Method for recovering DNA from soil

A method of recovery of DNA of a microorganism in soil is provided which comprises addition of nucleic acid to a liquid suspension containing the soil and the microorganism before lysis of the microorganism and recovery of the desired DNA.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method for recovering DNA of a 
microorganism in soil. 
2. Related Background Art 
A great variety of microorganisms exist in soil, and for most of them, even 
isolation can not be accomplished and culture conditions are not known. 
Identification of such microorganisms in soil, and investigation of the 
population thereof involve great difficulty, which hinders research of 
their microbiological ecology. Hitherto, studies on microbiological 
ecology have been made by improving methods of isolation and culture 
conditions. Although many ecologically important microorganisms are 
thought to exist in the soil, it is said that only a small number, 
probably 0.1% of the microorganism in soils can be isolated and cultivated 
by known methods. It is highly important to grasp the ecology of 
microorganisms which are hard to isolate and culture, not only for the 
progress of science but also for development of applied technology such as 
waste water treatment and environmental cleanup. 
In recent years, environmental pollution caused by hydrocarbons such as 
aromatic hydrocarbons, paraffins, and naphthenes, or organic chlorine 
compounds such as trichloroethylene and perchloroethylene has become a 
serious problem. Strongly desired are technologies for prevention of 
further expansion of such serious environmental pollution and for cleaning 
and recovering the environment. Conventional techniques for soil 
environment remediation includes physicochemical treatments, e.g., 
aeration, sun bleaching, vacuum vessel treatment, vacuum extraction, etc. 
However, these physicochemical treatments are not satisfactory due to 
their operation cost, operability, energy consumption, treatment capacity, 
their inability to decompose hardly-decomposing substances, and so forth. 
Therefore, much hope is laid on environment remediation techniques using 
microorganisms. 
There are a number of known microorganisms which can decompose the 
soil-polluting, hardly decomposable compounds such as aromatic 
hydrocarbons and organic chlorine compounds. Such microorganisms have been 
practically applied in polluted soil to decompose polluting substances. 
Further, field application of a recombinant microorganism of improved 
decomposition ability has been also studied. To generalize and fix the 
microbiological technology in the field of soil remediation or of 
agricultural production as a useful technology, it is highly important to 
grasp growth and survival of the microorganism in the environment to which 
it is applied, as well as to develop useful microorganisms. 
Conventionally, the growth and survival of a recombinant microorganism in 
the environment has been studied by estimating the number of viable cells 
into which a marker gene of antibiotic-resistance, pigment-productivity, 
etc. was introduced. This technique involves drawbacks such as falling-off 
of the marker gene, mutation, measurement limit of viable cell count and 
so forth. It is also pointed out that field application of an 
antibiotic-resistant microorganism involves epidemiological problems. 
Furthermore, few methods have been established to grasp the ecology of 
hard-to-isolate and hard-to-cultivate microorganisms. With recent 
development of molecular biology, DNA detection technique has become 
available for detection of recombinants or hard-to-isolate and 
hard-to-cultivate microorganisms. 
When DNA is used as a detection means, total DNA including the DNA of the 
intended microorganism should be recovered from the environmental sample. 
Two methods are known for recovering the microorganism-derived DNA from 
soil: Cell recovery method and Direct cell-lysis method. 
In the cell recovery method, microbial cells are collected from the soil 
and then DNA is isolated from the collected cells. In the direct 
cell-lysis method, microbial cells are lysed directly in the soil sample 
and then DNA is recovered. 
The cell recovery method has disadvantages that the recovery rate of the 
microorganism in soil greatly varies depending on the soil, e.g., about 
40% from one soil and about 10% from another soil, and that the amount of 
the recovered DNA is as small as from 1 .mu.g to 100 .mu.g per 100 g of 
soil. However, this cell recovery method has advantages in that the origin 
of the recovered DNA is authentic and that purity of DNA is high. 
