Chapter
3
Kennewick Man Ancient DNA Analysis: Final Report Submitted to the Department
of the Interior, National Park Service
By D. Andrew Merriwether, Graciela S. Cabana, and David M. Reed
On May 18, 2000, two bone samples from the Kennewick
collection - a right eighth rib [Catalog 97.I.12d(13)] and a left third
metacarpal (MC3) [Catalog 97.L.16(Mca)] - were transferred to the laboratory
of D. Andrew Merriwether for ancient DNA analysis. Three people with
extensive ancient DNA research experience were involved in the subsequent
analysis of these samples: the principal investigator, D. Andrew Merriwether,
a doctoral student, Graciela Cabana, and a laboratory technician, John
McDonough. Ancient DNA analysis of the Kennewick samples included five
separate extractions using four different extraction procedures and
multiple DNA amplification attempts using Polymerase Chain Reaction
(PCR) on these extractions. We were unable to successfully amplify any
ancient mitochondrial DNA (mtDNA) using standard methods. We conclude
that if any DNA remains in these particular samples, it is inaccessible
via current standard methods, but may be accessible and analyzable in
the future using novel methods.
Because we did not obtain positive results in this analysis, this report
consists primarily of a detailed description of the methods used and
explanation of the results obtained. We attach as appendices (1) a log
of electrophoresis gel images documenting the results of PCRs on these
samples (Appendix 1), (2)
a photographic log of procedures described in the text of this report
(Appendix 2), and electropherograms
documenting the contaminating sequences (Appendix
3).
DNA
Analysis: Methods
Below is a general description of the methods used
for the ancient DNA analysis of these Kennewick bone samples.
Contamination
Controls
Extraordinary care has been taken to control
for exogenous human DNA contamination. Contamination in ancient DNA
work, however, is inevitable and controls must be included at every
step of the process to monitor its effects. We followed these preventative
steps:
- All ancient DNA work is conducted in a physically
separate ancient DNA room. This room is always cleaned with a 10%
bleach solution before any procedure. The room is equipped with Ultra
Violet (UV) lights to cross-link any contaminating DNA on working
surfaces.
- Ancient DNA investigators change into a clean lab
coat, and exchange their street shoes for shoe covers in an ante-room.
Gloves and face masks are put on immediately inside the ancient DNA
laboratory. Investigators do not handle any samples or reagents for
ancient DNA use without this precautionary dress.
- All reagents are either bought as molecular grade,
DNase/RNase free directly from a manufacturer, or made under stringent
conditions in the ancient DNA lab. We use UV/UF filtered H2O
taken from a physically separate laboratory that does not work with
human DNA. All reagents made in the ancient DNA laboratory as well
as the UV/UF H2O are tested for exogenous
DNA contamination regularly.
- All reagents and samples are mixed or stored in DNase-free
Sarstedt brand conical tubes, or in glass media bottles that have
been decontaminated with HCl and subsequently left under UV light
for at least half an hour.
- Controls of all extraction reagents are run with
the samples during PCR.
Decontamination
of Bone Surfaces & Bone Extraction Preparation
The outside of Kennewick bone samples were
treated to remove any potential exogenous human DNA contamination by
first scrubbing the bone with a 10% bleach solution using a sterilized
brush. All bone surfaces were then subjected to UV light for half an
hour to cross-link any superficial DNA.
Bone extraction preparation varied according the type
of bone sample. The MC3 was sampled by drilling a fine bone powder using
a Dremel tool. The Dremel bit, parts and tool were cleaned with a 10%
bleach solution and UV-irradiated prior to use. The rib was either scraped
or ground because there was little actual bone accessible with a drilling
tool, and the inside of the rib was filled with a tough soil matrix.
For the first extraction of the rib (Extraction #3, below), the rib
sample was soaked in UV/UF H2O for
an hour in an attempt to loosen the soil matrix from the bone. The soil
matrix did not dissolve, so the cortex of the rib was then shaved off
with a sterile razor blade and only these shavings were used in the
subsequent extraction. This method had the advantage of not including
potential PCR inhibitors in the soil matrix, but also discarded any
internal rib bone pieces from the extraction. For the second and third
rib extraction (Extraction #4 and #5, below), a piece of rib with its
soil matrix was simply placed under a sterilized piece of bench paper
and ground with a sterilized hammer. The entire piece, matrix and all,
was used in the subsequent extraction to maximize the amount of ancient
human bone in the sample.
Extraction
Procedures
We used four different extraction procedures on
five separate occasions. Twice we attempted DNA extraction using a phenol-chloroform
method (e.g., Kaestle 1998), once with a phenol-chloroform-silica
method (modified from Baron et al. 1996), once with the QiaQuick
PCR Purification Kit (Qiagen) (Yang et al. 1998), and once with
a guanidine isothiocyanate - silica method (modified from Hoss & Paabo
1993). The first and second phenol-chloroform-based methods are known
to produce the highest DNA yields, but have the disadvantage of co-extracting
humic acids and possibly other elements typically present in soils that
react with reagents used during PCR. These may subsequently inhibit
PCR reactions and yield false negative results. The other two methods
produce cleaner results, meaning that inhibitory elements tend to be
removed. We used a variety of methods and attempted one method twice
because results do vary from one extraction to another, even with the
same protocol. The extraction methods used for the Kennewick samples
can be summarized as follows:
Phenol-Chloroform (e.g., Kaestle 1998)
- A sample of 0.10 to 0.25 g of bone powder is transferred
to a 15 ml conical tube with 0.5 M EDTA pH 8.0 (molecular grade).
