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

Molecular Cell Biology, Lodish

Wikipedia

• Right-hand helix

• One turn: 3.4 nm, ~10.5 bp

• Twist angle between bps θ=36

2nm

polymers 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

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.

Linking Number = Twist + WritheLk = Tw + Wr

Some Examples of Tw and WrLinear 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.

Charvin, Contemporary Physics,2004

Tw=2 Tw=0

Wr=0 Wr=2

Linking Number = Twist + WritheLk = Tw + Wr

Ex: Hold DNA out straight so that it has no Writhe, add of take out twist, then let fold up (Twist goes into Writhe).

= (Lk – Lko)/Lko =∆Lk/ Lko

Supercoiled DNA (): is the deviation from relaxed linking number.

Normal DNA is negatively supercoiled, -0.06 = 6 turns for every 100 taken out. Why?

Positively supercoiled

Helps unwind DNA– makes it easier to uncoil, separate strands. Enzymes which do this called Topoisomerases.

Makes DNA more stable

Why? What about archebacteria that lives in hot springs?

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 membrane-

bound 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

T. Strick et al., J. Stat. Phys., 93, 648-672, 1998

Extension vs. supercoiling at constant forceThree regimes

Low F: symmetric under - The shorteningcorresponds to the formation of plectonemes upon writhing.

When the force isincreased above 0.5 pN, the curve becomes asymmetric: supercoils still form for positive coiling while local denaturation adsorbs the torsional stress for negative .

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.

Playing with phone cord: can you explain graphs?

Over- and under-stretching

Rotation extension curvesfor different forces. At higher forces one cannot induce supercoils but denature the DNA molecule.

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 formplectonemes with a constant length reduction per turn is called buckling instability

DNA codes for genesGene: 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)

Nucleic Acids: DNA and RNAGeneral Properties to Specific Properties

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”

Count Start to Stop Codons: what fraction of 3 billion bases make up for proteins.

Can look at genome: how many proteins are coded for?Answer: about 23,000 genes

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

chromosome

cell nucleus

Double stranded DNA molecule

Individual nucleotides

Polymerase Chain Reaction.

Invented 1990; Nobel Prize in 1993: Kary Mullis

Target Region for PCRTarget Region for PCR

How to identify you based on your DNA

Make copies (extend primers)

Starting DNA Template

5’

5’

3’

3’

5’

5’

3’

3’

Add primers (anneal) 5’3’

3’5’

Forward primer

Reverse primer

DNA Amplification with the Polymerase Chain Reaction (PCR)

Separate strands

(denature)

5’

5’3’

3’

In 32 cycles at 100% efficiency, ?? copies are createdIn 32 cycles at 100% efficiency, ?? copies are created

PCR (Polymerase Chain Reaction) Copies DNA Exponentially through Multiple Thermal CyclesOriginal DNA target region

Heat

Cool

Heat

DNA Polymerase

dNTP

Oligo’s

1 copy2 copies

Cool4 copies

Heat …

1.07 billion

To work, what property of DNA polymerase has to have?

Heat stable so don’t have to add in new polymerase for every cycleThermostable organisms, e.g. living in Yellowstone Geysers have this.

New Scientists (1998)…Yellowstone's bugs land up in court ... Microorganisms from hot springs are especially valuable because their enzymes are not easily destroyed by heat. ...

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.