Polymerase Chain Reaction (PCR)

PCR method offers several benefits for wood samples, including:

  • PCR allows molecular genetic investigations of wood tissue. A successful DNA extraction from wood yielding appropriate DNA quality for PCR amplification allows molecular genetic investigations of wood tissue. Different DNA isolation techniques have been compared and optimized for wood samples from natural populations and wood processing enterprises. The quality of the DNA is tested by spectrophotometry, PCR amplification, and PCR inhibitor tests [5].

  • PCR-based methods have been developed for the early identification of several decay fungi from the wood of broadleaved trees. This method can help detect decay in standing trees, even at incipient stages [6].

  • Real-time PCR techniques offer more plant health test options than the traditional methods.

  • PCR-based assays can identify important decay fungi from wood of hardwood tree species in northern temperate regions. Multiplex PCR reactions were developed and optimized to detect fungal DNA and identify each taxon with a sensitivity of at least 1 pg of target DNA in the template. This assay correctly identified the agents of decay in 82% of tested wood samples [1][4].

 

PCR method for wood samples involves the following steps:

  1. DNA extraction: A successful DNA extraction from wood yielding appropriate DNA quality for PCR amplification allows molecular genetic investigations of wood tissue [1].

    Several different DNA isolation techniques have been compared and optimized for wood samples from natural populations and from wood processing enterprises [1].

  2. Quality testing: The quality of the DNA is tested by spectrophotometry, PCR amplification, and PCR inhibitor tests [1][5].

  3. PCR amplification: PCR-based methods have been developed for the early identification of several decay fungi from the wood of broadleaved trees [3].

    PCR-based assays can identify important decay fungi from the wood of hardwood tree species in northern temperate regions [4].

    Real-time PCR, also known as quantitative PCR (qPCR), can even specify the amount of fungus present in the sample by determining the cycle threshold value (Ct value) even in compromised samples from which little DNA can be extracted, thus safeguarding against false positives [1].

  4. Analysis: Sequencing the PCR amplicons can lead to reliable identification of logs and wood products to cultivar, ecotype, or even the falling population [4].

 

Function of primers

The function of primers in PCR for wood sample analysis is to target specific DNA sequences of interest for amplification. The primers are designed to bind to complementary sequences on opposite strands of the DNA template, flanking the region of interest. During PCR, the primers anneal to the template DNA and serve as a starting point for DNA polymerase to extend and synthesize new DNA strands. The amplified DNA fragments can then be analyzed for the presence or absence of specific target sequences, such as those from wood rot fungi or noncoding chloroplast DNA for wood identification. The specificity and sensitivity of the primers are critical for accurate and reliable PCR results, and can be optimized through careful primer design and testing.

The primers are designed to target specific DNA sequences of the fungi of interest.

The following are the ways in which primers help in the detection of fungi in wood samples using PCR:

  1. Specificity: The primers are designed to bind to complementary sequences on opposite strands of the DNA template, flanking the region of interest. This ensures that only the DNA of the target fungi is amplified, and not the DNA of other organisms that may be present in the sample.
  2. Sensitivity: The primers are designed to be highly specific to the target fungi, which increases the sensitivity of the PCR assay. This means that even small amounts of the target fungi can be detected in the wood sample.
  3. Multiplexing: Multiplex PCR can be used to detect multiple fungi in a single reaction using different sets of primers. This can save time and resources, and increase the efficiency of the assay.
  4. Quantification: Real-time PCR can be used to quantify the amount of DNA of the target fungi present in the wood sample. This can provide valuable information about the severity of the fungal infection.

 

To practise PCR use PraxiLabs

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