Samples rotate for 72-96 h at room temperature (~ 30 °C).
- Change EDTA every 24 h until EDTA is clear, but no
more than 3 times.
- Wash three times with UV/UF H2O
to thoroughly remove EDTA.
- Decalcify sample with 2 ml of Proteinase K buffer
and incubate overnight at 55 °C, with slight rotation.
Proteinase K Buffer: 50 mM (.05 M) Tris pH 8.0
1 mM CaCl2
1 mM DTT
0.5 % Tween 20
1 mg/ml Proteinase K
- Centrifuge samples at 2500 RPM for 10 min.
- Extract DNA using phenol, followed by phenol-chloroform
(1:1), and then chloroform : isoamyl alcohol 24:1. Use a volume for
each equal to the amount left after the Proteinase K digest.
- Concentrate samples with Centricon 100 filtration
units. Wash twice with molecular grade Tris-EDTA (TE) pH 8.0.
- Flip the Centricon column. Add 50 µl - 100 µl TE
pH 8.0 into the final (UV-irradiated) tube provided by the manufacturer.
- Transfer extract into new irradiated final 1.5 ml
tubes.
Phenol-Chloroform-Silica (modified from Baron et
al. 1996)
- A sample of 0.10 to 0.25 g of bone powder is transferred
to a 15 ml conical tube with 1.5 ml 0.5 M EDTA pH 8.0 (molecular grade).
Samples rotate for 96 h at room temperature (~ 30 °C).
- Add 500 µl of 20 mg/ml Proteinase K (molecular grade)
plus 1.5 ml of UV/UF H2O. Incubate
at 60 °C for 90 min, rotating gently.
- Centrifuge samples for 5 min at 3000 RPM.
- Extract DNA twice using 25:24:1 phenol : chloroform
: isoamyl alcohol, followed by chloroform. Use a volume for each equal
to the amount left after the Proteinase K digest.
- To precipitate DNA, add ~ 80 µl of 2M Sodium Acetate
(pH 4.5) and 3.3 ml 100 % isopropanol. Mix for 10 min.
- Add 5 µl glassmilk, a brand of silica. Mix for 10
min at room temperature.
- Centrifuge for 5 min at 3000 RPM.
- Wash twice with ice cold 80 % ethanol.
- Let samples dry for about 1 h in an incubator.
- Elute DNA with 50 µl molecular grade TE and transfer
to a smaller, 1.5 ml tube.
- Retain glassmilk (silica) in extract in subsequent
PCRs.
Silica-Based Extraction (Yang et al. 1998)
- Bone sample is dissolved in 8 ml extraction buffer:
0.5 M EDTA pH 8.0
0.5 % sodium dodecyl sulfate (SDS)
100 µg/ml Proteinase K
- Sample is incubated at 55 °C overnight.
- Sample is then incubated at 37 °C for 24 h.
- Sample is centrifuged at 2000 x g for 5 min.
- Sample is run through Centricon filters to reduce
volume to ~ 30 µl. Final collection tube is UV-irradiated.
- Five volumes of Qiagen PB Buffer is mixed with the
sample in the Centricon collecting tube.
- 750 µl of this mixture is loaded at a time onto the
Qiagen column and the column is centrifuged at 12,800 x g for 1 min.
Column is reloaded with extract until all extract has passed through
the columns.
- Sample is washed with 750 µl of Qiagen PE buffer
and column is centrifuged for 1 min.
- Qiagen columns placed into final irradiated tubes.
DNA is eluted by loading 100 µl of Qiagen's EB buffer.
Guanidine Isothiocyanate - Silica (modified from Hoss
& Paabo 1993)
- The following buffers are prepared and stored ahead
of time before possible exposure to the bone samples:
- Lysis Extraction Buffer
4.7 M GuScn
47 mM Tris-HCl pH 7.4
20 mM EDTA pH 8.0
5 % N-laurel sarcosine
- Extraction Buffer
4.7 M GuScn
47 mM Tris-HCl pH 7.4
- Prepared bone sample is transferred to a 15 ml conical
tubes with 10 ml of 0.5 M EDTA pH 8.0 and rotated at room temperature
for about 7 h.
- Sample is centrifuged at 3400 RPM for 10 min and
supernatant discarded.
- A 4 ml volume Lysis Extraction Buffer is added to
the original 15 ml tube, vortexed and rotated for about 13 h at 56
°C.
- Sample is centrifuged at 3400 RPM for 10 min.
- Sample supernatant is transferred to new, UV-irradiated
15 ml tubes.
- 50 µl of silica is added to each tube.
- Sample is incubated for 10 to 20 min at room temperature
with slight agitation.
- Silica is pelleted by centrifuging at 1500 RPM for
2 min and supernatant discarded.
- Silica is washed with 1.5 ml Extraction Buffer. Product
is transferred to a 1.5 ml tube.
- Silica is washed with 70 % ethanol, vortexed and
centrifuged.
- Silica is washed with acetone, centrifuged at 1300
RPM for 4 min and supernatant discarded.
- Sample is left to dry at room temperature for 15
min.
- Silica is resuspended with 130 µl TE (irradiated),
incubated at 56 °C for 10 min, then centrifuged at 13,000 RPM for
5 min to pellet the silica.
- Supernatant is pipetted out, leaving silica behind,
and transferred into UV-irradiated 1.5 ml final tubes.
