DNA

• DNA replication


  Production of exact copies of DNA with identical base sequences
• Replica


  • Exact copy of something
• DNA replication is the production of new strands of DNA with base sequences identical to existing strands
• The structure of DNA makes it suited to being replicated repeatedly, without any limit on how many times this is done
• Biological processes requiring DNA replication

  • Reproduction
  • Growth and tissue replacement in multicellular organisms
• DNA replication


  1. Separation of strands in the parent DNA molecule

  2. Complementary base pairing

  3. Synthesis of new strands
• Complementary base pairing ensures a high degree of accuracy when new strands are assembled on a template strand
• Complementary base pairing makes it possible to check the base sequence that has been assembled, recognize any mispairing, then cut out and replace the incorrect nucleotides
• A diploid human cell has DNA with approximately 6 billion base pairs, so on average there are only 0.6 errors when all the DNA is replicated prior to mitosis or meiosis
• This astonishing level of accuracy explains the genetic continuity that exists between generations
• Helicase


  Ring-shaped protein that separates the two strands of a DNA molecule so that they can each act as a template for the formation of a new strand
• DNA polymerase


  Assembles new strands of DNA, using the two original strands as templates
• DNA polymerase brings nucleotides into the position where hydrogen bonds could form, and if hydrogen bonds do not form and a complementary base pair is not formed, the nucleotide breaks away again
• Once a nucleotide with the correct base is in position and hydrogen bonds have formed between the two bases, DNA polymerase links the nucleotide to the end of the new strand
• Polymerase chain reaction (PCR)


  Automated method of DNA replication that follows a cycle of steps repeatedly, doubling the quantity of DNA with each cycle
• Only a very small quantity of DNA is required to start a PCR process - in theory, a single molecule
• PCR cycle


  1. Melting

  2. Annealing

  3. Elongation
• Gel electrophoresis


  Technique used to separate DNA molecules by length
• Gel electrophoresis


  1. Loading DNA samples into wells

  2. Applying voltage to create an electric field

  3. DNA molecules moving through the gel based on their length
• The right-hand lane in a gel electrophoresis is usually used to create a "ladder" of DNA fragments of known length, which is used to estimate the lengths of bands in other lanes
• Applications of PCR and gel electrophoresis


  • Testing for coronaviruses
  • Paternity testing
• PCR testing for coronaviruses

  1. Taking a nose or throat swab

  2. Converting RNA to DNA

  3. Amplifying viral base sequences

  4. Monitoring fluorescence to detect positive result
• Advantages of PCR testing for coronaviruses

  • Very sensitive - can detect miniscule quantities
  • Very specific - can detect only one strain
• Disadvantages of PCR testing for coronaviruses


  • Requires expensive materials and equipment
  • Results may not be immediately available
• DNA profiling for paternity testing


  Distinguishes between individuals using base sequences known as short tandem repeats
• PCR


  Polymerase Chain Reaction - a technique that amplifies small amounts of DNA
• PCR


  • Very sensitive - one molecule of viral RNA is amplified to produce about 35 billion molecules of DNA
  • Very specific - primers can be designed so only one strain of the virus is detected
• DNA profiling


  Technique that distinguishes between individuals using base sequences known as short tandem repeats
• Short tandem repeats


  • Sequences of between two and seven bases that are repeated consecutively
  • The number of repeats of each of these sequences varies considerably between individuals
• Stages of DNA profiling

  1. Obtain a sample of DNA

  2. Copy selected tandem repeats by PCR

  3. Separate the DNA produced by PCR according to length of fragment using gel electrophoresis

  4. Produce a pattern of bands of DNA that is unique to the individual
• Paternity testing


  Comparing the DNA profiles of the child, the child's mother and the man who might be the father to determine if the man is the biological father
• Reasons for paternity investigations

  • Men claiming they are not the father to avoid paying child support
  • Women with multiple partners wanting to identify the biological father
  • A child wanting to prove a deceased man was their father to show they are the heir
• Directionality of DNA


  • DNA strands have a 5' end and a 3' end
  • DNA polymerase adds nucleotides in the 5' to 3' direction
• DNA polymerase


  • Assembles new strands of DNA by linking together a strand of nucleotides with bases complementary to those of the template strand
  • Cannot initiate DNA replication, requires a 3' terminal to which a free nucleotide can be added
• Leading and lagging strands in DNA replication

  1. Leading strand - DNA polymerase adds nucleotides moving towards the replication fork, replication is continuous

  2. Lagging strand - DNA polymerase adds nucleotides moving away from the replication fork, replication occurs in short Okazaki fragments
• Enzymes involved in DNA replication

  • DNA primase - assembles RNA primers
  • DNA polymerase III - principal polymerase, adds DNA nucleotides and proofreads
  • DNA polymerase I - removes RNA primers and replaces with DNA
  • DNA ligase - connects gaps left by DNA polymerase I
• Transcription


  Synthesis of RNA using DNA as a template
• Transcription process


  RNA polymerase binds to DNA, unwinds the double helix, moves along the template strand, positions complementary RNA nucleotides, links them to form an RNA molecule, then detaches the RNA and allows the DNA to reform
• Complementary base pairing


  Cytosine pairs with guanine, thymine pairs with adenine, adenine pairs with uracil
• The DNA template strand is copied into an RNA molecule with the same base sequence, except uracil replaces thymine