Polymerase Chain Reaction (Edexcel International A Level Biology)

• Every person, with the exception of identical twins, has a unique DNA sequence which can be used to create a DNA profile
  • This is very useful in forensic science as it provides a way to identify individuals 
  • DNA profiling can also be used to determine the genetic relationships between different organisms e.g. 
    • Paternity and maternity testing
    • Ancestry kits
    • Determining evolutionary relationships between different species
• DNA profiles can be created using the following steps
  • Isolating a sample of DNA e.g. from saliva, skin, hair, or blood
  • Producing more copies of the DNA fragments in the sample using the polymerase chain reaction (PCR)
  • Carrying out gel electrophoresis on the DNA produced by PCR
  • Analysing the resulting pattern of DNA fragments

The polymerase chain reaction

• PCR is a common molecular biology technique used in most applications of gene technology e.g.
  • DNA profiling 
  • Genetic engineering
• It can be described as an in vitro method of DNA replication
• PCR produces many copies of a piece of DNA; this can be referred to as DNA amplification
  • It is used to produce large quantities of specific fragments of DNA or RNA from very small quantities; even just one molecule of DNA or RNA is enough to run PCR
  • By using PCR scientists can produce billions of identical copies of the DNA or RNA sample within a few hours
  • In each PCR cycle the DNA is doubled, so in a standard run of 20 cycles a million DNA molecules are produced.
• The process is carried out in a PCR machine, or thermal cycler, which automatically provides the optimal temperature for each stage and controls the length of time spent at each stage

• Each PCR reaction requires
  • DNA or RNA to be amplified
  • Primers
    • These are short sequences of single-stranded DNA that have base sequences complementary to the 3’ end of the DNA or RNA being copied; they define the region that is to be amplified, identifying where the DNA polymerase enzyme needs to bind
  • DNA polymerase
    • The enzyme used to build the new DNA or RNA strand.
    • The most commonly used polymerase is Taq polymerase, which comes from thermophilic bacterium Thermus aquaticus
      • Taq polymerase does not denature at the high temperature required during the first stage of the PCR reaction
      • The optimum temperature of Taq polymerase is high enough to prevent annealing of the DNA strands that have not been copied yet
  • Free nucleotides
    • Enable the construction of new DNA or RNA strands
  • Buffer solution
    • Ensures the optimum pH for the reactions to occur in

• There are three main stages of the PCR reaction
  
  • Denaturation
    • The double-stranded DNA is heated to 95 °C which breaks the hydrogen bonds that hold the two DNA strands together
  • Annealing
    • The temperature is decreased to 50-60 °C so that primers can anneal to the ends of the single strands of DNA
  • Elongation / Extension
    • The temperature is increased to 72 °C, as this is the optimum temperature for Taq polymerase to build the complementary strands of DNA to produce the new identical double-stranded DNA molecules
• These three stages make up a single PCR cycle, and many cycles can be completed
  • Each PCR cycle doubles the amount of DNA 

PCR can be used to amplify a fragment of DNA. Note that you don't need to know about forward and reverse primers

• After PCR is completed the DNA is treated with restriction endonuclease enzymes and a fluorescent tag can be added; both in preparation for gel electrophoresis
  • Restriction endonucleases break the DNA up into fragments of different length
  • Fluorescent tags enable the DNA fragments to be seen under UV light