A METHOD FOR PREPARATION OF FECAL DNA SUITABLE FOR PCR
A METHOD FOR PREPARATION OF FECAL DNA SUITABLE FOR PCR
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DOI：
10.1093/nar/23.18.3800
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发表时间：
1995-09-25
影响因子：
14.9
通讯作者：
MULLER, O
中科院分区：
文献类型：
--
作者：
DEUTER, R;PEITSCH, S;MULLER, O
The analysis of nuclear DNA extracted from fecal specimens is an important tool indifferentareasofmoleculargeneticresearch reaching from cancer diagnostics (1-3) to population genetic studies (4). Here we describe a short and simple method for the purification of chromosomal DNA that is ready to be used in PCR. Without further subcloning the resulting PCR products can be useddirectly for heteroduplex analysis, sequencing or other DNA analysing methods. Very often it is difficult to obtain chromosomal DNA from feces that is suitable for further analysis. Commonly two problems are encountered. First the DNA tends to degrade during longer periods of storage, secondly the DNA is not suitable for enzymatic manipulation such as PCR. Both problems can be traced to the presence ofcopurified excremental substances like bilirubin and bile salts (5). Thereforesubstantial reduction of these substances from the purified DNA is essential (6). Several dietary materials are knowntodecrease the concentration of fecal bile acids andtobind bile acids in vitro (5, 7, 8). We tested different adsorption matrices to remove bile acids during DNA extraction: immobilized BSA, cellulose, potato starch (all from Sigma, Munich, Germany) and potato flour (Honig, Postbus 45, 1540 AA Koog a/d Zaan, Netherlands) which is mainly an insoluble mixture of cellulose, starch, lipids and salts. Human stool specimens were collected, frozen and stored at-80 C. 200 mg stool was homogenized in 600 p1 stool lysis buffer (SLP: 500 mM Tris-HCl pH 9.0, 50 mM EDTA, 10 mM NaCl). To one fourth of the homogenate, 200, l SLP containing 100 mg of the adsorption matrix was added. The suspension was mixed vigorously and centrifuged twice at 5000 and 13 000 g each for 5 min to precipitate bacteria and other debris. After digesting the clear supernatant with proteinase Kat a concentra-tion of 2.5 mg/ml the DNA was purified using a DNA spin column recommended for DNA purification from blood and tissue (Qiagen, Hilden, Germany). Column loading and washing steps were performed as described by the manufacturer. The DNA was eluted from the spin column in a final volume of 150 p1 distilled water and stored at-20 C until further use. The yield of total DNA was determined by measuring the absorption at 260 nm. Allpreparations gave similar yields of 15-20 jig DNA. At first sight the use of different adsorption materials appeared to havenoinfluence on the yield or the quality of the extracted DNA asjudged by agarose gel electrophoresis (Fig. 1). However after storing theDNA at-20 C all the preparations showed degrada-
