U3 Biotechnology

• PCR (Polymerase Chain Reaction) is used to amplify the DNA sequence of interest.
• Restriction enzyme: a protein whose sole job is to cut DNA at 'restriction sites'
• restriction enzymes make two incisions through each strand's sugar phosphate backbone
• DNA Ligase is an enzyme that seals DNA strands together.
• DNA ligase catalyses the formation of phosphodiester bonds which seal DNA strands to one another
• Primers are used in DNA replication and PCR to mark the ends of a target sequence to be amplified
• Primers are required to provide a template when DNA is being synthesised, they are necessary because DNA polymerase can only extend existing strands of DNA
• anneal: join via hydrogen bonds
• amplify: replicate many times
• Machine used for PCR: thermal cycler
• Step 1 of PCR: denaturation at 95° which breaks hydrogen bonds & separates 2 strands
• Step 2 of PCR: annealing stage at 50-60°, allows primers to anneal to complementary sequences on opposite ends of each strand
• Step 3 of PCR: extending stage at 72° which is optimal for taq polymerase- this adds free nucleotides to the growing strand.
• Overall, DNA has a negative charge. it had directionality, and a double helix structure.
• What are the differences between RNA and DNA?

  RNA has a single-stranded structure, contains the sugar ribose, and uses uracil as a base instead of thymine. DNA has a double-stranded structure, contains the sugar deoxyribose, and uses thymine as a base.
• DNA replication occurs during S phase of cell cycle
• DNA is wound around nucleosomes like beads on a chain. When the nucleosome 'beads' are loose, there is more expression of the gene.
• Nucleosome: group of 8 histone proteins wrapped by DNA
• Chromatin: a complex of DNA and proteins found in the nucleus
• There are 2 chromosomes in each cell (2 copies of each gene)
• Locus: the position of a gene
• What do prokaryotes have concerning their DNA that eukaryotes do not?

  plasmids
• What are the four key functions of DNA?

  heritable, stores a code, used to compare organisms, replication
• The purpose of DNA replication is to replicate the code it carries, to then be passed to daughter cells
• PCR and DNA replication both:

  • create new copies of DNA
  • require a template
  • use free nucleotides
• PCR and DNA replication contrast in:

  • PCR uses a DNA primer, DNA replication uses a RNA primer
  • PCR uses taq polymerase (artificially occurring) DNAR uses a naturally occurring DNA polymerase
• Nitrogenous bases C and G have more hydrogen bonds
• Step 1 of DNAR: helicase unwind and seperate strands
• Step 2 of DNAR: primase attatches a short sequence of DNA called a primer
• Step 3 of DNAR: DNA polymerase adds complementary nucleotides in a 5' to 3' direction.
• Step 4: two identical DNA strands are created with one parent and one daughter strand. This process is therefore 'semi-conservative'.
• Lagging strands have primers added at short intervals to form a replication fork. DNA polymerase synthesises short strands of DNA called Okazaki fragments.
• DNA polymerase moves in opposite directions on the two anti-parallel parent strands.