Post-Extraction
"Clean-Up" Procedure using Proteinase K
To further remove inhibitors from extracted
samples, a Proteinase K clean-up method was used on Extractions #1 and
#2. The method follows:
- Make a Proteinase K buffer
50 mM KCL
15 mM Tris-HCl pH 7.5
2.5mM MgCl2
5 % Tween 20
1 mg/ml Proteinase K
- Place 10 µl of extract into PCR tube; add 40 µl
of Proteinase K buffer.
- Incubate for 1 h at 55 °C.
- Incubate at 95 °C for 10 min to inactivate the Proteinase
K.
- Use 1 µl of this Proteinase K/Extract solution per
1 µl PCR reaction size.
Polymerase
Chain Reaction (PCR) Amplification Procedures
- All reagents used in the PCR cocktails were
bought directly from the manufacturer as molecular grade, DNase/RNase
free specifically for this work. The only exception to this was the
UV/UF H2O, which, as described above,
was taken from another laboratory and is regularly tested for contaminants
in all PCR reactions.
- Individually capped PCR tubes in strips
of eight are always used so that each sample tube is covered when
not in use.
- PCR cocktails were made in volumes of 50
µl per sample, and included the following:
- 1x manufacturer's (molecular grade) buffer.
- 0.6 mM primers (Gibco-BRL) rehydrated
in molecular grade 1x TE and diluted in UV/UF H2O
(as above).
- 1.5 mM manufacturer's (molecular grade)
MgCl2.
- Taq polymerase from the manufacturer
(molecular grade). We use either Amplitaq Gold (Perkin-Elmer) or
Platinum-Taq (Gibco-BRL). These Taqs imitate "hot-start"
PCR, which reduces non-specific priming and amplification.
- 200 mM dNTPs (Gibco-BRL) rehydrated in
UV/UF H2O (as above).
- 1 mg/ml Bovine Serum Albumin (BSA), molecular
grade (Roche).
- UV/UF H2O
to bring the cocktail up to the total volume (e.g., 50 µl
per sample).
- The PCR cocktail minus the Taq was prepared and
UV-irradiated in a Stratagene for 15 min to eliminate any exogenous
DNA contamination. The Taq polymerase was then added to the mixture,
and the entire cocktail was then aliquoted into the separate PCR tubes.
We performed PCR amplifications under varying conditions
depending on the Taq manufacturer's specifications. In addition, we
used two cycling protocols, a standard PCR and a touch-down PCR.
The standard PCR cycling conditions began with an initial
4-10 min denaturation step at 94 °C, followed by 40 to 45 cycles in
which the DNA samples are denatured at 94 °C for 40 sec, annealed at
52 °C for 40 sec, and extended at 72 °C for 40 sec. This cycling procedure
is followed by a final extension step at 72 °C for 2 min.
The touchdown PCR involved an initial 2 min denaturation
step at 94 °C, followed by 50 cycles in which the DNA samples are denatured
at 94 °C for 14 seconds, annealed in the first of the 50 cycles at 58
°C for 30 seconds with a decrease in 0.1 °C in each successive cycle,
and extended at 72 °C for 15 seconds.
Most PCRs included a positive control of modern DNA
that is always added outside the ancient DNA room. This is to check
that the PCR reaction itself functioned, even in the case of no amplification
of the ancient samples.
DNA
Target
Our strategy was to begin by targeting DNA
from the maternally inherited mitochondrion, or mtDNA, for two reasons.
First, mtDNA is valuable for population studies, in that it allows for
the reconstruction of maternal lineages that often are correlated with
geographical groups. In the case of the New World, it is widely accepted
that most modern Native Americans fall into one of five mtDNA-based
maternal lineages, or haplogroups, labeled A, B, C, D and X (e.g.,
Smith et al. 1999; Merriwether et al. 1995; Schurr et
al. 1990). Studies of ancient samples of ancient Native Americans
confirm the presence of these maternal lineages before European contact
(e.g., O'Rourke et al. 1999; Stone and Stoneking 1998).
If DNA amplification had been successful in the case of the Kennewick
sample, such a haplogroup assignment might have been possible.
Second, because mtDNA exists in higher copy numbers
per cell relative to nuclear DNA, it is best-suited for ancient DNA
analyses. Positive amplification of mtDNA is a strong indicator of the
general presence of DNA in ancient samples. If no mtDNA amplifies, it
is unlikely that nuclear DNA remains in an ancient sample.
Moreover, in our experience, DNA from the displacement
loop, or D-loop, of the mitochondrion tends to amplify more readily
than DNA outside the mtDNA D-loop. Thus, our initial work targeted portions
of the D-loop and we designed our primer pairs used in PCR amplifications
accordingly. If amplification of the D-loop had occurred, then tentative
population-specific haplogroup assignments could have been made, since
certain D-loop mutations are known to be closely associated with population-specific
mtDNA haplogroups. At that point, the appropriate mutations outside
the D-loop would have been targeted to confirm the haplogroup assignment
based on D-loop sequences.
DNA
Visualization After PCR amplification, 10 µl of the PCR
product along with approximately 1 µl of sucrose loading dye was loaded
onto a 3 % NuSieve/1 % agarose gel stained with ethidium bromide. The
gel images were "photographed" on thermal paper using a UV
transilluminator attached to a digital imaging system. These gel images
were subsequently scanned and appear in Appendix
1.
DNA
Sequencing
When we successfully amplified DNA from PCRs that yielded
negative PCR controls as well as extraction controls (see PCRs 16 and
17), we sequenced the resulting PCR products using the following method:
- The PCR product is filtered to remove unincorporated
primers and salts using a silica-based spin column and manufacturer
reagents from the Qiagen PCR Purification kit.
