Extraction of Human DNA from Soil: Protocol Adaptations

Wera M Schmerer

Department of Biology, Chemistry and Forensic Science, School of Lifescience,
University of Wolverhampton, Wolverhampton, UK

<EMAIL_ADDRESS>
## Key words

DNA extraction, soil samples, humic acids, PCR inhibition, protocol
adaptation, human identification

## Abstract

PCR-based analysis of DNA is utilized in a wide variety of fields, including
Forensic Science. Aside from the more common ample sources, material analyzed
here can refer to specimen excavated from a soil environment, or a sampling of
the soil itself to recover DNA leached into the soil from decomposing human
remains or from body fluids intermingled with the soil in an outdoor crime
scene.

The common problematic of these types of sample is the presence of humic
acids, which are a component of any soil environment, and when the co-
extracted with the DNA, lead to inhibition of enzyme-based procedures
including PCR.

While a variety of methods exist for the extraction of DNA from excavated
skeletal remains, protocols for extraction of DNA from the soil directly are
usually targeting soil microorganism. To address the need for methodology
suitable for extraction of human DNA from soil, a selection of three published
protocols were adapted for this purpose, to be tested and evaluated using
standardized samples. The resulting protocols are presented here.

## 1 Introduction

PCR-based analysis of Deoxyribonucleic Acid (DNA) is common methodology in a
wide variety of fields, including Forensic Science. Here, the approach is
predominantly utilized to identify individuals or trace biological samples
back to their source individual. In tis effort, a wide variety of sample
material is analyzed, including soil samples with traces of human DNA,
respectively DNA-containing body fluids.

During the decomposition process, DNA is leaching into the ground from the
decomposing body (Emmonds 2015, Thomas et al 2019). Crime scenes in an outdoor
setting may include body fluids such as blood intermingled with soil as
evidence (Shahzad et al 2009, Batu-Boateng et al 2018). In either case, DNA
can survive in the soil environment, even as free extracellular DNA bound to
soil minerals (Willerslev et al 2003).

####

Samples of soil, or any material recovered from soil, such as excavated
skeletonized human remains, will inevitably be affected by the presence of
humic substances (humic acids), which are potent enzyme inhibitors (Pääbo et
al 1989, Kraeder 1996, Wilson 1997, Aleddini 2012) and may co-extract with the
DNA during extraction procedures (e.g. Hänni et al 1995). Where such occurs,
enzyme dependent analyses of DNA will be inversely affected due to the
inhibitory effect of humic acid (e.g. Pääbo et al 1989, Primorac et al 1996,
Kraeder 1996).

####

Skeletal remains in a (soil) burial, will be subject to physico-chemical
interaction and exchange with the soil environment over the burial interval
(Herrmann & Nesley 1982, Pate et al 1989), one effect of which is the influx
of humic substances from the soil into the bone tissue. That such occurs even
after a short period in a soil environment, can be shown by histological
analysis of the bone tissue (Schmerer 1999, 2000). The speed of this influx,
and thus the extend at any given point in time, may differ between soil
environments, dependent on their composition and characteristics (Schmerer
1999, 2000), but it will occur in each case, since humic substances are an
essential component of the organic matter portion of any soil (e.g. Schnitzer
1978, Tan 2014, Ukalska-Jaruga et al 2021).

In consequence, the presence of humic acids and co-extraction of these during
DNA extraction procedures resulting in inhibition of analyses is a generalized
problematic where samples of soil or matter recovered from soil is analyzed by
PCR-based methodology. This emphasizes the need for optimized protocols to
minimize the co-extraction of humic acids with the DNA.

####

To this point, there is a lack of published protocols optimized for the
extraction of human DNA having leached into the soil or is contained in body
fluids mixed with soil (Howarth et al 2022). The majority of existing
protocols are designed for extraction of DNA from the bacterial community in
the soil (e.g. Tsai & Olson 1991) or optimized for extraction of DNA contained
in skeletal remains recovered from soil burials (e.g. Hänni et al 1990, 1995,
Schmerer et al 1999, Schmerer 2003, 2021a), and thus optimized for a different
species of sample material, with its sample specific requirements.

