Polymerase Chain Reaction (Edexcel International A Level Biology)

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Polymerase Chain Reaction

  • 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 

Polymerase chain reaction (1)Polymerase chain reaction (2)

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

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Author: Marlene

Marlene graduated from Stellenbosch University, South Africa, in 2002 with a degree in Biodiversity and Ecology. After completing a PGCE (Postgraduate certificate in education) in 2003 she taught high school Biology for over 10 years at various schools across South Africa before returning to Stellenbosch University in 2014 to obtain an Honours degree in Biological Sciences. With over 16 years of teaching experience, of which the past 3 years were spent teaching IGCSE and A level Biology, Marlene is passionate about Biology and making it more approachable to her students.