DNA

• Two types of nucleic acids: DNA and RNA

  DNA = deoxyribonucleic acid, RNA = ribonucleic acid

  Nucleotide = monomer of nucleic acid. DNA monomer = deoxyribonucleotide. RNA monomer = ribonucleotide. Carbohydrates like starch are macromolecules that exist as polymers of glucose. Proteins such as insulin is a macromolecule that exists as a polymer of amino acids.
• Size:

  1. Steroid (a type of lipid)
  2. DNA (because it is shown as double helix)
  3. Protein (notice the alpha helix and beta sheet)
  4. Cells (e.g. animal cell, bacterial cell)
  5. Viruses
  6. Height of a clinical sample in a test tube
• Each nucleotide is composed of 5-C sugar, nitrogenous base & phosphate group(s). Phosphate group gives nucleic acids their (-) charge & acidity. 
• Nitrogenous bases:

  Purines:

  Adenine (A)

  Guanine (G)

  Pyrimidines:

  Cytosine (C)

  Uracil (U) in RNA

  Thymine (T) in DNA
• All nitrogenous bases have N duh & ring structures. Purines have 2 rings, pyrimidines have 1 ring. Each nucleotide contains 1/4 diff nitrogenous bases on C1. 
• Pentose sugar of DNA/ deoxyribonucleotide (DNA monomer) aka deoxyribose sugar. Pentose sugar of RNA/ ribonucleotide (RNA monomer) aka ribose sugar. 
• Wo phosphate, the nucleotide is called a nucleoside. No. of phosphate groups can vary from 1-3. Eg of ribonucleotide is ATP. If it was a deoxyribonucleotide it would be called deoxyadenosine triphosphate.
• Covalent bond linking 2 adj nucleotides aka phosphodiester bond. Consists of a phosphate group linked to 2 pentoses via 2 covalent ester linkages.
• Formation of nucleic acids from nucleotides:

  This rxn is catalysed by a polymerase. DNA polymerase is used if the monomers are deoxyribonucleotides ( ). Inorganic pyrophosphate (PPi) (2 lil phosphates on 3rd diagram) is removed. Phosphodiester bond is a covalent bond. OH group on C3 & phosphate group on C5 of another nucleotide react to form an ester linkage. Many nucleotides join to produce one long polynucleotide chain. A chain of polynucleotide  (DNA strand/ RNA strand)
• DNA and RNA are similar except in DNA, all pentose are deoxyribose but in RNA, all pentose are ribose. What varies btw diff nucleic acids is the seq of nitrogenous bases. 
• Free groups:

  • 5' end of a polynucleotide chain (C5) ends w free phosphate group
  • 3' end of a polynucleotide chain  (C3) ends with the free -OH group

  Each polynucleotide (DNA/ RNA strand) has directionality. 
• DNA has antiparallel orientation (One strand runs in the 5' to 3' direction, the complementary strand runs in the 3' to 5' direction). Sugar-phosphate backbones are outside of the molecule & nitrogenous bases are inside. Unique seq of nitrogenous bases allows store of genetic information. Sugar phosphate backbone with covalent bonds ensures store of information is stable & secure.
• Before mitosis (nuclear division) takes place, all DNA inside the nucleus of the cell must be replicated.
• 1. Conservative

  • 2 parental reassociate after acting as templates for new strand, thus restoring the og x2 helix
  • Daughter DNA consists of 2 newly synthesised strand 

  2. Dispersive

  • Parental DNA molecule is fragmented & dispersed
  • Daughter DNA contains mixture of old & newly synthesised parts

  3. Semi - Conservative

  • The 2 parent strands separate by breaking H bonds & each acts as a template for synthesis of new strand thru complementary base pairing
  • Daughter DNA is 1 parent + 1 newly synthesised
• Meselson and Stahl’s experiment:  Watch 

  1. Put DNA into 15N (heavier isotope) medium. Several generations ltr it should be all incorporated in the DNA
  2. Put 15N DNA into 14N medium 
  3. Take out the 1st & 2nd gen and put in test-tube
  4. Put DNA extracts of 1st gen into solu of CsCl & repeat for gen 2
  5. Centrifuge at 100,000 x g for a few days until density gradient is formed. Bands of DNA are visualised by UV absorption photography.
• Replication begins at a specific site called origin of replication, which has a specific sequence of nucleotides. 
• Role of RNA primer:

  The end of the RNA primer provides a free 3’ OH which is required for DNA polymerase III to initiate DNA synthesis. Use RNA primer cus DNA polymerase III can only add deoxyribonucleotides to a pre-existing chain w free 3’ OH group. 
• Role of DNA polymerase III:

  DNA polymerase III catalyses the formation of phosphodiester bonds btw adj DNA nucleotides of the newly synthesised strand. As DNA polymerase III moves along the parental strand, part of the enzyme "proof-reads'' the prev region to ensure that proper base pairing has taken place between the bases. If an incorrect DNA nucleotide is added, it will be removed by DNA polymerase III & replaced with the correct one. This is to ensure the fidelity of the DNA. 
• Since the parental strands are anti-parallel, the 2 new strands are synthesised in opp directions. DNA replication is continuous on the leading strand. The daughter strand is synthesised in 5’→3’ direction. DNA replication is synthesised discontinuously on the lagging strand. The fragments, known as Okazaki fragments, are synthesised in 5’→3’ direction. 
• Problem is lagging strand cus DNA polymerase adds DNA nucleotides only from the 5’→3’ direction, it will move in opp direction compared to the leading strand. So the synthesis of this strand is in a discontinuous fashion.
• Polymerase Chain Reaction (PCR) is a technique that allows amplification (making many copies) of a specified segment of DNA in a test tube.
• Each cycle of PCR results in a x2 in the number of DNA molecules being replicated. n cycles will yield 2n molecules of target DNA. The amount of desired sequence hence ↑ exponentially. ≈ repeated for 25 – 30 cycles in the thermocycler cus fewer cycles – not enough DNA for analysis & too many cycles – too many mistakes in replication
• ℃ control in 1 cycle

  1.  ℃ ↑ to 95℃ to denature DNA molecule (dsDNA → ssDNA)
  2. ℃ ↓ to 64℃ to allow DNA primers to anneal to their complementary DNA strands. The 2 primers define the region to be amplified.
  3. Temperature ↑ to 72℃ where Taq polymerase works optimally for strand elongation.
• ddNTP is to terminate DNA synthesis. The lack of the 3'-OH group means that it cannot form phosphodiester bond btw two nucleotides - thus terminating DNA. 
• Gel electrophoresis:

  Technique that separates DNA, RNA/ proteins mixtures acc to molecular size & charge via electric field. When placed in an electric field, DNA molecules which are (-) charged will move away from (-) electrode & towards (+) electrode. Shorter DNA fragments will move towards (+) electrode at faster rate than longer fragments. Gel consists of a mesh of filaments that resists the movement of molecules in a sample. If using ddTTP then only T fragments will be visible due to fluorescence of the fluorescence markers. Act DNA template seq complementary to results.