The History and Structure of DNA

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The History and Structure of DNA
These dogs have DNAand they are also super
amazing
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The molecule itself

• DNA is, without doubt, the most famous of all
  molecules.
• Its elucidation in 1953 by Watson and Crick is
  hailed as the greatest breakthrough in modern
  science.
• Q Why was this discovery such a big deal?

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Crazylike a fox?
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You should know the history

• Each of the following folks contributed
  significantly to our understanding of DNA. It is
  important to know their contributions.
• Frederick Griffith
• Oswald Avery
• Hershey and Chase
• Erwin Chargaff
• Wilkins and Franklin
• Watson and Crick
• Meselson-Stahl
• Okazaki

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Griffiths Experiment something is in there
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Hershey and Chases Experiment Viruses inject
DNA NOT protein
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Bacteriophage infecting a cell
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She got robbed!
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Nucleotides

• DNA is a nucleic acid (a polymer made of many
  monomers called nucleotides).
• Each nucleotide contains a ribose sugar (5 carbon
  sugar), a phosphate group, and a nitrogenous base.

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Nucleic Acids

• The sugar and phosphate make up the backbone of
  the molecule, while the nitrogenous bases point
  toward the center of the ladder and make up the
  rungs
• The backbone is linked with phosphodiester bonds.
• The rungs are connected with hydrogen bonds.

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Know your Bases!

• There are four types of nitrogenous bases
• Two pyrimidines (cytosine and thymine)
• Two purines (adenine and guanine)
• Chargaffs Rules A always binds with T and C
  always binds with G
• Q Why do they have this preferential bonding?
• A This is the only way they fit together!!!

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• Cytosine makes up 38 of the nucleotides in a
  sample of DNA from an organism. What percent of
  the nucleotides in this sample will be thymine?
• 12
• 24
• 31
• 38
• It cannot be determined from theinformation
  provided.

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• In an analysis of the nucleotide composition of
  DNA, which of the following is true?
• A C
• A G and C T
• A C G T
• A T G C
• Both B and C are true

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• In analyzing the number of different basesin a
  DNA sample, which result would be consistent with
  the base-pairing rules?
• A G
• A G C T
• A T G T
• A C
• G T

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The Double Helix

• The DNA strands themselves form a helical
  structure.
• The two strands of the helix are antiparallel
  they run in opposite directions. Their
  directions are determined by the orientation of
  their ribose molecules.

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PRIME TIME!5 and 3 (Five prime and Three prime)

• A ribose sugar can be labeled according to its
  carbons. These are what determine DNAs
  orientation.

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DNA vs. RNA
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Does DNA code for this?

• P.S. Worst example of ghost riding the whip EVER
• Next well talk about how DNA replicates! Yay!

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DNA Replication!!

• Watson and Crick, in their paper describing the
  structure of DNA, proposed the mechanism of
  replication.
• They suggested that since each strand is
  complementary to its partner, it could serve as
  the template for a newly synthesized strand.

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Meselson and Stahl

• The exact way this happens was demonstrated by
  Meselson and Stahl.
• Their experiments supported the idea of
  semiconservative replication.
• Q With a partner, briefly summarize how meselson
  and stahls experimental results eliminated
  conservative and dispersive replication as
  possible mechanisms.

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The possibilities
Conservative Semi-conservative Dispersive
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The experiment
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Quiz Question 1

• A space probe returns with a culture of a
  microorganism found on a distant planet. Analysis
  shows that it is a carbon-based life form that
  has DNA. You grow the cells in 15N medium for
  several generations and then transfer it to 14N
  medium. Which pattern in this figure would you
  expect if the DNA were replicated in a
  conservative manner?

e.
d.
c.
a.
b.
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Quiz Question 2

• Imagine the following experiment is done
  Bacteria are first grown for several generations
  in a medium containing the lighter isotope of
  nitrogen, 14N, then switched into a medium
  containing 15N. The rest of the experiment is
  identical to the Meselson and Stahl experiment.
  Which of the following represents the band
  positions you would expect after two generations?
  

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Replication in brief
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How it works the short version
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Electrophoresis

• Gel Electrophoresis is a process that separates
  different pieces of DNA based on their size.
• Fragments of DNA are pushed through the gel by an
  electrical field.
• Q Why would an electrical field have this effect
  on DNA?

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What separates the stuff?

• The gel is like a sieve or like a rainstick!
• DNA is negatively charged (phosphates!) so it
  goes toward the positive end.
• Q What size pieces move fastest?

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Summary How gel electrophoresis works
An electrical current pushes the DNA through a
sheet of gel that has lots of tiny holes in
it. Big pieces move slowly, little pieces move
fast and far
LARGE DNA PIECES
Small DNA pieces
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Restriction Fragment Analysis

• DNA fragments produced by restriction enzyme
  digestion of a DNA molecule are sorted by gel
  electrophoresis into a specific band pattern
  based on the sequence of DNA molecule
• The separated fragments can be recovered
  undamaged from gels, providing pure samples of
  individual fragments.

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RFLP Analysis
Restriction Fragment Length Polymorphism
difference in DNA sequence that leads to
different restriction enzyme cuts and therefore
different electrophoresis banding
patterns. Useful for mapping genes
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• Which of the following statements is consistent
  with the results below?

• B is the child ofA and C.
• C is the child ofA and B.
• D is the child ofB and C.
• A is the child ofB and C.
• A is the child ofC and D.

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• Which of the following statements is most likely
  true?

• D is the child ofA and C.
• D is the child ofA and B.
• D is the child ofB and C.
• A is the child ofC and D.
• B is the child ofA and C.

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• Which of the following are probably siblings?

• A and B
• A and C
• A and D
• C and D
• B and D

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• The segment of DNA shown in the figure below has
  restriction sites I and II, which create
  restriction fragments A, B, and C. Which of the
  gels producedby electrophoresis shown below
  would represent the separation and identity of
  these fragments?

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Sanger sequencing

• A method of sequencing segments of DNA using
  special nucleotides called dideoxyribonucleotides
  (ddNTPs)
• Remember DNA polymerase III has to have a free
  3 OH to add onto.
• Q What do you think dideoxy means?
• Q What will happen if we add a ddNTP?
• ddNTPs have had their 3 OH removed!

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Each type of ddNTP is flourescently
labeled. When the different length terminated
chains are run through a gel, they can be read
in order using their flourescent tag color!
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Shotgun Sequencing
Uses Sanger sequencing to analyze many fragments
of the same chromosome. The sequences are then
overlapped to place them in correct order.