Experimental experience shows, that a protocol can successfully be adapted to
be utilized for extraction of DNA for a variety of source materials, provided
sample-specific requirements are appreciated (Schmerer 2021a, 2021b). Such
usually entails adaptation of the sample preparation procedures prior to the
actual extraction process to the specific requirements of a given sample
material (see e.g. Walsh et al 1991, Schmerer 2022).

####

The aim of the study here is to develop and evaluate modified versions of
published protocols, which were adapted for the extraction of human DNA from
soil samples. Presented here are the outcomes of the protocol adaptation.

## 2 Adaptation of published protocols

Included in the adaptation process were three protocols, each of which
represents one of the major approaches to DNA extraction; organic solvent-
(phenol-chloroform), resin- (Chelex 100) and silica matrix-based procedures.

Based on established procedures for similar applications (Willerslev et al
2003), protocols were adapted to process equal amounts of 0.25g of soil. With
reference to the findings by Howarth et al. (2022), steps to retrieve the DNA
from the soil matrix were carried out uing phosphate buffered saline (PBS) to
stabilize the DNA, instead of the commonly used EDTA solution (see protocols
II & III).

### 2.1 Organic solvent (phenol/chloroform) based extraction – Protocol I

As representative for the organic extraction approach Tsai & Olson (1991) was
selected. This widely cited protocol was designed for direct extraction of
microorganism DNA from soil. Consequently, a number of steps involved in this
procedure are specific to retrieving microorganism despite their interaction
with the soil particles and lysis of a rather tough cell wall.

Sample preparation in original procedure (Tsai & Olson 1991) consist of the
soil sample being subjected to two washes in phosphate buffer, followed by
lysis of the pelleted material in the presence of NaCl, EDTA and lysozyme and
three freeze-thaw cycles following addition of a NaCl, Tris-HCl, SDS mixture
to release the DNA from the cell.

####

Where the extraction of DNA leached into the soil or body fluids intermingled
with soil is intended, the initial wash steps here would result in washing
out, and discarding, at least a portion of the target DNA and was thus
abandoned, as was the use of freeze-thaw cycles, which are redundant in case
of comparatively easily lyzable animal cell walls. These step were replaced by
direct lysis of the soil sample n the presence of a lysis buffer consisting of
NaCl, Tris-HCl, EDTA and SDS in the presence of proteinase K.

####

The extraction part of the Tsai & Olson (1991) protocol continues with
successive extractions using phenol, followed by a chloroform/isoamyl mixture
(24:1) and then pure chloroform.

In the adapted protocol, this was replaced by two successive extractions using
a phenol/chloroform/ isoamyl mixture (24:24:1), followed by chloroform (100%),
replacing the originally used reagents with updated formulations.

####

To purify the resulting DNA, Tsai & Olson (1991) use a precipitation in
isopropanol, vacuum drying, followed by re-suspension in TE buffer, RNase
treatment and additional column-based purification. For the purpose of
analyzing human DNA, RNase treatments are not required, which then likewise
removes the need for a column-based removal of associated reactants and
products. Instead, the adapted protocol utilizes a precipitation in cold
isopropanol in the presence of silica solution, followed by air-drying of the
pellet and re-suspension in PCR-grade water.

### Protocol I (phenol/chloroform approach):

Adaptation of Tsai & Olson (1991) to extraction of Human DNA from soil

####

Sample Preparation:

  1. 1.

0.25g soil is sampled and placed in a 2ml reaction tube (SafeSeal, Sarstedt),

  2. 2.

mixed with 500µl lysis buffer (0.15M NaCl, 10% sodium dodecyl sulfate (SDS),
0.1M Na2EDTA [pH 8.0])

  3. 3.

20µl proteinase K (10mg/ml) are added, followed by thorough mixing

  4. 4.

