. Seed Production
Why is genetic engineering becoming
such a popular science?
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DNA STRUCTURE AND FUNCTION
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We can learn a lot about
plants and the pathogens
that make them sick by
studying their DNA.
DNA is located within every
cell, and it contains the
genetic “code” necessary for
making a living thing
http://www.biopatent.com/DNA3.gif
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DNA STRUCTURE AND FUNCTION
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The DNA double
stranded helix was
described by Watson and
Crick in 1953
The DNA helix has a dual
backbone of deoxyribose
sugars and negatively
charged phosphate
molecules
From: www.accessexcellence.org/RC/VL/GG/dna_molecule.html
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DNA STRUCTURE AND FUNCTION
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There are pairs of
nucleotides or bases held
together with hydrogen
bonds between each
sugar-phosphate strand
The phosphate molecules
have a negative charge
that can be utilized to
separate small pieces of
DNA during
electrophoresis
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http://users.rcn.com/jkimball.ma.ultranet/
BiologyPages/D/DoubleHelix.html 6
DNA STRUCTURE AND FUNCTION
There are four different
bases – adenine (A),
thiamine (T), guanine (G)
and cytosine (C)

These are arranged in a
special order that tells the
cell what proteins to make

http://www.web-books.com/MoBio/Free/Ch3B.htm
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DNA STRUCTURE AND FUNCTION
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The strands are
“complementary”
Both strands thus
contain the same
genetic information and
can each serve as a
model or template for
the other strand
www.accessexcellence.org/RC/VL/GG/dna_molccule.html
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DNA STRUCTURE AND FUNCTION
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DNA is found in the cell nucleus
which is surrounded by a
membrane
DNA is packaged into
chromosomes
The nucleus is inside the cell
which is also surrounded by a
membrane and for plants by a
cell wall
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http://web.jjay.cuny.edu/~acarpi/NSC/13-cells.htm
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A chromosome contains five types of histones: H1 (or H5),
H2A, H2B, H3 and H4. H1 and its homologous protein H5
are involved in higher-order structures. The other four
types of histones associate with DNA to form nucleosomes.
Each nucleosome consists of 146
bp DNA and 8 histones: two
copies for each of H2A, H2B, H3
and H4. The DNA is wrapped
around the histone core, making
nearly two turns per nucleosome.
From http://www.web-books.com/MoBio/Free/Ch3D1.htm
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 Histones are basic proteins, bristling with positively
charged arginine and lysine residues.
 Histones are some of the most conserved molecules
during the course of evolution. Histone H4 in the calf differs
from H4 in the pea plant at only 2 amino acids residues in
the chain of 102.
 The arrows point to the
nucleosomes. You can see
why the arrangement of
nucleosomes has been
likened to "beads on a
string".
From http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/Nucleus.html
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DNA REPLICATION
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During cell division the DNA molecule replicates,
so that each new cell receives an exact copy of
genetic information.
This occurs by the hydrogen bonds between the
nucleotides breaking, allowing for the DNA
ladder to unzip. Then, the separated strands
unwind, and each strand becomes a template
for a new complementary strand.
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DNA STRUCTURE AND FUNCTION
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http://www.uvm.edu/~cgep/Pic/Replication.gif
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The result is that in both offspring of
a divided cell, each DNA molecule
has one original strand and one
newly synthesized strand.
This mechanism, called DNA
replication, ensures precise copying
of the nucleotide base sequences in
DNA.
On average, one mistake may exist
in every billion base pairs - the same
as typing out the entire Encyclopedia
Britannica five times and typing in a
wrong letter only once!
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
DNA replication must
be precise. Errors in a
particular codon could
lead to the wrong
amino acid being
specified at some point
in a protein molecule.
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/ReplicationFork.gif

This could result in a shape change large
enough to change the function of that protein.
Errors in replication, and those induced by
prolonged exposure to UV, X-rays, and
radioisotopes, are known as mutations
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Transcription of DNA
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Protein synthesis uses the genetic code
contained in the nucleotides as a blueprint
The region of DNA that contains genes is
“transcribed” or copied into another polymer
called RNA
Many of these RNA molecules undergo major
changes before leaving the nucleus to act as
messenger molecules (mRNA), that direct the
synthesis of proteins.
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Transcription of DNA
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RNA is similar to DNA but it has
ribose instead of deoxyribose
sugar in the backbone, uracil
(U) instead of thymine and is
single stranded
One of the two strands of DNA
acts as a template for the
synthesis of RNA.
Thousands of RNA copies may
be run off from the same DNA
segment
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http://www.accessexcellence.org/RC/VL/GG/RNA_trans.html
Translation of the code