- The resulting purified PCR product is sequenced
using the Big Dye Terminator Cycle Sequencing Ready Reaction kit v1.0
(Applied Biosystems).
- The sequenced product is loaded and visualized through
an ABI 377XL automatic sequencer.
Summary
of Extractions and Results
DAM = D. Andrew Merriwether
GSC = Graciela S. Cabana
JM = John McDonough
Extraction Log
Extraction #1: performed by DAM from 5/22/00 to 5/25/00.
A bone powder sample of the MC3 was taken, and the DNA extracted using
the Qiagen method, as above.
Extraction #2: performed by DAM from 5/29/00 to 5/31/00.
A bone powder sample of the MC3 was taken, and the DNA extracted using
the Phenol-Chloroform method, as above.
Extraction #3: performed by GSC from 6/14/00 to 6/16/00.
Bone scrapings were taken from the rib bone, and the DNA extracted using
the modified Guanidine Isothiocyanate - Silica method, as above.
Extraction #4: performed by GSC from 7/4/00 to 7/6/00.
A ground bone sample was taken from the rib bone, and the DNA extracted
using the Phenol-Chloroform method, as above.
Extraction #5: performed by GSC from 9/3/00 to 9/6/00.
The remaining rib bone was ground, and the DNA extracted using the Phenol-Chloroform-silica
method, as above.
PCR Log
This log complements the gel images shown in Appendix
1.
Most PCR gels have standard size 1 kilobase ladders flanking
the samples in both the left-most (lane 0) and right-most lanes.
PCR 1
(GSC)
Primers 16192 to 16322 (mtDNA D-loop)
PCR of Extraction #1 (Qiagen method)
Lane |
Sample |
Amplification status |
|
|
|
1-17 |
Other ancient samples |
|
18 |
Kennewick sample |
Negative |
19 |
EDTA + Extraction Buffer (1st wash) |
Positive |
20 |
EDTA + Extraction Buffer (2nd wash) |
Positive |
21 |
Proteinase K Buffer |
Negative |
22 |
PCR Negative Control |
Negative |
23 |
PCR Positive Control |
Negative |
24 |
PCR Negative Control |
Negative |
Result: Although the PCR negative controls did not show
amplification, the Kennewick extract controls did show contamination.
This does not mean, however, that the reagents used in the PCR did not
contain exogenous human contamination. This is because if very low levels
of exogenous DNA are in the PCR reagents, and there are more "samples"
than PCR negative controls, chances are the contamination from the PCR
reagents will show up in the samples. Thus, we typically wait to see
the results of several PCRs before determining that an extraction product
is itself contaminated. Indeed, subsequent PCRs on this extract suggested
to us that the this extract was not contaminated.
PCR 2
(GSC)
Primers 16268 to 16375 (mtDNA D-loop)
PCR of Extraction #1 (Qiagen method) and Extraction #2 (Phenol-Chloroform
method)
Lane |
Sample |
Amplification status |
|
|
|
1 |
Kennewick sample (Ext. #1) |
Negative |
2 |
EDTA + Extraction Buffer (1st wash) |
Positive |
3 |
EDTA + Extraction Buffer (2nd wash) |
Positive |
4 |
Proteinase K Buffer |
Negative |
5 |
Kennewick MC3 (Ext. #2) |
Negative |
6 |
Extract Control for MC3 |
Negative |
7 |
Kennewick MC3 |
Negative |
8 |
Kennewick MC3 |
Negative |
9 |
Extract Control for MC3 |
Negative |
10 |
Proteinase K buffer Control |
Negative |
11 |
Extract Control for MC3 |
Negative |
12 |
PCR Negative Control (+ H2O) |
Negative |
13 |
PCR Negative Control |
Negative |
14 |
PCR Positive Control |
Positive |
Result: No amplification except for positive control.
PCR 3
(GSC)
Primers 16112 to 16237 (mtDNA D-loop)
PCR of Extraction #1 (Qiagen method) and Extraction #2 (Phenol-Chloroform
method)
Lane |
Sample |
Amplification status |
|
|
|
1 |
Kennewick sample (Ext. #1) |
Negative |
2 |
EDTA + Extraction Buffer (1st wash) |
Negative |
3 |
EDTA + Extraction Buffer (2nd wash) |
Positive |
4 |
Proteinase K Buffer |
Positive |
5 |
Kennewick MC3 (Ext. #2) |
Positive |
6 |
Extract Control for MC3 |
Positive |
7 |
Kennewick MC3 |
Positive |
8 |
Kennewick MC3 |
Positive |
9 |
Extract Control for MC3 |
Positive |
10 |
Proteinase K Buffer Control |
Negative |
11 |
Extract Control for MC3 |
Negative |
12 |
PCR Negative Control (+ H2O) |
Positive |
13 |
PCR Negative Control |
Positive |
14 |
PCR Positive Control |
Positive |
Result: PCR contaminated by an unknown source. The PCR
reagents were discarded and samples were re-run with new reagents (below).
