Miescher (1869) - extracted nuclein from human pus cell nuclei, contains P, protease resistant
Kossel and Neumann (1894) - initial identification of the bases in DNA
Hammersten (1900) - DNA contains a pentose sugar
Levene (1929) - the pentose sugar is 2'deoxyribose
Todd and Levene (1920's) - determined the chemical nature of nucleic acids. Repeating units: nucleotides. Two forms: DNA and RNA.
Todd (1930's) - nucleotide linkages
Caspersson (1934) - DNA is a linear macromolecule
Chargaff (1950's) - constancy in base ratios from different species, A=T and G=C
Franklin and Wilkins (1952) - X-ray studies, DNA is helix and gave measurements for the helix
Watson and Crick (1953) - developed Franklin and Wilkins model - double helix
Nucleotide Building Blocks - Bases
- planar molecules
- two sorts; Pyrimidine & Purine
Purines are two ring bases.
Pyrimidines are one ring bases.
Nucleotide Building Blocks - Sugar
- Pentose sugar
- RNA = ribose
- DNA = deoxyribose
- (-OH vs. -H at 2'C)
Nucleotides and Nucleosides
Difference between nucleotide and nucleoside:
A nucleotide is a phosphorylated nucleoside.
Sugar + base = nucleoside
Sugar + base + P = nucleotide
In AMP and ATP, both nucleotide, the phosphate group(s) is bonded to the sugar at 5'C by an ester linkage.
Synthesis of DNA requires the triphosphate form.
- PPi is lost in the reaction
- Nucleotides are joined together to form a polynucleotide
- Join via P-sugar-P-sugar
Hence:- phosphodiester bond, sugar phosphate backbone, 3' and 5' ends.
Add dNTP ==> PPi released and base addedd 5' to 3' direction
RNA and DNA
- single stranded molecule
- ribose sugar
- P groups
- bases (purines/pyrimidines)
- phosphodiester links
- 5' ==> 3'
- double stranded molecule - two complementary anti-parallel strands with sugar-P backbone and protruding bases.
- deoxyribose sugar
- P groups
- bases (purines/pyrimidines)
- hydrogen bonds
- 5' ==> 3' for each strand
DNA as genetic material
Most DNA exists as a right handed double helix as predicted by the X-ray crystallgraphy of Wilkins and Franklin.
It is the B-form found in living cells, other forms can be generated in vitro.
- Base stacking/secondary structure and packaging.
- Copy strands using complementary base pairs as the template
- Uses the sequence of bases as the code. Reads bases in groups of three, 5' to 3'. Gives mechanism for mutations.
Transfer of information:
- RNA copy, U not T. Transcription transfers information
DNA - Linear or Circular?
Eukaryotic nuclear chromosomes - linear dsDNA, extremely long - H. sapiens, 46 chromosomes, 6.6 x 10^9 bp.
Eukaryotic organelle (mitochondron, chloroplast) - circular dsDNA, intermediate length - H. sapiens mitochondria, 16569 bp.
Bacterial genome - circular dsDNA, extremely long - E.coli, 4.6 x 10^6 bp.
Plasmids - circular dsDNA, relatively short - 2.6 x 10^3 bp.
Viruses - linear dsDNA or circular ssDNA, also RNA genome viruses.
When DNA is heated, the double stranded molecule becomes single stranded DNA as the hydrogen bonds break between the base pairs.
Base pairs separate A=T first, followed by G=C
Tm: melting temperature, increases with higher GC content.
The reaction is followed by measuring the absorbance at 260nm as ssDNA absorbs more light than dsDNA.
When ssDNA is cooled, the strands will reanneal
- due to complementary base pairing.
This does not have to be the original DNA strand it was separated from during heating, also referred to as hybridisation
- add another ssDNA (or RNA) to the reaction.
- DNA/DNA (RNA:DNA) hybrids formed
- relies on the sequences being complementary
- a sequence of DNA (or RNA) will form complementary base pairs with ANY likely sequence generating stem loop structures, hairpin loops giving secondary structures seen in many RNA molecules e.g. tRNA.
Determining GC content
Determination of GC content of a DNA molecule by...
- Buoyant Density.
- a linear function of GC content
- can be used to purify DNA
- 8M CsCl has about the same density as DNA
- Mix with the DNA sample of interest
- Centrifuge FAST (125 x g, 125 hours)
- Set up a density gradient in the centrifuge tube, the DNA will form a band at the point where its density is the same as CsCl.
- Protein will be found at the top of the gradient, RNA will pellet at the bottom of the tube
- This process can also be used to purify one DNA sample from another, for example plasmid DNA and bacterial cell genomic DNA will form separate bands.