The direct cell-lysis method has a remarkable advantage in that the amount 
of the recovered DNA is as high as 1 to 2 mg per 100 g of soil, 10 to 100 
times that of cell-extraction method. However, disadvantageously the 
source of the recovered DNA is not clear. That is, together with the DNA 
of the intended microorganism, all sorts of non-decomposed DNA are 
recovered from dead bacteria, mold, protozoa, etc. as well as from plants. 
A further important problem common to the both methods is the adsorption of 
cell-derived DNA to soil particles or soil organic substances during the 
recovery process. From soil of a low cell concentration, sometimes no DNA 
is recovered. 
Generally, from an nutrient-rich environment such as sediment and leaf mold 
where a huge number of microorganisms exist, a sufficient amount of DNA is 
recoverable by either of these methods, and specific microbial cells can 
be detected when the recovered DNA is suitably purified, and suitably 
detected. However, the quantitative estimation of the cells is considered 
to be biased to some extent because the adsorption of the DNA by the soil 
particles and organic substances is not estimated. The adsorption of the 
DNA is considered to affect also the detection limit. For instance, from 
the soil containing a small number of living cells, e.g., 10.sup.6 cells 
per 1 g of soil, the amount of recoverable DNA is extremely small (the 
amount of chromosomal DNA for 10.sup.6 cells being about 5 ng), and such a 
small amount of DNA may be adsorbed entirely by the soil particles and 
organic substances, resulting in no DNA recovery. 
In utilizing DNA for detection of a microorganism in soil, the technique of 
recovering DNA from microbial cells is essential, and the conventional 
methods involve disadvantages described above. 
SUMMARY OF THE INVENTION 
The present invention intends to provide a method for recovering the DNA of 
a microorganism in soil at a high recovery ratio without adsorption of the 
recovered DNA to the soil particles and soil organic compounds. The method 
of recovery of DNA of a microorganism in soil of the present invention 
comprises addition of an anionic substance to a liquid suspension 
containing soil and the microorganism. 
DESCRIPTION OF THE PREFERRED EMBODIMENT 
From the above viewpoint, the inventors of the present invention studied 
the recovery method of microbial DNA from soil, and found that anionic 
substances can prevent adsorption of microorganism-derived DNA to the soil 
particle or other substances in the sample dispersion and thereby the 
present invention has been completed. 
The present invention is described below more specifically. 
The main feature of the present invention is addition of an anionic 
substance to a sample suspension (a liquid suspension) containing soil and 
microbial cells. 
The anionic substance useful in the present invention includes inorganic 
ionic compounds such as hydrochloric acid, sulfuric acid, phosphoric acid, 
arsenic acid, nitric acid, and selenic acid; and organic ionic compounds 
such as nucleic acid. Of these ionic compounds, nucleic acid is preferred 
because of its similar physical and chemical properties to the microbial 
DNA. Since the organic ions like nucleic acid are larger in size and have 
more complicated structure than inorganic ions, the adsorption of an 
organic ion like nucleic acid onto soil particles or organic matters is 
effected by hydrogen bonding and Van der Waals bonding, and stereochemical 
properties in addition to the electrostatic bonding. Therefore, nucleic 
acid such as DNA or RNA having properties analogous to DNA, is preferred 
in effectively blocking the adsorption sites on soil particles for the 
microorganism-derived DNA. 
DNA as the anionic substance includes ordinarily used DNA such as those 
derived from calf thymus, salmon testes, herring sperm, E. coli, and so 
forth, but is not limited thereto. The RNA includes various ribosomal RNA, 
transfer RNA, etc.; more specifically including RNAs derived from baker's 
yeast, calf liver, tolura yeast, fetal calf thymus, wheat germ, bovine 
liver, E. coli, rabbit liver, brewer's yeast, and so forth, but is not 
limited thereto. 
When an ordinary method is employed for the DNA detection, RNA is 
preferably used as the anionic substance. 
The anionic substance is added preferably in an amount corresponding to the 
adsorption capacity of the sample soil to the anionic substance which can 
be preliminarily estimated. This amount depends on the characteristics of 
the sample soil, and the employed anionic substance. Therefore it is 
preferable to estimate the optimum amount preliminarily. 
