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Related Experiment Videos

New protocol for DNA extraction of stool.

B M Machiels1, T Ruers, M Lindhout

  • 1Maastricht University, The Netherlands.

Biotechniques
|February 23, 2000
PubMed
Summary

A new method isolates human nucleic acids from stool, regardless of sample age or collection method. This technique is suitable for large-scale screening of fecal samples, overcoming limitations of current DNA isolation procedures.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Clinical Diagnostics

Background:

  • Existing DNA isolation methods for stool samples present significant limitations.
  • These limitations include constraints on sample volume, reliance on lavage fluids, and the necessity for fresh stool specimens.
  • These drawbacks hinder the efficient and widespread use of stool DNA analysis.

Purpose of the Study:

  • To develop and present a novel method for isolating human nucleic acids from stool samples.
  • The primary goal was to create a method that is independent of the stool's collection time and storage conditions.
  • To enable large-scale screening studies using fecal DNA.

Main Methods:

  • Collected dry fecal samples from 75 patients.
  • Mixed two grams of stool with a phenol-containing lysis buffer for nucleic acid extraction.

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  • Human DNA presence and quantity were assessed using dot blots and nested PCR targeting the human K-ras gene, with confirmation via exonuclease-amplification coupled capture technique (EXACCT).

    • Main Results:

    • Variable DNA yields were obtained, ranging from 9-1686 micrograms/g of feces.
    • Human DNA was detected in 9 out of 75 cases via dot blot, with percentages ranging from 0.06%-46%.
    • In the remaining 66 cases, human genomic DNA was successfully identified using nested PCR and confirmed with EXACCT.

    Conclusions:

    • The developed DNA isolation method is effective for analyzing human nucleic acids from stool.
    • This method is independent of stool collection timing and method, making it versatile.
    • The technique is suitable for large-scale screening studies, overcoming previous limitations in fecal sample analysis.