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Three nucleotides
encode a single amino
acid
As mRNA moves through
the ribosome the
appropriate amino acids
are added to the
growing protein
molecule
http://web.mit.edu/esgbio/www/dogma/trl.html
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Table of Standard Genetic Code
T
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C
A
G
TTT Phe (F)
TTC Phe (F)
T TTA Leu (L)
TTG Leu (L)
TCT Ser (S)
TCC Ser (S)
TCA Ser (S)
TCG Ser (S)
TAT Tyr (Y)
TAC
TAA STOP
TAG STOP
TGT Cys (C)
TGC
TGA STOP
TGG Trp (W)
CTT Leu (L)
CTC Leu (L)
C CTA Leu (L)
CTG Leu (L)
CCT Pro (P)
CCC Pro (P)
CCA Pro (P)
CCG Pro (P)
CAT His (H)
CAC His (H)
CAA Gln (Q)
CAG Gln (Q)
CGT Arg (R)
CGC Arg (R)
CGA Arg (R)
CGG Arg (R)
ATT Ile (I)
ATC Ile (I)
ATA Ile (I)
A ATG Met (M)
START
ACT Thr (T)
ACC Thr (T)
ACA Thr (T)
ACG Thr (T)
AAT Asn (N)
AAC Asn (N)
AAA Lys (K)
AAG Lys (K)
AGT Ser (S)
AGC Ser (S)
AGA Arg (R)
AGG Arg (R)
GTT Val (V)
GTC Val (V)
G GTA Val (V)
GTG Val (V)
GCT Ala (A)
GCC Ala (A)
GCA Ala (A)
GCG Ala (A)
GAT Asp (D)
GAC Asp (D)
GAA Glu (E)
GAG Glu (E)
GGT Gly (G)
GGC Gly (G)
GGA Gly (G)
GGG Gly (G)
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Mutations
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Missense mutations
EXAMPLE: sickle-cell disease
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mutations.html#insertions
Nonsense
mutations
With a nonsense mutation, the new nucleotide
changes a codon that specified an amino acid
to one of the STOP codons (TAA, TAG, or
TGA).
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Mutations
e.g. Cystic fibrosis
Patient B, the
substitution of a T for a
C at nucleotide 1609
converted a glutamine
codon (CAG) to a STOP
codon (TAG). The
protein produced by
this patient had only
the first 493 amino
acids of the normal
chain of 1480 and could
not
function.
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http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mutati
ons.html#insertions
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Extraction of DNA
STRAWBERRY EXTRACTION PROCEDURE
 Add extraction buffer to the zipper bag with
strawberry. Close bag, let in as little air as
possible.
 Mush the strawberry thoroughly for 5
minutes, without breaking the bag.
 Place the zipper bags with fruit and extraction
solution into the hot water bath for about 1015 minutes. Occasionally shake the bag to
distribute heat.
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Extraction of DNA
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Put the mashed bags of strawberry and
solution into the ice bath for 1 minute.
Remove and mush the strawberry more.
Repeat this procedure 5 times.
Filter this mixture through the cheesecloth
filter. All students can combine their
solutions at this point. Let the solution
drain for 5 minutes.
Aliquot approximately 2 ml of the
strawberry solution into each test tube.
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Extraction of DNA
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Carefully, without disruption of the test
tube contents add approximately 2 ml of
ice-cold ethanol to each tube. Do this by
letting the drops run slowly down the side
of the test tube and rest on top of the
strawberry mixture.
Let the solution sit for 2 minutes without
disturbing it. The DNA will appear as
transparent, slimy, white mucus that you
can “hook” up with the bent paperclip.
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Extraction of DNA
WHAT IS THE PURPOSE OF EACH STEP?
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Why do we “mush” the strawberry fruit?
Crushing the strawberry fruit physically breaks
apart the cell walls.
Why do we use shampoo? After the cell
walls have been disrupted during mechanical
mashing of the fruit, the detergent in the
shampoo disrupts the cell and nuclear
membranes of each cell to release the DNA. It
does this by dissolving lipids and proteins that
hold the membranes together.
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Why do we need to cool the mixture? DNases
or enzymes that destroy DNA are present in the cell’s
cytoplasm. They are there to protect the cell from
invasion by viruses. Once the nuclear membrane is
destroyed by the soap the DNA is now susceptible to
the DNases and will quickly be degraded. However,
these enzymes are temperature sensitive and cooling
the solution slows down .the process of degradation.
What does the cold ethanol do? Everything
except the DNA will dissolve in ethanol. The ethanol
pulls water from the DNA molecule so that it then
collapses in on itself and precipitates. The DNA will
become visible as white mucous strands that can be
spooled with the wooden applicator stick.
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What does the salt do? The salt neutralizes
the negative charges on the DNA and thus
enables the DNA strands to stick together. It
also causes proteins and carbohydrates to
precipitate.
Why can’t we use room temperature
ethanol? The colder the ethanol is the greater
the amount of DNA that is precipitated. (You
could try having some of the students use room
temperature ethanol and see if the amount of
DNA they can spool is the same or less than that
for the groups using the ice-cold ethanol.)
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