L2 - dna replication

• Why grind down DNA sample (in mortar and pestle): to break down cell walls
• Why mix DNA sample with detergent: to break down the cell membrane, releasing cell contents into the solution
• Why add salt to the DNA sample: to break down the hydrogen bonds between the DNA and water molecules
• Why add protease enzyme to DNA sample: to break down the protein associated with the DNA in the nuclei
• Why add a layer of alcohol (ethanol) on top of the DNA sample: to cause the DNA to precipitate out of the solution
• Temperature should be kept low throughout the DNA extraction process to reduce enzyme activity and reduce the breakdown of DNA
• Detergent breaks down cell membranes as it disrupts the membrane structure and phospholipids form suspension in aqueous solution
• Semi conservative replication:

  -when describing the DNA double helix, the top and bottom of each strand are described as either the 3’ end or the 5’ end. The number refers to which carbon within the deoxyribose sugar of the nucleotide is closest to the top/bottom

  -an enzyme that catalyses DNA replication is complementary in shape to the 3’ end and can therefore only attach to the DNA at this location
• Stages of semi conservative replication:

  -before the DNA can be copied, the double helix must first unwind

  -the enzyme DNA helicase breaks the hydrogen bonds between the complementary bases of the two DNA polymers within the double helix

  -this causes the double helix to separate or unzip

  -these two separated strands both act as templates for DNA replication
• Stages of semi conservative replication:

  -the point at which the unzipping stops is called the replication fork

  -not all the DNA is unzipped in one go, as this increase the chances of copying errors resulting in mutations

  -within the nucleus, there are free floating DNA nucleotides

  -if a free floating DNA nucleotide aligns next to a complementary base on either template strand of DNA, then hydrogen bonds will form between them

  
• Stages of semi conservative replication:

  -the enzyme DNA polymerase is responsible for forming the phosphodiester bond between these nucleotides to create a new polymer chain of DNA

  -DNA polymerase can only attach at the 3’ end and therefore will move along the template strand in the 3’ to 5’ direction

  -when the enzyme is moving towards the replication fork, the new strand is referred to as the leading strand and can be created in one continuous go
• Stages of semi conservative replication:

  -on the anti parallel strand, the DNA polymerase still attached at the 3’ end and works down towards the 5’ end, but this is directly next to the replication fork

  -therefore, every time the replication fork unwinds further, the enzyme has to reattach to the 3’ end, and this creates small fragments of DNA

  -this strand is called the lagging strand and the small fragments are called okazaki fragments. The Okazaki fragments are later joined together together by the enzyme DNA ligase
• Stages of semi conservative replication:

  -both DNA template strands are now replicated, and this process continues until the entire length of DNA is replicated

  -this process is known as semi conservative replication because two copies of the original DNA molecule are produced, each copy conserving (replicating) the information from one half of the original DNA molecule

  -each copy contains one original strand and one newly synthesised strand
• Meselson and Stahl performed an experiment to prove semi conservative replication
• Meselson and Stahl:

  -bacteria was grown in a solution containing the 15N isotope, a heavier form of nitrogen

  -during replication, all the new DNA molecules incorporated will contains this isotope in the nitrogenous base

  -the DNA is therefore heavier, and this is demonstrated by centrifuging and seeing the DNA band settling at a lower point in the test tube
• Meselson and Stahl:

  -a sample of this bacteria is then transferred to a solution containing only the 14N lighter isotope of nitrogen and left to replicate once only

  -all the newly synthesised DNA will now be lighter. After centrifugation, all the DNA settles in the middle of the test tube, which shows that in DNA replication, 50% of the old DNA is always conserved and 50% of the DNA is new
• Meselson and Stahl:

  -the bacteria are left to replicate for a second time in the light 14N medium. After another round of semi conservative replication, the results can be seen above. There will now be two double helices composed of completely light DNA and two helices that contain one heavy strand and one light strand

  
• Meselson and Stahl results: