DG2- Replication of DNA

• DNA replication occurs prior to cell division so that when the cell divides, each daughter cell will inherit an identical copy of the DNA
• DNA is unwound and hydrogen bonds between bases are broken to form two template strands
• DNA is replicated by the enzyme DNA polymerase
• DNA polymerase needs primers to start replication
• A primer is a short strand of nucleotides which binds to the 3' end of the template DNA strand allowing polymerase to add DNA nucleotides
• DNA polymerase adds DNA nucleotides, using complimentary base pairing, to the deoxyribose 3' end of the new DNA strand which is forming
• DNA polymerase can only add DNA nucleotides in one direction (3' to 5')
• The leading strand is replicated continuously
• The lagging strand is replicated in fragments
• Fragments of DNA are joined together by the enzyme ligase
• The requirements for DNA replication are:

  • original DNA template
  • free DNA nucleotides
  • primers
  • DNA polymerase
  • ligase
  • ATP
• The two new strands twist to form a double helix
• The replication of DNA is semi-conservative
• Each identical daughter DNA molecule ends up with one of the original strands and one newly synthesised strand
• Polymerase chain reaction (PCR) amplifies a target sequence of DNA
• 1. The DNA is heated to between 92 to 98 to separate the strands (break the hydrogen bonds between complimentary bases)
• 2. The DNA is then cooled to between 50 to 65 allow primers to bind to target sequences
• The primers used in PCR are short strands of nucleotides which are complimentary to specific target sequences at the two ends of the region of DNA to be amplified
• 3. The DNA is heated to between 70 to 80 for heat tolerant DNA polymerase to replicate the region of DNA
• PCR:

  • Heat 92-98c
  • Cool 50-65c
  • Heat 70-80c
  • Repeat
• PCR requires polymerase to operate at high temperatures
• Repeated cycles of heating and cooling amplify the target region of DNA
• PCR is performed automatically by a thermal cycler
• Each cycle doubles the amount of DNA
• The requirements for PCR are:

  • original strand of DNA which needs amplified
  • complimentary primers
  • heat-tolerant DNA polymerase
  • supply of 4 different DNA nucleotides
  • thermal cycler- equipment that varies the temperature of the reaction
  • buffer solution to maintain optimum pH
• PCR is used in forensic science, allowing minute quantities of DNA from a crime scene to be amplified, sequenced and compared to DNA sequences from suspects
• PCR can be used to settle paternity suits by amplifying and comparing a child's DNA to their potential father
• PCR has medical applications:

  • diagnosis of genetic disorders
  • infection detection
  • early warning of cancer