PCR 4 (GSC)
Primers 16112 to 16237 (mtDNA D-loop)
PCR of 1:20 dilutions of Extraction #1 (Qiagen method) and Extraction
#2 (Phenol-
Chloroform method)
Lane |
Sample |
Amplification status |
|
|
|
1 |
Kennewick sample (Ext. #1) |
Negative |
2 |
EDTA + Extraction Buffer (1st wash) |
Negative |
3 |
EDTA + Extraction Buffer (2nd wash) |
Negative |
4 |
Proteinase K Buffer |
Negative |
5 |
Kennewick MC3 (Ext. #2) |
Negative |
6 |
Extract Control for MC3 |
Negative |
7 |
Kennewick MC3 |
Negative |
8 |
Kennewick MC3 |
Negative |
9 |
Extract Control for MC3 |
Negative |
10 |
Proteinase K buffer Control |
Negative |
11 |
Extract Control for MC3 |
Negative |
12 |
PCR Negative Control (+ H2O) |
Negative |
13 |
PCR Negative Control |
Negative |
14 |
PCR Positive Control |
Positive |
Result: No amplification except for positive control.
PCR 5
(GSC)
Primers 16268 to 16375 (mtDNA D-loop)
PCR of 1:100 dilutions of Extraction #1 (Qiagen method) and Extraction
#2 (Phenol-Chloroform method)
Lane |
Sample |
Amplification status |
|
|
|
1 |
Kennewick sample (Ext. #1) |
Negative |
2 |
EDTA + Extraction Buffer (1st wash) |
Negative |
3 |
EDTA + Extraction Buffer (2nd wash) |
Negative |
4 |
Proteinase K Buffer |
Negative |
5 |
Kennewick MC3 (Ext. #2) |
Negative |
6 |
Extract Control for MC3 |
Negative |
7 |
Kennewick MC3 |
Negative |
8 |
Kennewick MC3 |
Negative |
9 |
Extract Control for MC3 |
Negative |
10 |
Proteinase K buffer Control |
Negative |
11 |
Extract Control for MC3 |
Negative |
12 |
PCR Negative Control (+ H2O)
|
Negative |
13 |
PCR Negative Control |
Negative |
14 |
PCR Positive Control |
Positive |
Result: No amplification except for positive control.
PCR 6
(GSC)
Primers 16268 to 16375 (mtDNA D-loop)
PCR of Extraction #3 (modified Guanidine Isothiocyanate - Silica method)
Lane Sample Amplification
status 1-14 Other ancient samples 14 Kennewick (Ext. #3) Negative
15 Lysis Extraction Buffer Negative 16 Extraction Buffer Negative
17 Extract Control Negative 18 PCR Negative Control (+ H2O)
Negative 19 PCR Negative Control Negative 20 PCR Positive Control Positive
Result: No amplification except for positive control.
PCR 7
(DAM)
Primers 8195 to 8317 (Haplogroup B)
PCR of Extraction #1 (Qiagen method), Extraction #2 (Phenol-Chloroform
method) and Extraction #3 (modified Guanidine Isothiocyanate - Silica
method)
Lane |
Sample |
Amplification status |
|
|
|
1 |
Kennewick sample (Ext. #2) |
Negative |
2 |
Kennewick sample (Ext. #2) |
Positive |
3 |
Kennewick sample (Ext. #2) |
Positive |
4 |
Kennewick sample (Ext. #1) |
Positive |
5 |
Kennewick sample (Ext. #3) |
Positive |
6 |
Extract Control for MC3 (Ext. #2) |
Positive |
7 |
Proteinase K Buffer (Ext. #2) |
Positive |
8 |
Extract Control for MC3 (Ext. #2) |
Negative |
9 |
Proteinase K buffer Control (Ext. #2) |
Positive |
10 |
Extract Control for MC3 (Ext. #2) |
Positive |
11 |
Lysis Extraction Buffer (Ext. #3) |
Positive |
12 |
Extraction Buffer (Ext. #3) |
Negative |
13 |
Extract Control (Ext. #3) |
Negative |
14 |
PCR Negative Control (+ H2O)
|
Positive |
15 |
PCR Negative Control |
Negative |
16 |
PCR Negative Control |
Positive |
17 |
PCR Positive Control |
Positive |
18 |
PCR Positive Control |
Positive |
Result: PCR contaminated by an unknown source. The PCR
reagents were discarded and samples were re-run with new reagents (below).
PCR 8
(GSC)
Primers 8244 to 8313 (Haplogroup B)
PCR of a Post-Extraction Clean-up Procedure (Proteinase K) of Extraction
#1 (Qiagen method), Extraction #2 (Phenol-Chloroform method) and Extraction
#3 (modified Guanidine Isothiocyanate - Silica method)
Note: this gel was made by melting down a previously-used gel, hence
the stray band in the gel image.
Lane |
Sample |
Amplification status |
|
|
|
1 |
Proteinase K of Kennewick sample (Ext. #2) |
Negative |
2 |
Proteinase K of Kennewick sample (Ext. #2) |
Negative |
3 |
Proteinase K of Kennewick sample (Ext. #2) |
Negative |
4 |
Proteinase K of Kennewick sample (Ext. #1) |
Negative |
5 |
Proteinase K of Kennewick sample (Ext. #3) |
Negative |
6 |
Proteinase K of Extract Control for MC3 (Ext. #2) |
Negative |
7 |
Proteinase K of Proteinase K Buffer (Ext. #2) |
Negative |
8 |
Proteinase K of Extract Control for MC3 (Ext. #2) |
Negative |
9 |
Proteinase K of Proteinase K buffer Control (Ext.
#2) |
Negative |
10 |
Proteinase K of Extract Control for MC3 (Ext. #2)
|
Negative |
11 |
Proteinase K of Lysis Extraction Buffer (Ext. #3) |
Negative |
12 |
Proteinase K of Extraction Buffer (Ext. #3) |
Negative |
13 |
Proteinase K of Extract Control (Ext. #3) |
Negative |
14 |
Negative Control of Proteinase K buffer (+ H2O)
|
Negative |
15 |
Negative Control of Proteinase K buffer |
Negative |
16 |
PCR Negative Control (+ H2O) |
Negative |
17 |
PCR Negative Control |
Negative |
18 |
PCR Positive Control |
Positive |
Result: No amplification except for positive control.