The lid of the 2ml reaction tube should be sealed using a thin strip of
Parafilm (Bemis Corp.)

  5. 5.

For cell lysis, samples are incubated at 56°C for 2h under constant agitation
(e.g. shaking waterbath)

Phenol/chloroform extraction:

  1. 1.

An equal volume (520µl) of phenol mixture (phenol:chloroform:isoamylalcohol in
the ratio 24:24:1) is added

  2. 2.

The mixture should be briefly vortex to obtain an emulsion, followed by

  3. 3.

centrifugation at 6000rpm (ca. 3000 x g, Micro Centaur, MSE), for 10min to
reach phase separation

  4. 4.

The aquatic phase is carefully transferred to a new 2ml tube

  5. 5.

Followed by a repetition of the phenol step by adding an equal volume of
phenol mixture (steps 1.-4.)

  6. 6.

An equal volume (520µl) of chloroform (100%) is added,

  7. 7.

proceeding as previously with the phenol mixture by repeating steps 2.-4.

DNA purification:

  1. 1.

For precipitation of the DNA, an equal volume (520µl) of cold isopropanol
(abs.) and 5µl of silica solution (Ancient DNA Glasmilk™, MP Biomedicals) are
added

  2. 2.

The mixture is incubated at -20°C (freezer) for 1h, followed by centrifugation
at 6000rpm (ca. 3000 x g), for 10min

  3. 3.

The supernatant is decanted

  4. 4.

The pellet is air-dried,

  5. 5.

and re-suspended in 100µl PCR-grade water (UltraPure™, Invitrogen)

  6. 6.

Extract is transferred to a new 2ml tube

####

A more detailed version of the protocol with additional procedural instruction
can be seen in Fig 1

####

Fig 1: Protocol I - Phenol/chloroform-based protocol (Tsai & Olson 1991),
adapted for extraction of human DNA from soil (Schmerer 2024)

###  2.2 Resin (Chelex 100) based extraction – Protocol II

The resin-based approach to DNA extraction is represented by the Chelex-100
based protocol by Schmerer (2021a). This protocol is already optimized for
limiting the co-extraction of humic acids, but was originally developed with
focus on the extraction from historical skeletal remains excavated from
burials in a soil environment. Subsequent tests on a variety of sample
materials showed that with material-specific modification of the sample
preparation steps, this protocol can be successfully applied to a wide variety
of sample materials aside from the one it was designed for, including material
containing high concentrations of humic acids (Schmerer 2021a, b).

####

Sample preparation in original procedure (Schmerer 2021a) is specific to
extraction from bone samples and consists of decontamination, pulverization,
decalcification for several days in 0.5M EDTA solution, and lysis of the
supernatant in the presence of proteinase K for 60-90min depending on the
preservation of the sample.

####

To adapt this protocol to the extraction of human DNA from soil, bone matrix-
specific reagents and procedures were replaced with a soil matrix specific
sample preparation, the focus of which is to physically retrieve the DNA,
respectively DNA-containing cells from the soil matrix, while stabilizing the
DNA to prevent degradation during processing.

For this purpose, an extended incubation in phosphate buffered saline (1x PBS)
is utilized. Following centrifugation, the supernatant is subjected to the
remainder of the procedure.

####

Since the retrieval step here utilizes a significantly smaller volume compared
to the decalcification step in the original protocol, all volumes for the
subsequent steps were adjusted accordingly, which allows for use of 2ml
reaction tubes throughout. Aside from the reduction in volume, the adapted
protocol follows the original procedure for extraction and purification of
DNA.

### Protocol II (Chelex 100 approach):

Adaptation of Schmerer (2021a) to extraction of Human DNA from soil paragraph

Sample Preparation:

  1. 1.

0.25g soil is sampled and placed in a 2ml reaction tube (SafeSeal, Sarstedt),

  2. 2.

mixed with 500µl PBS (1X PBS: 137mM sodium chloride, 2.7mM potassium chloride,
and 10mM phosphate buffer)

  3. 3.

