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BCH5425 Molecular Biology and Biotechnology

BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber
blaber@sb.fsu.edu


Lecture 22

Polymerase Chain Reaction (PCR)


The steps of:

  1. Template denaturation
  2. Primer annealing
  3. Primer extension

comprise a single "cycle" in the PCR amplification methodology.

After each cycle the newly synthesized DNA strands can serve as templates in the next cycle.

Note that half of the newly synthesized strands from the second round of replication have 5' and 3' termini which are defined by the annealing location of the primer oligonucleotides.

Summary of products at the end of each PCR cycle:

57 - (6 + 1) = 50 x copies of each defined length fragments

(i.e. 50 x duplexes of defined length)

[(2n - (n + 1)) - (n + 1)] x

or

(2n - 2(n + 1))

(this is often abbreviated to a simple rule of thumb for the amplification: (2n - 2n) x)

  1. Competition of complementary daughter strands with primers for reannealing (i.e. two daughter strands reannealing results in no amplification).
  2. Loss of enzyme activity due to thermal denaturation, especially in the later cycles
  3. Even without thermal denaturation, the amount of enzyme becomes limiting due to molar target excess in later cycles (i.e. after 25 - 30 cycles too many primers need extending)
  4. Possible second site primer annealing and non-productive priming

PCR was invented in 1985 by Kary Mullis, working for Cetus corporation somewhere near Berkeley, California. The original method of PCR used the Klenow fragment of E. coli DNA polymerase I. This enzyme, however, denatures at temperatures lower than that required to denature most template duplexes. Thus, after each cycle, fresh enzyme had to be added to the reaction. This was quite tedious. In addition to this problem with the enzyme, the samples had to be moved from one temperature bath to another to allow the individual steps of denaturation, annealing and polymerization (which all required different temperatures. This was pretty tedious too.

  1. The use of thermostable DNA polymerases, which resisted denaturation (inactivation) at high temperatures. Thus, an initial aliquot of polymerase could last throughout the numerous cycles of the protocol. The first thermostable DNA polymerase to be used was isolated from the bacterium Thermus aquaticus. It was isolated from a hot spring in Yellowstone National Park where it lived happily (i.e. it replicated its DNA) at temperatures in excess of 85 C
  2. The development of temperature baths which could shift their temperatures up and down rapidly and in an automated programmed manner. These are known as thermal cyclers or PCR machines.

These two developments let to the automation of PCR. The PCR process is covered by patents owned by Hoffmann-La Roche Inc. (a faceless conglomerate you can trust).

Thermal cycling parameters

  1. denaturation of template
  2. annealing of primers
  3. extension of the primers

A representative temperature profile for each cycle might look like the following:

Template denaturation


Primer annealing temperature

Primer extension

Number of cycles