Thermal Cyclers

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Thermal Cyclers

Background – Polymerase Chain Reaction (PCR)

The PCR process is a fast and inexpensive in vitro method for enzymatically, synthesising and amplifying defined DNA sequences.
Significant amounts of a sample of DNA are necessary for molecular and genetic analyses and this is almost impossible without DNA amplification.
Once amplified, the DNA produced by PCR can be used in many different laboratory procedures such as most mapping techniques in the Human Genome Project. PCR is also valuable in a number of laboratory and clinical techniques including DNA fingerprinting, detection of bacteria or viruses and diagnosis of genetic disorders.
In order to amplify a segment of DNA using PCR, the sample is first heated so the DNA denatures i.e separates into two pieces of single stranded DNA. Then, an enzyme called Taq polymerase synthesises (builds) two new strands of DNA, using the original strands as templates.
This process results in the duplication of the original DNA, with each of the new molecules containing one old and one new strand of DNA. Each of these strands can then be used to create two new copies, and so on, and so on. The cycle of denaturing and synthesising new DNA is repeated as many as 30 or 40 times, leading to more than one billion exact copies of the original DNA segment.
At this stage the amplified nucleic acid may then be analysed for size, quantity, sequence, etc. or used in further experimental procedures (e.g. cloning).

The basic PCR steps consist of:

  1. Initial step denatures the target DNA by heating it to 95°C or higher for 15 seconds to 2 minutes.
  2. The temperature is reduced to 40 - 60°C, at which temperature the oligonucleotide primers anneal with the separated target DNA strands and serve as primers for DNA synthesis by a thermostable DNA polymerase (e.g. TAQ polymerase). This step lasts approximately 30 – 60 seconds.
  3. The synthesis of new DNA begins when the reaction temperature is raised to the optimum for the thermostable DNA polymerase (approx. 72°C for most thermostable DNA polymerases). Extension of the primer by the thermostable polymerase lasts approximately 1 – 2 minutes.
  4. Step 3 completes one cycle, and the next cycle begins with a return to 95°C for denaturation.
  5. The number of target DNA copies (amplicons) approximately doubles every cycle, therefore:
    10 cycles can multiply the amplico by a factor of about 1000
    20 cycles can multiply the amplicon by a factor of about 1,000,000 in a matter of     hours
    After 20 – 40 cycles the amplified nucleic acid may then be analysed.

Although the PCR concept is simple, successful performance of a PCR reaction depends on a number of factors. PCR has several characteristics that vary qualitatively: specificity, sensitivity, efficiency and fidelity (error rate). The design of reaction determines which of these which of these characteristics is most important. For example, diagnostic testing requires sensitivity, specificity and reproducibility. For preparative PCR, used for synthesis of probes or template for sequencing, the efficiency of the reaction determines its overall success. By modifying the physical conditions and chemical components of the reaction, the PCR characteristics can be optimosed. For instance, the elimination or reduction of non-specific reactions can optimise both the sensitivity and specificity for diagnostic testing by removing competing side reactions.

PCR optimisation depends on a combination of the following factors:

  1. Primer design
  2. Magnesium concentration: free magnesium ion Mg++ is required for Taq DNA polymerase to be active
  3. Enzyme: several choices are available for selection of thermostable DNA polymerase
  4. Enzyme concentration: the optimal enzyme concentration may vary between 1.0 – 5 units per reaction and it is especially important o titrate the Mg++ concentration and the amount of enzyme required per assay.
  5. Template considerations: successful amplification of the region of interest is dependent on the amount and quality of the template DNA with the amount of template required being dependent upon the complexity of the DNA sample.

The entire cycling process of PCR is automated and can be completed in just a few hours using a thermal cycler (thermocycler), which is programmed to alter the temperature of the reaction every few minutes to allow DNA denaturing and synthesis.

Thermal cycler features Progen Scientific sells a range of single block thermal cyclers (thermocyclers) to suit most requirements:

  1. SensoQuest Thermal Cyclers:
  • High specification thermal cyclers which can be upgraded
  • Choice of 3 thermoblocks which can be easily changed in seconds (48 x 0.5ml.; 96 x 0.2ml. or 96 well microtitre plates; 384 well microtitre plates
  • Gold coated silver thermoblocks with own microprocessor for fast temperature ramp rates (up to 4.2°C per second) and cooling rates (up to 3.2°C per second) plus excellent temperature homogeneity in the range -5°C to +99.9°C
  • Very efficient and user friendly user interface via touchscreen (the heart of a good thermal cycler) with illuminated and colour graphic display. Context sensitive online help menu means you will hardly ever require the operating manual
  • Heated lid with programmable temperature and pressure (for different size tubes and plates)
  • LabCycler Basic is standard format (i.e. same programmed temperatures in each well) whereas LabCycler Gradient can also be used with a temperature gradient (up to a maximum temperature differential of 40°C across the block) so that reactions can be optimised to the optimal temperature conditions. The LabCycler Basic can be upgraded at a later date to the LabCycler Gradient if required.

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  1. Labnet MultiGene Gradient thermal cycler:
  • Cost effective gradient thermal cycler (up to a maximum temperature differential of 24°C across the block)
  • Aluminium thermoblock with temperature ramp rate of up to 2.2°C per second and cooling rate of up to 1.5°C per second in the range +4°C to +99°C
  • Heated lid (105°C)
  • Currently 96 x 0.2ml. or 96 well microtitre plate block only but others planned for release. Blocks can be removed / changed
  • Small footprint

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  1. Labnet MultiGene II Personal thermal cyclers:
  • Cost effective, smaller block capacity, standard thermal cycler. Excellent for teaching laboratories as well as for researchers with fewer sample requirements
  • Choice of two models with different thermoblocks: 25 x 0.2ml. or 16 x 0.5ml.
  • Aluminium thermoblocks with temperature ramp rate of up to 3°C per second and cooling rate of up to 2°C per second in the range +4°C to +99.9°C
  • Heated sliding lid which automatically adjusts to tube or plate  height
  • Currently 96 x 0.2ml. or 96 well microtitre plate block only but others planned for release. Blocks can be removed / changed
  • Very small footprint

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