If the anionic substance does not affect the detection of the recovered 
DNA, the anionic substance may be added in excess without preliminary 
estimation of the absorption capacity. 
The anionic substance in the present invention is more preferably added to 
the liquid suspension containing the microorganism and soil before the 
microbial cells are disrupted, thereby remarkable effect being achieved. 
Presumably, the anionic substance added to the sample suspension prior to 
disruption of the microorganism is adsorbed by the soil particles and 
other adsorbent, thus prevents the adsorption of the DNA of the 
microorganism released by cell disruption. 
The disruption of the microbial cells may be conducted by any known method 
including physical methods (e.g., French press, ultrasonifier, vortex 
mixer, etc.), chemical methods (e.g., surfactant), and enzymatic cell 
lysis. Such a method is combined with a known recovery method such as 
direct cell-lysis and cell recovery method to recover the DNA. The direct 
cell-lysis and the cell recovery method are described in detail in the 
report by Steffan (Steffan, R. J., J. Goksoyr, A. K. Bej, and R. M. Atlas: 
"Recovery of DNA from Soils and Sediments", Appl. Environ. Microbiol. 54, 
2908-2915 (1988)).

The present invention is described below in more detail without limiting 
the invention thereby. 
EXAMPLE 1 
(A) Measurement of anionic substance adsorption capacity of sample soil 
Aliquots of 0.5 gram of loam soil sterilized by autoclaving were placed in 
2 ml-Eppendorf tubes. To each tube, a certain amount of DNA suspended in 1 
ml of TE buffer solution (pH: 8.0) was added (Table 1)(salmon testes, 
Sigma Co.). After mixing by a vortex mixer, 0.1 ml of 10% SDS (sodium 
dodecylsulfate) was added thereto and vortexed to mix again. The mixture, 
after standing at 70.degree. C. for one hour, was centrifuged at 15,000 
rpm at 4.degree. C. for 10 minutes in a microcentrifuge. The supernatant 
was collected and thereto 0.25 ml of 7.5M ammonium acetate solution was 
added. After standing at room temperature for 5 minutes, the mixture was 
further centrifuged under the above conditions. The supernatant was 
collected. Thereto 0.8 ml of isopropanol was added with sufficient 
agitation, and left standing for 10 minutes at room temperature. The 
mixture was centrifuged at 15,000 rpm at 15.degree. C. for 10 minutes. The 
obtained pellet was air-dried. Thereto 50 .mu.l of TE buffer solution (pH: 
8.0) was added to dissolve the DNA. 
An aliquot of 10 .mu.l of the above DNA solution was subjected to agarose 
gel electrophoresis. The amount of recovered DNA was estimated by 
comparison with the agarose gel electrophoresis pattern of 
HindIII-digested .lambda.-DNA. The results are shown in Table 1. 
TABLE 1 
______________________________________ 
Recovery of DNA 
Added DNA (.mu.g) 
0.5 1.0 2.0 4.0 8.0 16.0 
Recovered N.D. N.D. N.D. N.D. N.D. 1.0 
DNA (.mu.g) 
______________________________________ 
N.D. : No DNA detected ( &lt;5 ng ) 
The above result shows that the adsorption sites of the sample soil can be 
sufficiently blocked by adding 15 .mu.g of DNA or more as the anionic 
substance. 
(B) Measurement of Quantity of DNA Derived from E. coli Strain HB101 
In 2-ml Eppendorf tubes, was placed respectively a prescribed amount (Table 
2) of HB101 strain of E. coli (Takara Shuzo Co.) suspended in 1 ml of 0.1M 
phosphate buffer solution (pH: 8.0). After mixing by a vortex mixer, 0.1 
ml of 10% SDS (sodium dodecylsulfate) was added thereto, and the mixture 
was agitated by a vortex mixer, and kept standing at 70.degree. C. for one 
hour. Then the mixture was centrifuged at 15,000 rpm at 4.degree. C. for 
10 minutes in a microcentrifuge. The supernatant was collected, to which 
0.25 ml of 7.5 M ammonium acetate solution was added. The mixture was left 
standing at room temperature for 5 minutes, and then centrifuged under the 
above conditions. To the separated supernatant, 0.8 ml of isopropanol was 
added. The mixture was mixed sufficiently, and left standing at room 
temperature for 10 minutes, and centrifuged at 15,000 rpm at 15.degree. C. 
