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Magnetic Traps– measuring
Twist
Last time: WLC very good theory for DNA bending
This time: Twist & Writhe
General Properties of DNA to Specific: PCR
HW due next Monday; also quiz #1 on Chpt 1 of Phillips et al
DNA Structure
• Right-hand helix
• One turn: 3.4 nm,
~10.5 bp
Molecular Cell Biology, Lodish
• Twist angle
between bps θ=36
polymers
Wikipedia
2nm
DNA
DNA will resist twisting
DNA in Eukaryotes is wrapped around Histones (Proteins)
Chromatin = Complex of DNA + Protein (histones + non-histones)
8/17/06
DNA may be Supercoiled:
Has both Twist and Writhe
Twist
Linking Number = Twist + Writhe
Lk = Tw + Wr
s Writhe
Relaxed DNA: Twist =1 turn/10.4 bp. Writhe = 0. ∆Tw=0
Can’t just twist up DNA and have it all go into twist.
Example: Phone cord.
Pull on DNA and writhe comes out.
Measure relaxed (non super-coiled) DNA and figure out length vs. force.
Some Examples of Tw and Wr
Linear DNA with Constrained Ends
Twist (Tw), Writhe (Wr), Linking Number (Lk)
Tw: # of times the two strands
wrap around each other
Wr: # of times C crosses itself.
Linking Number = Twist + Writhe
Lk = Tw + Wr
Supercoiled DNA (s): is the deviation from
relaxed linking number.
Tw=2
Tw=0
Wr=0
Wr=2
Charvin, Contemporary Physics,2004
s = (Lk – Lko)/Lko =∆Lk/ Lko
Ex: Hold DNA out straight so that it has no Writhe, add of take out twist,
then let fold up (Twist goes into Writhe).
Normal DNA is negatively supercoiled, -0.06 = 6 turns for every 100 taken out. Why?
Helps unwind DNA– makes it easier to uncoil, separate strands. Enzymes
which do this called Topoisomerases.
Why? What about archebacteria that lives in hot springs? Positively supercoiled
Makes DNA more stable
Archea—live hot!
The Archaea are a group of single-celled microorganisms. A single
individual or species from this domain is called an archaeon (sometimes
spelled "archeon"). They have no cell nucleus or any other membranebound organelles within their cells.
Archaea are divided into four recognized phyla, but many more
phyla may exist.
Finding Archaea : The hot springs of Yellowstone National Park, USA, were among the
first places Archaea were discovered. At left is Octopus Spring, and at right is Grand
Prismatic Pool. Each pool has slightly different mineral content, temperature, salinity, etc.,
so different pools may contain different communities of archaeans and other microbes. The
biologists pictured above are immersing microscope slides in the boiling pool onto which
some archaeans might be captured for study.
http://www.ucmp.berkeley.edu/archaea/archaea.html
Archaea— the 3rd tree of life!
different superhelicity
Why? What about archebacteria that lives in hot springs? Positively supercoiled
Makes DNA more stable
Torsionally stressed single DNA molecule
Playing with phone cord: can you explain graphs?
When the force is
increased above 0.5
pN, the curve becomes
asymmetric: supercoils
still form for positive
coiling while local
denaturation adsorbs
the torsional stress for
negative s.
Low F: symmetric
under s  - s
The shortening
corresponds to the
formation of
plectonemes upon
writhing.
At forces larger than 3
pN no plectonemes are
observed: the torsional
stress is adsorbed not
by writhe but in local
structural changes of
the molecule.
Extension vs. supercoiling at constant force
T. Strick et al., J. Stat. Phys., 93, 648-672, 1998
Three regimes
Over- and under-stretching
Upon twisting a DNA molecule it
takes a number of turns, before
the DNA length reduces
significantly and plectonemes are
formed. The point (Nbuckling)
where DNA starts to form
plectonemes with a constant
length reduction per turn is called
buckling instability
Rotation extension curves
for different forces. At higher
forces one cannot induce
supercoils but denature the
DNA molecule.
Nucleic Acids: DNA and RNA
General Properties to Specific Properties
DNA codes for genes
Gene: length of DNA which codes for a protein
Code first goes through RNA.
RNA: every 3 bases codes for amino acid.
Peptide = linear string of amino acids (from 10kD
(≈ 100aa) to 1 MD or more)
Coding of DNA/RNA:
3 bases are codon amino acid
43= 64 possible amino acids, but there are only 20 aa.
Degeneracy aa = AT1, AT2, AT3…
Plus stop and start codons (where proteins stop, start).
All genes start with ATG  proteins start with Methionine
All Genes stop with:
•TAG: "They Are Gone"
•TAA: "They Are Away"
•TGA: "They're Going Away”
Can look at genome: how many proteins are coded for?
Answer: about 23,000 genes
Count Start to Stop Codons: what fraction of 3 billion bases
make up for proteins. Only about 10% is used for genes.
90% used to be called “junk” DNA.
“Junk” DNA codes for important
regulatory elements
• Produce RNA, RNA then feeds back onto DNA and turns
it on/off in some unclear manner.
“Rethinking ‘Junk’ DNA” NYTimes 9/5/12
“Bits of Mystery DNA, Far From ‘Junk,’ Play Crucial Role”
• Cancer is one result.
“Cancer’s Secrets Come Into Sharper Focus: NYTimes
• Some junk DNA is truly “junk”—evolutionary baggage.
DNA in the Cell
How to identify you based on your
DNA
chromosome
cell nucleus
Double stranded
DNA molecule
Target Region for PCR
Individual
nucleotides
Polymerase Chain Reaction.
Invented 1990; Nobel Prize in 1993: Kary Mullis
DNA Amplification with the
Polymerase Chain Reaction (PCR)
5’
3’
5’
3’
Starting DNA
Template
3’
3’
5’
5’
Separate
strands
(denature)
Forward primer
5’
3’
5’
3’
Make copies
Add
primers
(extend
primers)
5’
(anneal)
3’
3’
5’
Reverse primer
PCR (Polymerase Chain Reaction) Copies DNA
Exponentially through Multiple Thermal Cycles
Original DNA
target region
1 copy
Heat
Oligo’s
DNA
Polymerase
dNTP
Cool
In 32 cycles at 100% efficiency,
Heat
Cool
2 copies
4 copies
Heat
…
?? billion copies are created
1.07
To work, what property of DNA polymerase has to have?
New Scientists (1998)…Yellowstone's bugs land up in court ... Microorganisms from hot
Heat
so don’t
have
to add
new polymerase
for every
cycle
springsstable
are especially
valuable
because
theirinenzymes
are not easily destroyed
by heat.
...
Thermostable organisms, e.g. living in Yellowstone Geysers have this.
Applications of PCR
(next time)
Class evaluation
1. What was the most interesting thing you learned in class today?
2. What are you confused about?
3. Related to today’s subject, what would you like to know more about?
4. Any helpful comments.
Answer, and turn in at the end of class.