Introduction to DNA Cloning

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Introduction to DNA Cloning

• Nucleotides and DNA Structure

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Learning Objectives

• Understand the double helix structure and
  dimensions of DNA molecule.
• Understand the chemical bonds of the DNA molecule
  (which are covalent and which are hydrogen).
• Understand complementary base-pairing rules of
  the DNA molecule and be able to predict the
  opposite strand.
• Understand the antiparallel nature of DNA
  molecule.

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DNA History

• Deoxyribonucleic acid, or DNA, was discovered in
  the late 1860s.
• It was ignored because it seemed too simple A,C,
  G, T.
• Thats because they degraded it when they
  purified it.
• In the 1940s scientists discovered that
  chromosomes, which carry hereditary information,
  consist of DNA and proteins.
• Experiments conducted throughout the 1940s showed
  that DNA actually seemed to be the genetic
  material.

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Happy 51th Birthday DNA Structure
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The first X-ray photograph of crystalline DNA in
the A form. Taken by Rosalind Franklin, 1952
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Evidence for a Double Helix

• Rosalind Franklin, working with Maurice H.F.
  Wilkins, studied isolated fibers of DNA by using
  the X-ray diffraction technique, a procedure in
  which a beam of parallel X rays is directed on a
  regular, repeating array of atoms.
• Watson saw pictures when Wilkins showed them at a
  talk, without Franklins knowledge.

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Franklins Data

• The diffraction patterns obtained by directing
  X-rays along the length of drawn-out fibers of
  DNA indicated that the molecule is organized in a
  highly ordered, helical structure.
• The data showed DNA was a helical structure which
  had two distinctive regularities of 0.34 nm and
  3.4 nm along the axis of the molecule.
• It looked like a double helix.

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Watson and Crick

• Linus Pauling built a model of an alpha helix for
  protein structure and won the Nobel Prize.
• Watson and Crick were inspired that Pauling used
  his imagination and molecular models to deduce
  this protein structure.
• They believed if an eminent scientist such as
  Pauling could model a structure with little
  experimentation, then they might be able to do
  the same with DNA.

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What Watson and Crick Knew

• Main components of DNA
• Phosphates
• Sugars
• Four nitrogenous bases
• Adenine
• Thymine
• Cytosine
• Guanine
• Using wire and pieces of metal, Crick and Watson
  began building scale models of DNA.

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Chargaffs Rules

• Chargaff had found that the amounts of adenine
  and thymine were approximately equal and the
  amounts of guanine and cytosine were also
  approximately equal.
• This information gave Watson and Crick the idea
  that the bases might be paired in a specific way.
  

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Base Composition Can Differbut Chargaffs Rules
Still Hold.
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Failure at First

• At first, Watson imagined that the bases paired
  like with like, for example adenine with adenine,
  and cytosine with cytosine.
• The resulting model did not fit Franklins X-ray
  data.
• Then Watson and Crick discovered that thymine and
  guanine could adopt a slightly different chemical
  shape, and their models used the wrong version of
  the bases.

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Success

• Using the new forms, Watson discovered that he
  could make two base pairs, one consisting of
  adenine and thymine, and the other consisting of
  guanine and cytosine, that had exactly the same
  size.
• They built the model and wrote the paper.

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• Watson and Crick discovered the structure (or
  solved it) without direct experimentation
  themselves.
• They read, thought and talked their way to a
  Nobel Prize.

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The Solution

1. The DNA molecule consists of two polynucleotide
   chains wound around each other in a right-handed
   double helix.
2. Viewed on end, the two strands wind around each
   other in a clockwise (right-handed) fashion.

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2. The diameter of the helix is 2 nm or 20 Å
2 nm
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The Solution (cont)

• 3. The two chains are antiparallel
• ( show opposite polarity).
• The two strands are oriented in opposite
  directions with one strand oriented in the 5' to
  3' way, while the other strand is oriented 3' to
  5'.

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Three components of each nucleotideFour
different nucleotides in DNA
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Chemists name carbon atoms within the ring
structures of organic molecules C1, C2, C3 etc.
When there are two rings in a structure, they
name one ring prime. In nucleotides, the base
is one ring (C1, C2 C3 etc) and the sugar was
named prime. The 5 carbon has the phosphate and
the 3 carbon has an OH group on the carbon.
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The Solution (cont)

• 4. The sugar-phosphate backbones are on the
  outsides of the double helix, while the bases are
  oriented toward the central axis.
• The bases of both chains are flat structures
  oriented perpendicularly to the long axis of the
  DNA that is, the bases are stacked like pennies
  on top of one another (except for the "twist" of
  the helix).

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The Solution (cont)

• 5. The bases of the opposite strands are bonded
  together by relatively weak hydrogen bonds.
• The only specific pairings are A with T (two
  hydrogen bonds) and G with C (three hydrogen
  bonds).
• The A-T and G-C base pairs are the only ones that
  can fit the physical dimensions of the helical
  model.
• The specific A-T and G-C pairs are called
  complementary base pairs, so the nucleotide
  sequence in one strand dictates the nucleotide
  sequence of the other.

