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New protocol for DNA extraction of stool
B M Machiels1, T Ruers, M Lindhout
1Maastricht University, The Netherlands.
Abstract:
Present methods for DNA isolation of stool have various limitations such as the amount of stool used, the requirement of lavage fluids or the use of fresh stool. In this paper, a new method is described for the isolation of human nucleic acids from stool, which is independent from the moment of collection. Fecal samples as dry as possible were collected from 75 patients; two grams of stool were mixed with a lysis buffer containing phenol. DNA yields of crude stool were variable and ranged from 9-1686 micrograms/g of feces. With dot blots in 9 of the 75 cases, the human DNA was identified and ranged from 0.06%-46%. In the remaining 66 cases, human genomic DNA was detected by nested PCR, using human K-ras gene amplification as an example. Amplification products were confirmed for human K-ras with the exonuclease-amplification coupled capture technique (EXACCT). In conclusion, the developed DNA isolation method can be used for the study of large numbers of stool samples, is independent of the age or method of stool collection and is suitable for large-scale screening studies.
Insights
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.
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.
- 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.