for 10 minutes in an microcentrifuge. The pellet was recovered and 
air-dried. Thereto 50 .mu.l of TE buffer solution (pH: 8.0) was added to 
dissolve the DNA. 
A 10 .mu.l aliquot of the above prepared DNA solution was subjected to 
agarose electrophoresis, and the amount of recovered DNA was measured. The 
results are shown in Table 2, from which the amount of DNA in each sample 
can be estimated. 
(C) Recovery and Detection of DNA of E. coli Injected to Sample Soil (1): 
Of loam soil sterilized by autoclaving, 0.5 gram aliquots were placed in 2 
ml-Eppendorf tubes respectively. To each tube, was added a prescribed 
amount (Table 2) of E. coli strain HB101 (product of Takara Shuzo Co.) 
suspended in 1 ml of 0.1M phosphate buffer solution (pH: 8.0). Further 
thereto, 50 .mu.l (25 mg RNA) of a solution of RNA (produced by Sigma Co., 
derived from bakers yeast, 500 mg/ml) was added to each tube as the 
anionic substance of the present invention and mixed by a vortex mixer. 
0.1 ml of 10% SDS (sodium dodecylsulfate) was added thereto, and vortexed. 
The mixture, after standing at 70.degree. C. for one hour, was centrifuged 
at 15,000 rpm at 4.degree. C. for 10 minutes in a microcentrifuge. The 
supernatant was collected and thereto 0.25 ml of 7.5M ammonium acetate 
solution was added. After standing at room temperature for 5 minutes, the 
mixture was centrifuged under the above conditions. The supernatant was 
collected and thereto 0.8 ml of isopropanol was added with sufficient 
agitation, and was left standing for 10 minutes at room temperature. The 
mixture was centrifuged at 15,000 rpm at 15.degree. C. for 10 minutes. The 
obtained pellet was air-dried. Thereto 50 .mu.l of TE buffer solution (pH: 
8.0) was added to dissolve the DNA. 
A 10 .mu.l aliquot of the above DNA solution was subjected to agarose gel 
electrophoresis. The amount of the recovered DNA was estimated in the same 
manner as in the above item (A). The results are shown in Table 2. 
The results are almost the same as the results obtained in the above item 
(B), and it was shown that DNA can be nearly completely recovered from 
even a small number of E. coli cells according to the method of the 
present invention. 
COMATIVE EXAMPLE 1 
Recovery and Detection of DNA of E. coli Injected to Sample Soil (2) 
0.5 Gram aliquots of loam soil sterilized by autoclaving were placed in 2 
ml-Eppendorf tubes respectively. To each tube, was added a prescribed 
amount (Table 2) of HB101 strain of E. coli (product of Takara Shuzo Co.) 
suspended in 1 ml of 0.1 M phosphate buffer solution (pH: 8.0). After 
mixing it by a vortex mixer, 0.1 ml of 10% SDS (sodium dodecylsulfate) was 
added thereto, and vortexed. The mixture, after kept standing at 
70.degree. C. for one hour, was centrifuged at 15,000 rpm at 4.degree. C. 
for 10 minutes in a microcentrifuge. The supernatant was collected and 
thereto 0.25 ml of 7.5M ammonium acetate solution was added. After 
standing at room temperature for 5 minutes, the mixture was centrifuged 
under the above conditions. The supernatant was collected and thereto 0.8 
ml of isopropanol was added with sufficient agitation, and was left 
standing for 10 minutes at room temperature. The mixture was centrifuged 
at 15,000 rpm at 15.degree. C. for 10 minutes. The obtained pellet was 
air-dried. Thereto 50 .mu.l of TE buffer solution (pH: 8.0) was added to 
dissolve the DNA. 
