ka2: replication of DNA

• Replication of DNA by DNA polymerase and primers.

  • Prior to cell division, DNA is replicated by a 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 complementary base pairing, to the deoxyribose (3’) end of the new DNA strand which is forming.
• DNA is unwound and hydrogen bonds between bases are broken to form two template strands. DNA polymerase can only add DNA nucleotides in one direction resulting in the leading strand being replicated continuously and the lagging strand replicated in fragments.
• Fragments of DNA are joined together by ligase.
• Polymerase chain reaction (PCR) amplifies DNA using complementary primers for specific target sequences.

  • In PCR, primers are short strands of nucleotides which are complementary to specific target sequences at the two ends of the region of DNA to be amplified.
• Repeated cycles of heating and cooling amplify the target region of DNA.

  • DNA is heated to between 92 and 98°C to separate the strands. It is then cooled to between 50 and 65°C to allow primers to bind to target sequences. It is then heated to between 70 and 80°C for heat-tolerant
  • DNA polymerase to replicate the region of DNA.
• Practical applications of PCR.

  • PCR can amplify DNA to help solve crimes, settle paternity suits, and diagnose genetic disorders.
• DNA is replicated prior to cell division (mitosis or meiosis) by a DNA polymerase.  DNA replication produces exact copies of the parent DNA molecule.  This ensures that a complete set of genetic information is passed on to new cells and from generation to generation.
• DNA replication will not occur unless all of the following are available:

   

  • Parent DNA to act as a template.
  • A supply of new nucleotides.
  • DNA Polymerase and ligase enzymes.
  • ATP to provide energy.
  • A DNA Primer - 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.
• Prior to cell division, DNA is replicated by a DNA polymerase.  DNA polymerase adds nucleotides and thus form the sugar phosphate bonds.  It requires a primer to start replication

  
• DNA polymerase can only add complementary nucleotides to the deoxyribose (3’) end of a DNA strand i.e. in a 5’ to 3’ direction. This results in one strand being replicated continuously and the other strand being replicated in fragments.  Fragments must be primed and are joined together by an enzyme called ligase.
• Process of DNA replication

   1. DNA unwinds.

   2. Hydrogen bonds break to form 2 template strands.

   3. DNA polymerase adds nucleotides, complementary base pairing occurs and new hydrogen bonds form between bases.

   4.Strong chemical bonds form between sugar and phosphate.

   5. Two identical DNA strands formed then rewind.

   

  This process occurs at several locations on a DNA molecule.

  
• PCR is the amplification of DNA using complementary primers for specific target sequences at the two ends of the region to be amplified
• The specific sections of DNA are amplified in vitro [replicated out with a cell in a test tube (in vitro = in glass)].  Millions of copies of a specific piece of DNA can be created in a few hours in a thermocycler.
• Step 1: The DNA is heated to between 92°C and 98°C to separate the strands.

   

  Step 2: The separated strands are then cooled to between 50°C and 65°C for primer

        binding. Cooling allows primers to bind to target sequences (see below).

  
• Step 3: The strands are then heated to between 70°C and 80°C to allow heat-tolerant

  DNA polymerase to replicate the region of DNA (see below).

  
• Step 4: Repeated cycles of heating and cooling amplify this region of DNA
• DNA Profiling

  • PCR helps to rapidly identify people
  • Specific areas of DNA known to vary between individuals are amplified
  • Giving different sized fragments in different people
  • An essential tool in solving crime
• Disease detection

  • DNA sequences that are known to indicate certain genetic disorders or diseases are amplified using PCR for the purposes of diagnosis
• Archaeological analysis

  • Ancient DNA, degraded over the years, can be amplified and used in archaeological, paleontological and evolutionary research
• Settle Paternity Suits

  • DNA samples can be analysed to help identify a child's father when unknown
  • Similarities in the DNA sequences of the child and potential father(s) would indicate they are related
• Sequencing

  • DNA sequences can be determined to help determine relatedness between species