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Related Concept Videos

DNA Isolation01:24

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
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Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...

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Methods for optimizing DNA extraction before quantifying oral bacterial numbers by real-time PCR.

Mangala A Nadkarni1, F Elizabeth Martin, Neil Hunter

  • 1Institute of Dental Research, Westmead Millennium Institute and Westmead Centre for Oral Health, Wentworthville, NSW, Australia. mangala_nadkarni@wmi.usyd.edu.au

FEMS Microbiology Letters
|May 23, 2009
PubMed
Summary

Optimized genomic DNA extraction for oral bacteria real-time PCR was achieved by overcoming Porphyromonas gingivalis nuclease activity. This method accurately quantifies oral bacteria, crucial for understanding oral flora dynamics.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genomics

Background:

  • Accurate enumeration of oral bacteria is essential for understanding oral flora and associated diseases.
  • Real-time PCR (polymerase chain reaction) offers a sensitive method for bacterial quantification.
  • Challenges exist in genomic DNA extraction from complex oral microbial communities, particularly due to bacterial nucleases.

Purpose of the Study:

  • To develop and optimize a method for genomic DNA extraction suitable for real-time PCR-based enumeration of oral bacteria.
  • To address challenges in DNA extraction caused by bacterial nuclease activity, specifically from Porphyromonas gingivalis.
  • To validate the optimized real-time PCR approach against traditional bacterial quantification methods.

Main Methods:

  • Development of an in vitro oral flora model including Lactobacillus acidophilus, Streptococcus mutans, Parvimonas micra, Porphyromonas gingivalis, Prevotella melaninogenica, and Fusobacterium nucleatum.
  • Comparison of traditional methods (colony counting, DNA content estimation) with real-time PCR for bacterial enumeration.
  • Investigation and mitigation of Porphyromonas gingivalis nuclease activity using diethyl pyrocarbonate or ethanol treatment.
  • Incorporation of a final DNA purification step to remove PCR inhibitors.

Main Results:

  • Porphyromonas gingivalis nuclease activity was identified as a significant inhibitor of double-stranded DNA extraction.
  • Treatment with 20 mM diethyl pyrocarbonate or 70% ethanol effectively neutralized P. gingivalis nuclease activity.
  • The optimized DNA extraction protocol, including nuclease inhibition and purification, enabled accurate real-time PCR quantification of oral bacteria.
  • Real-time PCR results were validated against traditional quantification methods.

Conclusions:

  • An optimized genomic DNA extraction protocol using mutanolysin, with specific treatments to inhibit Porphyromonas gingivalis nuclease activity, is effective for real-time PCR.
  • This method allows for accurate enumeration of oral bacteria in complex samples.
  • The developed protocol enhances the reliability of real-time PCR for studying oral microbiome composition and dynamics.