An 10 .mu.l aliquot of the above DNA solution was subjected to agarose gel 
electrophoresis. The amount of the recovered DNA was estimated in the same 
manner as in the above item (A) of Example 1. The results are shown in 
Table 2. 
E. coli-derived DNA could be recovered from the sample soils only when the 
sample contained a large amount of E. coli as much as 10.sup.9 /ml. 
EXAMPLE 2 AND COMATIVE EXAMPLE 1 
PCR Detection of E. coli DNA Added to Sample Soil 
It was used DNA solutions prepared in Example 1 (B), Example 1 (C), and 
Comparative Example 1 and also DNA solutions prepared in the same manner 
as in Example 1 (B), Example 1 (C), and Comparative Example 1 except that 
the bacterial cell concentrations were reduced to 10.sup.3, 10.sup.4, and 
10.sup.5 cells/ml. The DNA derived from E. coli was detected in these 
samples by PCR method employing a gene encoding 16S ribosomal RNA of E. 
coli. The employed primers were the two shown below: 
Primer #1: 
5'-AAGGGAGTAAAGTTAATACCTTTG-3'SEQ ID No: 1 
Primer #2: 
5'-GGCACATTCTCATCTCTGAAA-3'SEQ ID No: 2 With these two primers, an 
amplification product of about 0.6 Kb can be obtained. 
PCR was conducted with the total volume of 50 .mu.l in a 0.5 ml-Eppendorf 
tube. The reaction mixture was overlaid with mineral oil. The thermal 
cycler employed was Model PJ-1000 manufactured by Perkin Elmer Cetus Co. 
The Taq DNA polymerase was AmpliTaq DNA polymerase supplied by Takara 
Shuzo Co. The buffer solution was sold with the enzyme in a set, and used 
in the concentration according to the supplier's manual. each dNTP was 
used at a concentration of 200 .mu.M, and the above two primers were used 
at a concentration of 0.1 .mu.M respectively. An 1 .mu.l aliquot of the 
above-prepared DNA solution was added to the above reaction mixture, and 
further thereto 1.0 unit of Taq DNA polymerase was added, and the mixture 
was subjected to PCR for 30 cycles of 90.degree. C. for one minute, 
55.degree. C. for one minute, and 72.degree. C. for one minute in one 
cycle. The sample after the PCR was subjected to agarose gel 
electrophoresis to confirm the presence of the PCR amplification product. 
The results are shown in Table 2. 
In Example 2, DNA from the samples with or without soil could be detected 
for the bacterial cell concentration of as low as 10.sup.3 cells/ml. On 
the contrary, in Comparative Example 2, the DNA could not be detected for 
the samples of the bacterial concentrations lower than 10.sup.7 cells/ml. 
From the above results, according to the present invention, bacterial cells 
can be detected satisfactorily at a cell number of as low as 10.sup.3 
cells/ml by combination of PCR amplification method. 
TABLE 2 
__________________________________________________________________________ 
Concentration of E. coli (cells/ml) 
0 10.sup.3 
10.sup.4 
10.sup.5 
10.sup.6 
10.sup.7 
10.sup.8 
10.sup.9 
__________________________________________________________________________ 
Example 1 (B) 
Recovered DNA (.mu.g) 
x -- 
-- 
-- 
0.005 
0.05 
0.4 
3.5 
Example 2 (B) 
PCR amplification 
x o o o o o o o 
product 
Example 1 (C) 
Recovered DNA (.mu.g) 
x -- 
-- 
-- 
0.005 
0.05 
0.35 
3.6 
Example 2 (C) 
PCR amplification 
x o o o o o o o 
product 
Comparative 
Recovered DNA (.mu.g) 
x -- 
-- 
-- 
x x x 3.2 
Example 1 
Comparative 
PCR amplification 
x x x x x o o o 
Example 2 
product 
__________________________________________________________________________ 
Example 2 (B): cells only, Example 2 (C) cells + soil 
Agarose electrophoresis method x: No DNA detected (&lt;5 ng) PCR method o: 
Amplification product found; x: No amplification product found 
EXAMPLE 3 
Recovery and Detection of DNA Derived from E. coli Injected to Soil Sample 
(3) 
0.5 Gram aliquots of loam soil sterilized by autoclaving were placed in 2 
ml-Eppendorf tubes respectively. To each tube, was added a prescribed 
amount (Table 3) of HB101 strain of E. coli (product of Takara Shuzo Co.) 