The lid of the 2ml reaction tube should be sealed using a thin strip of
Parafilm (Bemis Corp.)

  4. 4.

Sample is incubated at 40°C for 4h under constant agitation (e.g. shaking
waterbath)

  5. 5.

Soil is pelleted by centrifugation at 6000rpm (3000 x g, Micro Centaur, MSE)
for 5min

  6. 6.

Supernatant is transferred to a new 2ml reaction tube

Chelex-based extraction:

  1. 1.

The supernatant from the previous step is mixed with 500µl Chelex-100 solution
(5% (w/v) in PCR-grade water (UltraPure™, Invitrogen),

  2. 2.

20µl proteinase K (10mg/ml) are added, followed by thorough mixing

  3. 3.

The lid of the 2ml reaction tube should be sealed using a thin strip of
Parafilm (Bemis Corp.)

  4. 4.

Samples are incubated at 56°C for 1h under constant agitation (e.g. shaking
waterbath),

  5. 5.

then incubated at 95°C for 8min (e.g. boiling water bath)

  6. 6.

Samples are left to cool to RT

  7. 7.

Chelex beads are pelleted by centrifugation at 4000rpm (ca. 1300 x g), 6min

  8. 8.

The aqueous supernatant is carefully transferred to a new 2ml tube

DNA purification:

  1. 1.

For precipitation of the DNA, an equal volume (1ml) of Isopropanol (abs., RT),
20µl sodium acetate buffer (2M, pH 4.5) and 5µl of silica solution (Ancient
DNA Glasmilk™, MP Biomedicals) are added,

  2. 2.

followed by incubation at RT for 30min for precipitation of the DNA,

  3. 3.

and centrifugation at 4000rpm (ca. 1300 x g), for 5min

  4. 4.

The supernatant is decanted

  5. 5.

The pellet is washed in 500µl ethanol (abs.)

  6. 6.

air-dried, and resuspend in 100µl PCR-grade water (UltraPure™, Invitrogen)

  7. 7.

The resulting extract is transferred to a new 2ml tube

####

A more detailed version of the protocol with additional procedural instruction
can be seen in Fig 2

####

Fig 2: Protocol II – Chelex-based protocol (Schmerer2021a), adapted for
extraction of human DNA from soil (Schmerer 2024)

### 2.3 Silica column based extraction – Protocol III

The method selected for the silica-based extraction (Schmerer 2022) is an
approach for a streamlined parallel extraction of DNA from a variety of source
materials, where individually timed, sample specific preparations are followed
by using one common DNA extraction procedure. This approach in itself
demonstrates that by utilizing a material-specific sample preparation step, a
protocol may be adapted to be suitable for a variety of different sample
sources.

####

To adapt this protocol to the extraction of DNA from human soil, the material-
specific sample preparation utilized in the adaptation of the Chelex approach
(Schmerer 2021a, see 2.2) was combined with the “common extraction protocol”
aspect of the procedure as published, which consists of a modified version of
a silica-kit based extraction protocol (Schmerer 2022). The protocol utilized
is a modification of the manufacturer instruction for the E.Z.N.A.® Blood DNA
Mini Kit using the ”Buccal Swabs Protocol” (Omega Bio-Tek 2019); without
addition of PBS to the sample, an extended proteinase K digest (30min instead
of 10min), elution in the minimum volume of pre-heated PCR-grade water instead
of using Elution Buffer, and with reapplication of the eluate to the silica
column for a second elution, instead of using a further aliquot of water.

A pre-digestion step, as featured in the protocol overview (see Fig 3), can be
included but is not essential.

### Protocol III (Silica kit based approach):

Adaptation of Schmerer (2022) to extraction of Human DNA from soil paragraph

Sample Preparation:

  1. 1.

0.25g soil is sampled and placed in a 2ml reaction tube (SafeSeal, Sarstedt),

  2. 2.

mixed with 500µl PBS (1X PBS: 137mM sodium chloride, 2.7mM potassium chloride,
and 10mM phosphate buffer)

  3. 3.

