PCR

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PCR (Polymerase Chain Reaction) is a widely used technique for the detection of microbes including those involved in bioleaching. It has primarily been used for the detection and identification of organisms. PCR is the primary step in many molecular tools such as DGGE, T-RFLP, CE-SSCP etc. The use of PCR techniques for the monitoring of microorganisms is largely related to identification of bioleaching organisms rather than monitoring their presence and functioning in relation to process parameters and leaching performance.

Steps in PCR. By courtesy of Anna Kaksonen
Steps in PCR. By courtesy of Anna Kaksonen
PCR-machine. By courtesy of Anna Kaksonen
PCR-machine. By courtesy of Anna Kaksonen

Contents

Principle

PCR is based on the exponential amplification of a small part (sequence; specific fragment, template, target) of the DNA of an organism. The isolated DNA is used as template for the amplification of a specific gene product. Through amplification the target sequence can be obtained in such high quantities that it can be made visible on an agarose gel.

  1. Denaturation. The double-stranded target DNA is denatured by heating (94°C, 5 min) into two separated single strands
  2. Primer annealing. Two primers (short synthetic oligonucleotides that match both sides of the target sequence) bind to the DNA.
  3. Amplification - DNA synthesis. The primers are extended with nucleotides complementary to the target strand by a DNA-polymerase, resulting in the duplication of the DNA fragment (72°C for 1 min per kb of product length).

This three-step sequence is repeated 25-40 times resulting in billions of copies of the original target. Finally, the DNA is allowed to become double-stranded again (renaturing) in a final extension step for 10 min at 72°C.

The final product is separated by size on an agarose gel and is visualized by binding to a dye. The presence of a PCR-product (band) of the right size confirms the presence of the target. The correct identification of this target can be confirmed by performing sequence analysis on this product.

The specificity of the reaction can be determined by the design of the primers. This makes it possible to detect organisms on the species level. By targeting functional genes it is also possible to detect all organisms carrying a specific feature in their DNA. The tool provides information on the presence and identity of known organisms, and can be used in a semi-quantitative way when applied to a dilution series of target material.

Advantages

  • Very sensitive, detects 10 cells per sample or more
  • High specificity
  • Robust
  • Fast
  • Easy to develop high throughput system

Disadvantages

  • Not quantitative
  • Limited number of targets in the same reaction possible
  • Detects DNA from dead as well as living cells

See also

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