3. DNA forms and denaturation

A

A-DNA

right handed, no major/minor grooves, 11 bases/turn low humidity, high salt

B-DNA

right handed, moderate depth grooves, 10.5 bases/turn, high humidity, low salt

Z-DNA

left handed, single groove, high nacl or ethanol, in the presence of methylated cytosine: high humidity and low salt

sliped or cruciform structures can form if there are

repeated sequences in the DNA

triple helix DNA

formed when purines make up one strand and pyrimidines the other, then a third strand can be accomodated, in test tube, but also likely in vivo during DNA recombination or repair

• gene therapy possibilities

pitch/turn of helix Bform

34

rise/base pair along helix axis armstrong

3.4

factors that denature dna

1. heat

2. low ionic strength (promotes repulsion between negative phosphate back bones (low salt))

3. high pH: stripping of H+ shared between electronegative centers

agents that influence H-bonds

1. competition: have functional groups that can form H- bonds w electronegative centres

2. covalent modifications: modify electronegative centers and blofk the formation of H-bonds

3. agents that enhance the solubility of hydrophobic substances

progress of denaturation can be monitored by examining the properties of the molecule that can change when the strands separate

1. viscosity - rarely used, difficult

2. abosrbance (260nm) commonly used in lab

tm melting temp

temp at which 50% of the DNA is denatured

in dsDNA the bases are stacked and

absorbance is lower (hypochromic)

in denatured ssDNA the bases are unstacked and

absorbance increases (hyperchromic)

Tm is a function of the gC content

more gc = higher tm needed, bc at regions separate first during denaturation

the tm of dna increases by 0.4 C with every

1% increase in G-C content under normal condition, higher salt = higher Tm

renaturation dependant on

1. dna concentration

2. salt concentration

3. temperature

4. time

5. size of dna fragment

6. complexity - simple sequences renature faster than complex

rate of renaturation =

measure of complexity of DNA/genome

re-association kinetics

speed at which a single strand sequence is able to find a complementary sequence and base pair with it

increase in genome size. =

increase in complexity

Cot

starting concentration x reaction time

cot 1/2

when 50% renaturation has occured

units of complexity measured in terms

of nucleotides

if a genome contains unique sequences and some repetitive sequences

# of unique nucleotides + total # of nucleotides form one copy of each repetitive sequence

if two dna sequences do not have reptitive sequences and have similar C-G content

their sizes are proportional to their cot1/2

how is cot analysis carried out

1. control dna + unknown

2. sheared into small pieces ~200 bp

3. denatured, reanneal

4. sub-samples removed, ds & ss DNA measured

5. data points plotted as a proportion of ssDNA out of the total DNA

e.coli genome

no repetitive sequences, difficult for sequences to find complementary sequences, once found fast reassociation

calf genome

lots of hihgly repetitive sequences, fast reassociation, some moderately repeptitive sequences slower re-association at the begnning, slowest those unique sequences are comparable to ecoli genome

reassociation is inversely proportional

genome dna size