PCR 9
(DAM)
Primers 16192 to 16375 (mtDNA D-loop)
PCR of a Post-Extraction Clean-up Procedure (Proteinase K) of Extraction
#1 (Qiagen method), Extraction #2 (Phenol-Chloroform method) and Extraction
#3 (modified Guanidine Isothiocyanate - Silica method)
Lane |
Sample |
Amplification status |
|
|
|
1 |
Proteinase K of Kennewick sample (Ext. #2) |
Positive |
2 |
Proteinase K of Kennewick sample (Ext. #2) |
Positive |
3 |
Proteinase K of Kennewick sample (Ext. #2) |
Positive |
4 |
Proteinase K of Kennewick sample (Ext. #1) |
Positive |
5 |
Proteinase K of Kennewick sample (Ext. #3) |
Positive |
6 |
Proteinase K of Extract Control for MC3 (Ext. #2) |
Positive |
7 |
Proteinase K of Proteinase K Buffer (Ext. #2) |
Positive |
8 |
Proteinase K of Extract Control for MC3 (Ext. #2) |
Positive |
9 |
Proteinase K of Proteinase K buffer Control (Ext.
#2) |
Negative |
10 |
Proteinase K of Extract Control for MC3 (Ext. #2) |
Positive |
11 |
Proteinase K of Lysis Extraction Buffer (Ext. #3) |
Positive |
12 |
Proteinase K of Extraction Buffer (Ext. #3) |
Positive |
13 |
Proteinase K of Extract Control (Ext. #3) |
Positive |
14 |
Negative Control of Proteinase K buffer (+ H2O) |
Positive |
15 |
Negative Control of Proteinase K buffer |
Positive |
16 |
PCR Negative Control (+ H2O) |
Positive |
17 |
PCR Negative Control |
Positive |
18 |
PCR Positive Control |
Positive |
19 |
PCR Positive Control |
Positive |
20 |
PCR Negative Control |
Positive |
Result: PCR contaminated.
PCR 10
(JM)
Primers 16112 to 16237 (mtDNA D-loop)
PCR of a Post-Extraction Clean-up Procedure (Proteinase K) of Extraction
#1 (Qiagen method), Extraction #2 (Phenol-Chloroform method) and Extraction
#3 (modified Guanidine Isothiocyanate - Silica method)
Lane |
Sample |
Amplification status |
|
|
|
1 |
Proteinase K of Kennewick sample (Ext. #2) |
Negative |
2 |
Proteinase K of Kennewick sample (Ext. #2) |
Negative |
3 |
Proteinase K of Kennewick sample (Ext. #2) |
Negative |
4 |
Proteinase K of Kennewick sample (Ext. #1) |
Negative |
5 |
Proteinase K of Kennewick sample (Ext. #3) |
Negative |
6 |
Proteinase K of Extract Control for MC3 (Ext. #2)
|
Negative |
7 |
Proteinase K of Proteinase K Buffer (Ext. #2) |
Negative |
8 |
Proteinase K of Extract Control for MC3 (Ext. #2) |
Negative |
9 |
Proteinase K of Proteinase K buffer Control (Ext.
#2) |
Negative |
10 |
Proteinase K of Extract Control for MC3 (Ext. #2) |
Negative |
11 |
Proteinase K of Lysis Extraction Buffer (Ext. #3)
|
Negative |
12 |
Proteinase K of Extraction Buffer (Ext. #3) |
Negative |
13 |
Proteinase K of Extract Control (Ext. #3) |
Negative |
14 |
Negative Control of Proteinase K buffer (+ H2O)
|
Negative |
15 |
Negative Control of Proteinase K buffer |
Negative |
14 |
PCR Negative Control (+ TE) |
Negative |
15 |
PCR Negative Control (+ H2O) |
Negative |
16 |
PCR Negative Control |
Negative |
17 |
PCR Negative Control |
Negative |
18 |
PCR Positive Control |
Positive |
Result: No amplification except for positive control.
PCR 11
(JM)
Primers 16268 to 16375 (mtDNA D-loop)
PCR of a Post-Extraction Clean-up Procedure (Proteinase K) of Extraction
#1 (Qiagen method), Extraction #2 (Phenol-Chloroform method) and Extraction
#3 (modified Guanidine Isothiocyanate - Silica method)
Lane |
Sample |
Amplification status |
|
|
|
1 |
Proteinase K of Kennewick sample (Ext. #2) |
Negative |
2 |
Proteinase K of Kennewick sample (Ext. #2) |
Negative |
3 |
Proteinase K of Kennewick sample (Ext. #2) |
Negative |
4 |
Proteinase K of Kennewick sample (Ext. #1) |
Negative |
5 |
Proteinase K of Kennewick sample (Ext. #3) |
Negative |
6 |
Proteinase K of Extract Control for MC3 (Ext. #2) |
Negative |
7 |
Proteinase K of Proteinase K Buffer (Ext. #2) |
Negative |
8 |
Proteinase K of Extract Control for MC3 (Ext. #2) |
Negative |
9 |
Proteinase K of Proteinase K buffer Control (Ext.
