Use of Taq DNA polymerase to produce multiple copies of DNA rapidly by the polymerase chain reaction (PCR)

• Polymerase chain reaction?(PCR) is a common molecular biology technique used in most applications of gene technology, for example, DNA profiling (eg. identification of criminals and determining paternity) or genetic engineering
  • PCR is also used in routine?COVID-19 testing?to detect and amplify small amounts of viral RNA

  It can be described as the in vitro?method of 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)
• By using PCR scientists can have?billions of identical copies?of the DNA or RNA sample within a few hours
• The PCR process involves?three key stages?per cycle
• In each cycle, DNA is doubled so, in a standard run of 20 cycles, 1 million DNA molecules are produced. The three stages are undertaken in a PCR instrument (or?thermal cycler) which automatically provides the?optimal temperature?for each stage and controls the?length of time?spent at each stage

The process of PCR

• Each PCR reaction requires:
  • Target DNA?or?RNA?that is being amplified
  • Primers?(forward and reverse) – these are short sequences of single-stranded DNA that define the region that is to be amplified by showing the DNA polymerase?where to begin building?the new strands
  • DNA polymerase?– is the enzyme used to build the new DNA or RNA strand.
    • The most commonly used polymerase is?Taq?polymerase?as it comes from a thermophilic bacterium?Thermus aquaticus
    • This bacterium lives in?hot springs?in geothermal areas
    • Taq polymerase?does not denature?at the high temperature involved during the first stage of the PCR reaction
    • The enzyme's optimum temperature is?high enough to prevent annealing?of the DNA strands that have not been copied yet

    Free nucleotides?– used in the construction of the DNA or RNA strands

  • Buffer solution?– to provide the optimum pH for the reactions to occur in

  The three stages are:

  • Denaturation?– the double-stranded DNA is heated to 95°C for 15 seconds, which breaks the hydrogen bonds that hold the two DNA strands together
  • Annealing?– the temperature is decreased to between 50 - 60°C so that primers (forward and reverse ones) can attach to the ends of the single strands of DNA by hydrogen bonding
  • Elongation / Extension –?the temperature is increased to 72°C for at least a minute
    • This is the optimum temperature for?Taq?polymerase to build the complementary strands of DNA
    • To produce the new identical double-stranded DNA molecules

    The three stages of a cycle take 2-3 minutes, so many cycles can be completed in a short space of time

The substances required for the Polymerase Chain Reaction to occur and the three key stages of the reaction

Production of human insulin in bacteria as an example of the universality of the genetic code allowing gene transfer between species

• Prior to the mid-1980s, some insulin-dependent diabetics would need to inject?pig or cattle insulin?as a substitute for human insulin to control their blood sugars
  • Some diabetics developed an?allergy?so could not use it

  Human insulin can be produced by other organisms by transferring the human insulin gene to them?for?large-scale expression?of that gene

• DNA can be transferred from a human to a prokaryote (eg.?E. coli), a eukaryote single-celled organism (eg. yeast) or a plant (eg. safflower species)
  • All these organisms express the human insulin gene
  • The insulin produced can be?harvested for medical use

  The fact that DNA can be transferred from one organism to another across kingdoms?(and can still do the same job) demonstrates the?universality of the genetic code

  • The presence of nucleotides is a marker between living and non-living entities

  This was an early successful example of genetic modification

Human and Bacterial DNA working together

• In 1982,?insulin?was the first genetically engineered human protein to be approved for use in?diabetes?treatment
• Bacterial?plasmids?are modified to incorporate the human insulin gene
• These genetically modified plasmids are then?inserted into?Escherichia coli
• The newly-adjusted bacteria are isolated, purified and placed into large scale?fermenters?that provide?optimal conditions
• The genetically engineered bacteria multiply by binary fission, and express the human protein - insulin, which is eventually extracted and purified
• The advantages for scientists to use genetically engineered insulin are:
  • It is?identical to human insulin, unless modified to have different properties (eg. act faster, which is useful for taking immediately after eating or to act more slowly)
  • There is a?reliable supply available?to meet demand (no need to depend on the availability of meat stock)
  • Fewer ethical,?moral?or?religious?concerns (proteins are not extracted from cows or pigs)
  • Fewer rejection problems?or?side effects?or?allergic reactions
  • Cheaper?to produce in large volumes
  • That it is useful for people who have?animal insulin intolerance

The production of human insulin; the combination of? DNA from two widely different organisms demonstrates the universality of the genetic code