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PCR Research GMO

One commonly used method for determining GMOs is based on the polymerase chain reaction (PCR). All PCR assays require that the sequence of the target DNA be known. Also important is the isolation and purification of the DNA in the sample. PCR technology has become the only reliable method to determine the presence of a specific DNA sequence from samples that contain little or poor quality DNA.
PCR-based GMO detection methods have been classified according to their level of specificity:

  1. Broadly used sequences such as P-35S (CaMV 35S promoter), T-35S (CaMV 35S terminator), T-Nos(nopaline synthase gene terminator), bla (β-lactamase), andnptII (neomycinphosphotransferaseII);
  2. Sequences within the gene of interest (the transgene itself);
  3. Construct-specific sequences, an example being the connection between the promoter sequence and the transgene itself;
  4. Random-specific sequences, such as the integration site of the transgene.

Quantitative real-time PCR (QRT-PCR) is the most powerful tool for quantifying GM cultures.
It works by continuously monitoring the amplification reaction, using the strength of the fluorescence signal to indicate the amount of amplicon present.
The specificity of QRT-PCR depends on both the chemistry used to control the amplification and the instrumentation used to control the signal.
List of equipment and supplies to complete the PCR laboratory
Sample preparation area:

  1. Homogenizer
  2. Analytical scales up to 200 g / 0.1 mg 
  3. Analytical scales up to 300 g / 1.0 mg
  4. Set of variable volume pipettes 10, 200, 1000 µl
  5. Refrigerator + freezer

ZONE 1.
For DNA extraction from samples:

  1. Laminar flow box, Biosafety Class II .
  2. Eppendorf-type thermostat for test tubes from 25 to 100 °C.
  3. Microcentrifuge for Eppendorf type tubes up to 16,000 rpm.
  4. Vortex
  5. Vacuum medical suction device with vacuum trap for supernatant removal.
  6. A set of variable volume pipettes of 10, 200, 1000 µl.
  7. Disposable polypropylene screw-on or screw-out 1.5ml microtubes.
  8. Supplied 1.5ml microtube racks and tips.
  9. Disposable tips for variable volume pipettes with aerosol barrier up to 200 and 1000 µl.
  10. Disposable tips for variable volume pipettes up to 200 µl.
  11. Refrigerator from 2 to 8 °C with freezer not exceeding minus 16 °C.
  12. Separate gown and disposable gloves.
  13. A container with disinfectant solution.
  14. A set of tools for treating the workplace.


ZONE 2.

  1. For PCR amplification as well as hybridization-fluorescence detection of PCR amplification products:
  2. Amplifier .
  3. PCR Box.
  4. Vortex.
  5. Set of variable volume pipettes 10, 200, 1000 µl.
  6. For the instruments, depending on the model:
    • 0.2 ml disposable polypropylene microtubes for PCR (flat lid, non-striped).
    • 0.2 ml disposable polypropylene PCR microtubes
    • Sheared tubes with domed lids or 96-well PCR plates equipped with thermostable optically transparent films.
  7. Disposable tips for variable volume pipettes with an aerosol barrier up to 200 µl.
  8. Tip racks and 0.2ml microtubes.
  9. Refrigerator from 2 to 8 °C.
  10. Separate gown and disposable gloves.
  11. Disinfectant solution container.
  12. Set of tools for treating the workplace.