#2) |
Negative |
10 |
Proteinase K of Extract Control for MC3 (Ext. #2) |
Negative |
11 |
Proteinase K of Lysis Extraction Buffer (Ext. #3)
|
Negative |
12 |
Proteinase K of Extraction Buffer (Ext. #3) |
Negative |
13 |
Proteinase K of Extract Control (Ext. #3) |
Negative |
14 |
Negative Control of Proteinase K buffer (+ H2O) |
Negative |
15 |
Negative Control of Proteinase K buffer |
Negative |
16 |
PCR Negative Control (+ H2O)
|
Negative |
17 |
PCR Negative Control |
Negative |
18 |
PCR Negative Control |
Negative |
19 |
PCR Positive Control |
Positive |
Result: No amplification except for positive control.
PCR 12
(JM)
Primers 16192 to 16322 (mtDNA D-loop)
PCR of a serial dilution of Extraction #1.
Lane |
Sample |
Amplification status |
|
|
|
1 |
1:5 dilution of Kennewick sample (Ext. #1) |
Negative |
2 |
1:10 dilution of Kennewick sample (Ext. #1) |
Negative |
3 |
1:20 dilution of Kennewick sample (Ext. #1) |
Negative |
4 |
1:50 dilution of Kennewick sample (Ext. #1) |
Negative |
5 |
1:10 dilution of Extract Control #2 (Ext. #1) |
Negative |
6 |
1:10 dilution of Extract Control #3 (Ext. #1) |
Negative |
7 |
1:10 dilution of Extract Control #4 (Ext. #1) |
Negative |
8 |
PCR Negative Control (+ H2O) |
Negative |
9 |
PCR Negative Control (+ H2O)
|
Negative |
10 |
PCR Positive Control |
Positive |
Result: No amplification except for positive control.
PCR 13
(GSC)
Primers 642 to 708 (Haplogroup A)
PCR of a small fragment (66bp) of Extraction # 3 and #4.
Lane |
Sample |
Amplification status |
|
|
|
1-4 |
Other ancient samples |
|
5 |
Kennewick sample (Ext. #3) |
Positive |
6 |
Extract Control for Ext. #3 |
Positive |
7 |
Kennewick sample (Ext. #4) |
Negative |
5 |
Extract Control for Ext. #4 |
Positive |
8 |
PCR Negative Control (+ H2O) |
Negative |
9 |
PCR Negative Control |
Negative |
10 |
PCR Positive Control |
Positive |
Result: One Kennewick DNA sample and 2 extraction controls
amplified. PCR product was subsequently digested with the Hae
III enzyme (below).
Digest 1
(GSC)
Product of PCR 13 digested the Hae III enzyme to detect the mutation
defining Haplogroup A.
Lane |
Sample |
Digestion status |
|
|
|
1 |
No sample |
|
2-4 |
Other ancient samples |
|
5 |
Kennewick sample (Ext. #3) |
No digest |
6 |
Extract Control for Ext. #3 |
No digest |
5 |
Extract Control for Ext. #4 |
No digest |
Result: No Kennewick sample or extract control digested.
PCR 14
and 15 (GSC)
Primers 16112 to 16237 (PCR 14) and Primers 16192-16322 (PCR 15) (mtDNA
D-loop).
PCRs of Extraction # 5 (modified Phenol-Chloroform-silica method).
Lane |
Sample |
Amplification status |
|
|
|
PCR 14 |
|
|
1 |
Kennewick sample (Ext. #5) |
Negative (with smear) |
2 |
Extract Control for Ext. #5 |
Negative |
3 |
PCR Negative Control (+ H2O) |
Negative |
4 |
PCR Negative Control |
Negative |
PCR 15 |
|
|
5 |
Kennewick sample (Ext. #5) |
Negative (with smear) |
6 |
Extract Control for Ext. #5 |
Negative |
7 |
PCR Negative Control (+ H2O)
|
Negative |
8 |
PCR Negative Control |
Negative |
Result: The Kennewick samples did not yield specific
amplification products in that no distinct bands at the appropriate
levels on the gel appeared. However, a smear of non-specific DNA appeared
in the Kennewick sample lanes that is typical when bacterial and fungal
DNA are also present. This is not unlikely given that the rib sample
included a soil matrix. The co-extraction of bacterial and fungal DNA
also suggests that humic acids and other potential inhibitors present
in soils might be affecting our ability to detect any human DNA. One
way to lessen their effect is to dilute the extract with the hopes of
diluting inhibitory elements. PCRs 16 and 17 (below) represent PCRs
based on these dilutions.
PCR 16
(GSC)
Primers 16112 to 16237 (mtDNA D-loop).
PCRs of dilutions of Extraction # 5 (modified Phenol-Chloroform-silica
method).
Lane |
Sample |
Amplification status |
|
|
|
1 |
1:5 dilution of Extract Control for Ext. #5 |
Negative |
2 |
1:5 dilution of Kennewick sample (Ext. #5) |
Negative |
3 |
PCR Negative Control (+ H2O) |
Negative |
4 |
PCR Negative Control |
Negative |
5 |
1:50 dilution of Extract Control for Ext. #5 |
Negative |
6 |
1:50 dilution of Kennewick sample (Ext. #5) |
Positive |
7 |
PCR Negative Control |
Negative |
Result: Positive amplification of Kennewick sample (Lane
#6), while PCR and extraction negatives showed no DNA amplification.
The resulting PCR product was sequenced, and found to match the sequence
of JM.
PCR 17
(GSC)
Primers 16192 to 16322 (mtDNA D-loop).
PCRs of dilutions of Extraction # 5 (modified Phenol-Chloroform-silica
method).
