Techniques Used in Molecular Biology

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Techniques Used in Molecular Biology Based on DNA
Replication
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Figure 21.3
Cloning
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Polymerase Chain Reaction (PCR)
What you need for PCR?

• DNA Template
• Two Primers
• Taq DNA Polymerase
• dNTPs
• Appropriate buffers and cofactors

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The discovery of PCR is here presented as a great
American epiphany - its recipient struck by a
flash of inspiration on the road, not to
Damascus, but to Mendocino. Sometimes Mullis says
the creative spirit came on an evening in April
of 1983, sometimes in May. Anyway, the buckeyes
were in bloom Mullis's little silver Honda Civic
was purring through the vineyards and redwoods of
the Anderson Valley and his mind wandered. Life
is sweet, he thought 'I am a big kid with a new
car and a full tank of gas. I have shoes that
fit. I have a woman sleeping next to me and an
exciting problem, a big one.' At mile-marker
46.58 on Highway 128 - he had both the presence
of mind and the sense of history to note the
exact spot, if not the month - the epiphany
arrives. 'Holy shit,' Mullis cries out, and his
girlfriend almost, but not quite, wakes up. He
pulls the Honda to the side of the road to write
down his ideas and check his calculations. Within
feverish minutes, the problem is solved, and
Mullis is left with the mop-up operation of
getting PCR actually to work. This takes almost
two years, and the original report was famously
rejected by both Nature and Science. Mullis was
not fazed '"F--- them," I said.'
Nobel Savage Steven Shapin
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How can you fit all the DNA replicated by a cell
in a small nucleus?
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Genomic DNAs are much longer than the cells or
viruses that contain them!
Length to width ratio of DNA in human is 10 mill
to 1. DNA Packaging Problem More Acute for
Eukaryotes!
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Eukaryotic Nucleus Typically 10 mm in diameter
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Chromatin

• DNA proteins ( RNAs ?)
• Histones
• Non-histone chromosomal proteins
• Two main types of chromatin
• Euchromatin - dispersed appearance by EM,
  transcriptionally active
• Heterochromatin dense appearance by EM,
  transcriptionally repressed, includes highly
  repetitive regions such as telomeres and
  centromeres

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Tobacco meristem cell Nucleus with large
Nucleolus, and Euchromatin. Stars indicate
heterogeneity in the nucleolus.
Euchromatin
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Narcissus flower cell with a lot of
heterochromatin in the nucleus.
Heterochromatin
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Eukaryotic Chromatin
Electron microscopy of a chromatin spread.
Nucleosomal beads-on-a-string structure.
Beads are Nucleosomes.
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Nucleosomes contain Histones
21,000 Daltons 15,000 11,000
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Nucleosome core octamer of histones (2 each of
H2A, H2B, H3, H4) 2 wraps (145 bp) of DNA
Packing ratio 5
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Figure 13.3a
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Figure 13.3c
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Figure 13.4a
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Figure 13.4b
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Figure 13.4c
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The role of histone H1
1 mM NaCl
5 mM NaCl
H1 binds to the nucleosome where the DNA enters
and exits the core.
- H1
H1
H1 is needed to form the zig-zag structure.
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Role of H1 (cont.)

• Evidence indicates that H1 also functions in
  forming the 30 nm fiber, and that it interacts
  with other H1 molecules

H1-H1 interaction proven by cross-linking
experiments.
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Figure 13.8
H1
H1
-H1
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Chromatin Condenses into 30 nM Fiber at near
physiological ionic strength
100 mM NaCl
Packing ratio 6-8-fold for this step
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30 nM fiber forms Solenoid - 6 nucleosomes per
turn
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Solenoid attaches to scaffold, generating loops
Packing ratio 25 for this step 1000 overall
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Figure 13.13
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700 nm fiber
Beads on a String
Solenoid or Condensed Fiber
Loops Snake the Solenoid PR 25
Double Helix
Probably involve SARS DNA scaffold attachment
regions
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DNA Supercoiling
Eukaryotic chromosomal DNA also has supercoiled
regions.
Fig. 13.14
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Right handed
Writhing structures
Writhing double helical strand passing over
itself
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Writhing structures
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Equation for Supercoiling
Lk Tw Wr
Lk Linking number ( times that strands
cross) Tw Twist ( of times that strands
cross, excluding writhe,
or of helical turns) Wr Writhe ( of
times that double helix crosses itself)
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Topoisomerase I relaxes DNA one link at a time
Reduces Lk one unit at a time.
Topo I
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Type I Topoisomerases change Ln by 1, breaks 1
strand.
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Type II Topoisomerases change L by 2, cuts both
strands
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Questions
1. Catenane structure can be resolved using a.
Topoisomerase I b. DNA gyrase c. Topoisomerase
IV d. Type II topoisomerase e. c-d 2. Describe
different levels of DNA packaging. 3.
True/False. Heterochromatin is more transcribed
than euchromatin.
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• What was the most important lesson that you
  learned
• today?
• 2. What was the most difficult point that needs
  more discussion?
• 3. Any suggestion for improving learning in the
  class?