Primary Structure
Nucleotide- base, pentose, phosphate group (a + g purines, c, t and u pyrmidines)
Ribose binds to base via covalent glycosidic bond
Phosphate group attaches to the 5’ C of ribose group
There can be 1-3 phosphate groups attached to the nucleotide via an ester bond
Nucleotide monophosphates joined together via a phosphodiester linkage, one phosphate connects to 3’C of one ribose group to the 5’C of the next. Created 5’ to 3’ direction
Secondary structure
Anti parallel, opposing strand is 3’ to 5’ phosphodiester bonds
RNA rarely stays linear- often forms hairpin loops
Tertiary structure
Rod like- worm like- random coil
RNA subject to more contortions
Charge properties
Bases- NH2 (pKa 2.5-4.5) and OH groups (pKa 9-10) @pH 7 both uncharged
Phosphate group pKa approx 1.0, therefore at pH 7 overall structure will be neg charged
Exterior backbone is polar, interior is hydrophobic
DNA
Heredity material found in cytoplasm of prokaryotic cells and nucleus of eukaryotic cells
Makes up the genome of an organism
Absorbance
DNA and protein absorb light at similar wavelengths, need to be aware of potential contamination.
DNA- 260nm
Protein- 280nm
Ratio of absorbance at 260 (A260) to absorbance at 280 (A280) should be greater than 1.8-2.0
A260/ A280
Hyperchromicity
ssDNA absorbs at same wavelength as dsDNA but has higher absorbance
Viscosity
DNA is viscous
A. Rigidity of double helix when compared to random cool of ssDNA
B. Extraordinary length of some types of DNA
Denaturation
G and C- 3 hydrogen bonds
A and T- 2 hydrogen bonds
Renaturation
Two steps
- Nucleation: slow diffusion controlled rate- limiting step where some initial bases hydrogen bond and pair
- zippering: once enough bases have bound to keep the two strands stably interlocked, the remaining bases can easily and quickly base pair. Temp for annealing for renaturation must be below the melting temp.
Hydrolysis
A. pH
Depurination can break glycosidic bond bw ribose and base pairs A and G with mild acidic conditions
Mild basic conditions do not cause depurination
B. Mechanical shearing
DNA prone to break when pressure is applied such as by swirling, pipetting etc. DNA will shear halfway when pressure applied, so successive halting of long DNA during shearing.
C. Enzymatic Specific enzymes exist which deliberately hydrolyse nucleic acid: RNA- ribonucleases DNA- deoxyribonucleases Exonuclease Endonuclease - cut at random sites - cut at specific sequences eg restriction enzymes
Size and shape
Can be linear or circular
Majority is found in chromosomes, the genetic repository of the cell
Bacteria contain plasmids - small circular dsDNA
Extra chromosomal DNA in mitochondria (mtDNA)
Chloroplasts also contain DNA
Packaging of DNA
- DNA
- Chromatin
- Nucleosome (DNA wraps around histone), consists of the core. H1 and linker DNA. The H1 histone is not located within nucleosome but acts as a loop fastener by binding to the DNA in the linker region
- nucleosomes packaged into a solenoid, aka 30nm fibre. 6 nucleosomes make up one turn of the solenoid, with H1 histone a in the centre of the fibre
- Solenoid packaged around nuclear scaffold. Not all parts are like this, but those which are are looped in domains around a protein scaffold of unknown composition.
- chromosomes
Histone
Protein scaffold. Interact with neg charged DNA via basic amino acid side groups.
Nucleoid
Where bacterial chromosome is packaged
Supercoils
The condensing of plasmid circular DNA. Formed due to an extra (or one less) right handed turn in the DNA. The DNA folds in on itself into a smaller coiled form. Supercoiling occurs in cells as a result of the action of topoisomerases
Conjugation
The sharing of plasmids between organisms.
Bacteria propagation
Able to survive external changes due to conjugation.
In lab, bacteria can be modified to take up DNA (transformation). These bacteria are called competent cells.
Restriction digestion
Restriction enzymes cut as specific sites.
If a disease results from a mutation at a restriction site, an enzyme will no longer be able to cut there, and so, the length of DNA would be different
Banding pattern
Unique combination of DNA fragments
- cleave chromosomal DNA with restriction endonucleases
- separate DNA fragments by agarose gel electrophoresis