Lane |
Sample |
Amplification status |
|
|
|
1 |
1:5 dilution of Extract Control for Ext. #5 |
Negative |
2 |
1:5 dilution of Kennewick sample (Ext. #5) |
Positive |
3 |
PCR Negative Control (+ H2O) |
Negative |
4 |
PCR Negative Control |
Negative |
5 |
1:50 dilution of Extract Control for Ext. #5 |
Negative |
6 |
1:50 dilution of Kennewick sample (Ext. #5) |
Positive |
7 |
PCR Negative Control |
Negative |
Result: Positive amplification of Kennewick sample (Lanes
#2 and 6), while PCR and extraction negatives showed no DNA amplification.
The resulting PCR product was sequenced, and found to match the sequence
of JM (see Appendix 3 which
contains electropherograms of the sequences from PCRs 16 and 17, with
the sites differing from the published reference sequence circled).
Note that for the region sequenced, all three mutations match the sequence
of JM and there are no positions that do not match JM. All are consistent
with a European origin of the sequence. The observed sequence is not
found in genebank or HVRbase, only in JM from the Merriwether lab. The
16145 mutation has not been observed before. Thus the best explanation
for this sequence is contamination of the extract by unknown means by
the DNA from JM.
Conclusion
We were unable to obtain reliable ancient DNA amplification
results from the Kennewick samples. This means that either (1) no original
DNA was preserved in the bone samples transferred to our laboratory,
or (2) original DNA was preserved in the bone samples but we were unable
to extract it. The latter could occur either because the extraction
methods used in this analysis were inadequate, or because we were unable
to overcome PCR inhibitors once the DNA was extracted. However, we used
a variety of methods to overcome PCR inhibition, including an additional
Proteinase K clean-up step to further break down proteins in the samples,
dilutions of the extractions reduce the proportion of inhibitors relative
to DNA, and the use of BSA in the PCR reaction (BSA may bind to inhibitors,
thus inhibiting the inhibitors).
Moreover, any DNA amplification we obtained seems to have been a product
of exogenous human contamination. This is immediately clear in two cases:
(1) when the PCR negative controls are contaminated, and (2) when the
PCR product is sequenced and matched to an investigator (e.g.,
an excavator or other analyst). The first case implies that at the very
least the reagents used in the PCR are contaminated, and reagents become
easily contaminated through multiple uses. In this case, we threw out
the old PCR reagents and performed a new PCR with fresh reagents. The
second case demonstrates that even under the most stringent conditions,
modern contaminating DNA is problematic in ancient DNA analysis.
Finally, it is possible that an amplified DNA product is the result
of exogenous human contamination despite the fact that negative controls
yield no amplification. If very low levels of exogenous DNA exist in
the PCR reagents, and there are more "samples" than PCR negative
controls, chances are the contamination from the PCR reagents will show
up in the samples, rather than in the PCR controls. For similar sampling
reasons, low levels of exogenous DNA in DNA extracts and disposable
pipette tips, tubes, etc., also will amplify sporadically, from one
PCR to another. Thus, in addition to the use of controls, we monitor
the results of positive amplifications by sequencing the amplification
products. For example, PCRs 14, 15, 16, and 17 showed no positive amplification
of PCR or extract controls, yet the sequences (shown in Appendix
3) were an exact match of our laboratory technician.
To conclude, we attempted a variety of techniques to both extract
DNA from the sample and reduce the effects of potential PCR inhibitors.
When DNA amplified, it was always from an exogenous source. This, combined
with the fact that even very low levels of contaminating DNA will preferentially
amplify when the sample itself contains little or no DNA of its own,
leads us to conclude that little or no DNA remained in the Kennewick
samples transferred to our laboratory. However, our conclusion should
not preclude further DNA testing using future novel methods on other,
perhaps more DNA-rich, bone samples from the Kennewick remains. We are
still of the belief that teeth are the best source for clean DNA free
of exogenous contamination and would still argue for testing of the
teeth (even with the caveat that Xrays can damage DNA and all but two
of the teeth were apparently X-rayed).
References
Cited
Baron, H., S. Hummel, B. Herrmann
(1996) Mycobacterium tuberculosis
Complex DNA in Ancient Human Bones, Journal of Archaeological Science,
23:667-671
Hoss, M., and S. Paabo
(1993) DNA Extraction from Pleistocene
Bones by a Silica-Based Purification Method. Nucleic Acids Research
21(16): 3913-3914
Kaestle, F. A.
(1998) Molecular Evidence for Prehistoric
Native American Population Movement: The Numic Expansion. Ph.D., University
of California, Davis.
Merriwether, D. A, F. Rothhammer, R. E. Ferrell
(1995) Distribution of the Four
Founding Lineage Haplogroups in Native-Americans Suggests a Single Wave
of Migration for the New-World. American Journal of Physical Anthropology
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O'Rourke, D. H., R. L. Parr and S W. Carlyle
(1999) Molecular Genetic Variation
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Schurr, T. G., S. W. Ballinger, Y. Gan, J. A. Hodge,
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C. Wallace
(1990) Amerindian Mitochondrial
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Smith, D. G., R. S. Malhi, J. Eshleman, J. G. Lorenz,
and F. A. Kaestle
(1999) Distribution of mtDNA Haplogroup
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110(3):271-284.
Stone, A. C. and M. Stoneking
(1998) MtDNA Analysis of a Prehistoric
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Yang, D. Y., B. Eng, J. S. Waye, J. C. Dudar and S.
R. Saunders
(1998) Technical Note: Improved
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Journal of Physical Anthropology 105:539-543
Kennewick
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