suspended in 1 ml of 0.1 M phosphate buffer solution (pH: 8.0). For a 
control, a sample containing no E. coli was also prepared. Further 
thereto, 50 .mu.l (DNA: 15 .mu.g) of a solution of DNA (Salmon testes DNA, 
product of Sigma Co., 300 .mu.g/ml) was added and mixed by a vortex mixer. 
Thereto 0.1 ml of 10% SDS (sodium dodecylsulfate) was added, and vortexed. 
The mixture, after kept standing at 70.degree. C. for one hour, was 
centrifuged at 15,000 rpm at 4.degree. C. for 10 minutes in a 
microcentrifuge. The supernatant was collected, and thereto 0.25 ml of 
7.5M ammonium acetate solution was added. After standing at room 
temperature for 5 minutes, the mixture was further centrifuged under the 
above conditions. The supernatant was collected and thereto 0.8 ml of 
isopropanol was added with sufficient agitation, and was left standing for 
10 minutes at room temperature. The mixture was centrifuged at 15,000 rpm 
at 15.degree. C. for 10 minutes. The obtained pellet was air-dried. 
Thereto 50 .mu.l of TE buffer solution (pH: 8.0) was added to dissolve the 
DNA. (A) A 10 .mu.l aliquot of the above DNA solution was subjected to 
agarose gel electrophoresis. The amount of the recovered DNA was estimated 
in the same manner as in Example 1 (A). The results are shown in Table 3. 
In this Example, DNA was added as the anionic substance and the excess DNA 
was also recovered, which made the recovery of DNA larger. (B) The 
presence of DNA derived from E. coli was confirmed by PCR method employing 
a gene encoding the 16S ribosomal RNA of E. coli. The employed primers 
were the two shown below: 
Primer #1: 
5'-AAGGGAGTAAAGTTAATACCTTTG-3'SEQ ID No: 1 
Primer #2: 
5'-GGCACATTCTCATCTCTGAAA-3'SEQ ID No: 2 With these two primers, an 
amplification product of about 0.6 Kb can be obtained. 
PCR was conducted with the total volume of 50 .mu.l in a 0.5 ml-Eppendorf 
tube. The reaction mixture was overlaid with mineral oil. The thermal 
cycler employed was Model PJ-1000 manufactured by Perkin Elmer Cetus Co. 
The Taq DNA polymerase was AmpliTaq DNA polymerase supplied by Takara 
Shuzo Co. The buffer solution was sold with the enzyme in a set, and used 
in the concentration according to the supplier's manual. Each dNTP was 
used at a concentration of 200 .mu.M, and the above two primers were used 
at a concentration of 0.1 .mu.M respectively. An 1 .mu.l aliquot of the 
above-prepared DNA solution was added to the above reaction mixture, and 
further thereto 1.0 unit of Taq DNA polymerase was added, and the mixture 
was applied to PCR for 30 cycles of 90.degree. C. for one minute, 
55.degree. C. for one minute, and 72.degree. C. for one minute in one 
cycle. The sample after the PCR was subjected to agarose gel 
electrophoresis to confirm the presence of the PCR amplification product. 
The results are shown in Table 3. 
From the results, it was confirmed that DNA from a small number of E. coli 
can be detected using PCR to detect E. coli-specific DNA, even when DNA is 
used as the anionic substance. 
TABLE 3 
______________________________________ 
Concentration of 
E.coli cells/ml 
0 10.sup.6 
10.sup.7 
10.sup.8 
10.sup.9 
______________________________________ 
Example Recovered 1.0 1.0 1.1 2.4 &gt;5 
3 (A) DNA (.mu.g) 
Example PCR amplifi- 
x .smallcircle. 