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The Solution (cont)

• 6.The base pairs are 0.34 nm apart in the DNA
  helix.
• A complete (360 degrees) turn of the helix takes
  3.4 nm therefore, there are 10 base pairs per
  turn.
• Each base pair, then, is twisted 36 degrees
  clockwise with respect to the previous pair.

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0.34 nm 2 bp distance
3.4 nm, 10 base pairs per turn
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The Solution

• 7. Because of the way the bases bond with each
  other, the two sugar-phosphate backbones of the
  double helix are not equally spaced along the
  helical axis.
• This results in grooves of unequal size between
  the backbones called the major groove (the wider
  groove of the two) and the minor groove (the
  narrower groove of the two).
• Both of these grooves are large enough to allow
  protein molecules to make contact with the bases.
  

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DNA and RNA as Chemicals

• Chemical Bonds
• Covalent (permanent)
• Ionic (salt)
• Hydrogen (sharing H, very weak)
• Van der Waals Bonds
• Hydrophobic Interactions

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Charge in Molecules

• Water is perhaps the most obvious example of a
  molecule with partial charges. The symbols delta
  and delta- are used to indicate partial charges.

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Covalent Bonds

• Covalent Bonds are the strongest chemical bonds,
  and are formed by the sharing of a pair of
  electrons.
• Once formed, covalent bonds rarely break
  spontaneously.
• Covalent bonds dont fall apart when heated or
  dissolved in a solvent like water.

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Ionic Bonds

• Ionic bonds are formed when there is a complete
  transfer of electrons from one atom to another,
  resulting in two ions, one positively charged and
  the other negatively charged.

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Hydrogen Bonds

• Hydrogen bonds are formed when a hydrogen atom is
  shared between two molecules.

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Double strands of DNA are held together by
hydrogen bonds.

• The DNA molecule is usually double-stranded, with
  the sugar-phosphate backbone on the outside of
  the helix.
• In the interior are pairs of nucleotide bases,
  holding the two strands together by hydrogen
  bonds.
• Hydrogen bonding between the bases is specific.
  The adenine base can pair only with the thymine
  base, and the guanine base can only pair with the
  cytosine base.

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Van der Walls Interactions

• Van der Walls interactions are very weak bonds
  formed between nonpolar molecules or non-polar
  parts of a molecule. The weak bond is created
  because a C-H bond can have a transient dipole
  and induce a transient dipole in another C-H
  bond.

H H CH3 CH3
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Hydrophobic Interactions

• Nonpolar molecules cannot form H-bonds with H2O,
  and are therefore insoluble in H2O.
• These molecules are known as hydrophobic (water
  hating), as opposed to water loving hydrophilic
  molecules which can form H-bonds with H2O.

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If thymine makes up 15 percent of the bases in a
certain DNA sample, what percentage of bases must
be cytosine?
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If thymine makes up 15 percent of the bases in a
certain DNA sample, what percentage of bases must
be cytosine?

• thymine 15, then adenine 15
• A T 30, then G C 70
• So, cytosine is 1/2 of 70 35

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A certain segment of DNA has the following
nucleotide sequence in one strand 5
ATTGGCTCT 3What must be the sequence of the
other strand (label its 5 and 3 ends)?
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A certain segment of DNA has the following
nucleotide sequence in one strand 5
ATTGGCTCT 3What must be the sequence of the
other strand (label its 5 and 3 ends)?

• Writing in the same direction 3 TAACCGAGA 5
• Writing 5 to 3 5 AGAGCCAAT 3

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How many bases are there in 2 kb (2000 base
pairs) of DNA?
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How many bases are there in 2 kb (2000 base
pairs) of DNA?

• 4000 bases in all.

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For the DNA strand 5'-TACGATCATAT-3' the correct
complementary DNA strand is A
3'-TACGATCATAT-5' B 3'-ATGCTAGTATA-5' C
3'-AUGCUAGUAUA-5 D 3'-GCATATACGCG-5 E
3'-TATACTAGCAT-5'
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Correct Answer is
B 3'-ATGCTAGTATA-5'
5-TACGATCATAT3 3'-ATGCTAGTATA5
This choice has the correct polarity and
complementarity.
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DNA Structure as a Ladder

• The curving sides of the ladder of DNA represent
  the sugar phosphate backbone.
• The rungs are the base pairs.
• The spacing between the base pair rungs is 3.4 Å
  (Angstroms are 1-10 or 1/10,000,000,000 of a
  meter or 1/10 nanometer).
• The helix repeat distance is 34 Å, meaning there
  are 10 base pairs per turn of the helix.
• The strands are antiparallel. If one has 5 to
  3 polarity from top to bottom, the other must
  have 3 to 5 polarity from top to bottom.
• The helix is 20 Å across at the base.

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