The lid of the 2ml reaction tube should be sealed using a thin strip of
Parafilm (Bemis Corp.)

  4. 4.

Sample is incubated at 40°C for 4h under constant agitation (e.g. shaking
waterbath)

  5. 5.

Soil is pelleted by centrifugation at 6000rpm (3000 x g, Micro Centaur, MSE)
for 5min

  6. 6.

Supernatant is transferred to a new 2ml reaction tube

Pre-Digestion:

  1. 1.

After adding 20µl proteinase K (10mg/ml), and thorough mixing

  2. 2.

the sample is incubated at 56°C for 1h under constant agitation (e.g. shaking
waterbath or thermomixer)

Silica kit-based extraction:

  1. 1.

After addition of 25µl Proteinase K Solution (kit component) and 500µl BL
Buffer (kit component), the sample is thoroughly mixed

  2. 2.

Incubation at 65°C for 30min

  3. 3.

The proteinase K digest is terminated by addition of 100µl of Ethanol (abs.)
and thorough mixing

  4. 4.

The sample can be briefly centrifuged to avoid liquid remaining in the lid of
the tube

  5. 5.

A silica column (kit component: HiBind® DNA Mini Column) is placed in a 2ml
collection tube (kit component)

  6. 6.

750µl of the sample is transferred to the column

  7. 7.

After centrifugation at 10,000 x g for 1min, the filtrate is discarded

  8. 8.

Steps 6 and 7 are repeated until the remainder of the sample has been
transferred completely, after which the column is placed into a new collection
tube

  9. 9.

Following addition of 500µl HBC Buffer (kit component), the column is
centrifuged at 10,000 x g for 1min and the filtrate is discarded

  10. 10.

After adding 700µl DNA Wash Buffer (kit component), the column is centrifuged
at 10,000 x g for 1min and the filtrate is discarded

  11. 11.

The wash step is repeated with a second aliquot of 700µll DNA Wash Buffer

  12. 12.

To remove any residual ethanol, the column matrix is dried by further
centrifugation at maximum speed (at least 10,000 x g) for 2min, after which
the column is transferred to a 2ml (SafeSeal, Sarsted)

  13. 13.

To elute the DNA from the silica matrix, 100µl PCR-grade water (UltraPure™,
Invitrogen), pre-heated to 65°C, are added to the column, and incubated for
5min at RT, followed by centrifugation at 10,000 x g for 1min

  14. 14.

The elution step is repeated by re-applying the eluate to the column, followed
by incubation and centrifugation as described in step 13, to elute any DNA
remaining bound to the silica matrix after the first elution

Fig 3: Protocol III – Silica column-based protocol (Schmerer 2022), adapted
for extraction of human DNA from soil (Schmerer 2024)

## 3\. Evaluation of adapted protocols

To test and evaluate the adapted protocols, the study utilizes standardized
samples consisting of 0.25g homogenized soil mixed with identical amounts of a
well-mixed saliva sample. Saliva is used as DNA source to avoid adding further
inhibitory substances, as present in other DNA-containing body fluid such as
blood (Wilson 1997).

Soil blanks are included to determine amounts of background DNA from the soil
microorganisms. With this type of control in place, DNA yield can be
determined by UV spectrophotometry, which likewise allows for quantification
of the concentration of humic acids to monitor the level of co-extraction of
this inhibitor. Since humic acids do not have a distinctive absorbtion band,
but show increased UV absorbtion at the lover wavelenths (Kumada 1955),
measurements are taken at 320nm and 360nm, to avoid the absorbtion ranges of
most proteins.

Quality of the extracted DNA is evaluated by fragmentation assay (agarose gel
electrophoresis of extracts) and PCR-based amplification of common STR loci
(see Schmerer et al. 1999). The latter approach likewise allows for relative
quantification of amplifyable DNA.

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