.smallcircle. 
.smallcircle. 
.smallcircle. 
3 (B) cation product 
______________________________________ 
Amplification product - .smallcircle. : detected, x : not detected 
EXAMPLE 4 
Recovery of DNA from Actual Soil Sample (1) 
Samples (0.5 gram) of various soil were placed in 2 ml-Eppendorf tubes. To 
each tube, 1 ml of 0.1M phosphate buffer solution (pH: 8.0) was added. 
Further thereto, 50 .mu.l of solution (500 mg RNA/ml) of RNA (Baker's 
yeast RNA, Sigma Co.) was added, and the mixture was agitated by a vortex 
mixer. Thereto 0.1 ml of 10% SDS (sodium dodecylsulfate) was added, and 
vortexed. The mixture, after kept standing at 70.degree. C. for one hour, 
was centrifuged at 15,000 rpm at 4.degree. C. for 10 minutes in a 
microcentrifuge. The supernatant was collected, and thereto 0.25 ml of 
7.5M ammonium acetate solution was added. After standing at room 
temperature for 5 minutes, the mixture was centrifuged under the above 
conditions. The supernatant was collected and thereto 0.8 ml of 
isopropanol was added with sufficient agitation, and the mixture was left 
standing for 10 minutes at room temperature. It was centrifuged at 15,000 
rpm at 15.degree. C. for 10 minutes. The obtained pellet was air-dried. 
Thereto 50 .mu.l of TE buffer solution (pH: 8.0) was added to make a DNA 
solution. 
A 10 .mu.l aliquot of the above DNA solution was subjected to agarose gel 
electrophoresis. The amount of the recovered DNA was estimated in the same 
manner as in Example 1 (A). DNA was recovered from the samples as in Table 
4. 
COMATIVE EXAMPLE 3 
Recovery of DNA from Actual Soil Sample (2) 
0.5 Gram samples of various soil were placed in 2 ml-Eppendorf tubes. To 
each tube, 1 ml of 0.1 M phosphate buffer solution (pH: 8.0) was added, 
and the mixture was agitated by a vortex mixer. Thereto 0.1 ml of 10% SDS 
(sodium dodecylsulfate) was added, and the mixture was agitated by a 
vortex mixer. The mixture, after kept standing at 70.degree. C. for one 
hour, was centrifuged at 15,000 rpm at 4.degree. C. for 10 minutes in a 
microcentrifuge. The supernatant was collected, and thereto 0.25 ml of 
7.5M ammonium acetate solution was added. After standing at room 
temperature for 5 minutes, the mixture was further centrifuged under the 
above conditions. The supernatant was collected and thereto 0.8 ml of 
isopropanol was added with sufficient agitation, and the mixture was left 
standing for 10 minutes at room temperature. It was centrifuged at 15,000 
rpm at 15.degree. C. for 10 minutes. The obtained pellet was air-dried. 
Thereto 50 .mu.l of TE buffer solution (pH: 8.0) was added to make a DNA 
solution. 
A 10 .mu.l aliquot of the above DNA solution was subjected to agarose gel 
electrophoresis. The amount of the recovered DNA was estimated in the same 
manner as in Example 1 (A). No DNA was recovered from the samples as shown 
in Table 4. 
TABLE 4 
______________________________________ 
Recovery of DNA from actual soil sample 
Sample No. 1 2 3 4 
______________________________________ 
Example 4 Recovered 0.45 0.75 0.14 0.52 
DNA (.mu.g) 
Comparative 
Recovered x x x x 
Example 3 DNA (.mu.g) 
______________________________________ 
x : No DNA detected (&lt;5 ng) 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (Synthesized Polynucleotide) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AAGGGAGTAAAGTTAATACCTTTG24 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (Synthesized Polynucleotide) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GGCACATTCTCATCTCTGAAA21 
__________________________________________________________________________