Process for purifying, stabilising or isolating nucleic acids fr

Details Process for purifying, stabilising or isolating nucleic acids fr Process for purifying, stabilising or isolating nucleic acids fr

1998-02-11

2000-07-04

Wilson, James O.

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

536 271, 435 6, 435 91, C07H 2100, C07H 1900

Patent

active

060840919

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

This invention relates to a method of stabilizing, purifying and/or isolating nucleic acids contained in biological matter by removing contaminants, eg, substances which damage nucleic acids and inhibit enzymatic reactions. The method is especially suitable for analyzing, detecting or isolating nucleic acids in stool specimens. A reagents kit for implementing the method of the invention is also disclosed.
Numerous examples from various fields of research underline the impedance of analyzing nucleic acids contained in biological matter in which there are contaminants that damage nucleic acids during storage and inhibit the enzymatic manipulation thereof, eg, by amplification. If the nucleic acids contained in the biological matter are to be used for further analyses, it is thus important that these contaminants are only present in very low concentrations or are removed entirely from the specimen.
The analysis of nucleic acids contained in fecal specimens is of particular importance. The principal medical application is the detection of tumor-specific changes in nuclear DNA from fecal matter. Such changes can serve as a parameter for the early diagnosis of tumors in the digestive tract. The identification of bacterial and viral pathogens contained in fecal specimens by means of test methods based on nucleic acids is likewise becoming increasingly more important.
A method of isolating nucleic acids from fecal matter is disclosed in WO 93/20235. This method, however, results in only small yields of nucleic acids, and DNA-damaging and/or PCR-inhibiting substances are not removed. This means that the isolated DNA cannot be stored for long, and the amplification of specific gene sections to be analyzed does not lead to reproducible results. An especially serious drawback of the known method is that PCR amplification does not provide any intact DNA fragments of uniform sequence, which are necessary for further analysis. Obtaining these requires time-consuming cloning of the amplified gene sections.
Yet another drawback of the method described in the prior art is the necessity of using the solvents phenol and chloroform, which pose a severe health risk.
One of the objects of this invention was thus to provide a method with which nucleic acids contained in biological matter can be stabilized against degradation and with which substances inhibiting the enzymatic manipulation of nucleic acids can be removed. In particular, a method was to be provided with which DNA can be reliably isolated from fecal specimens.


SUMMARY OF THE INVENTION

This object is established by means of a method of purifying, stabilizing and/or isolating nucleic acids contained in biological matter, wherein an adsorption matrix is added to a nucleic-acid-containing specimen of biological material in order to bind contaminants and the nucleic acids are subsequently separated if necessary from the bound contaminants. The nucleic-acid-containing specimen is brought into contact with the adsorption matrix either directly or after taking up the specimen in a liquid.
With the method of the invention the usability of nucleic acids - especially DNA--isolated from biological matter is improved markedly. Besides, the addition of the adsorption matrix means that both substances which damage nucleic acids and substances which inhibit the enzymatic manipulation thereof are largely removed. The nucleic acids stabilized by the method of the invention can thus be stored for extended periods of time. A further advantage is that amplification, eg, by means of PCR, of the nucleic acids treated by addition of an adsorption matrix leads to reproducible results. This reproducibility is essential with regard to the informative value of the results of the nucleic-acid analysis. The high quality of the nucleic acids purified by means of the method of the invention is evident, for example, in that they can be examined directly by way of sequencing or heteroduplex analysis. Cloning is unnecessary, and there is no need to use solvents which po

REFERENCES:
QIAGEN Product Lit., 1995.
Fearon and Vogelstein, Cell, vol. 61, pp. 759-766, 1990.

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