Brian Kemp’s Ancient DNA Protocol (updated Summer 2008)
This protocol is useful in extracting and analyzing DNA from ancient skeletal remains. I
have also used it to successfully extract and analyze DNA from coprolite samples.
I am currently working on publishing on some of the below mentioned ideas, so
please contact me if you would like to use the ideas for your publication or
presentation.
Some improvements over previous protocols I have employed:
1. I use much less skeletal material than before. Currently I try to use less that 500 mg
and have been successful with as little as 110 mg.
2. The use of high concentration bleach for decontaminating the surfaces of bones and
teeth.
3. The disuse of PTB and BSA. I have never had real success working with these
substances. In side-by-side comparisons (with and without PTB and BSA, respectively),
I have not found that PTB or BSA aids in the removal of inhibitors (from bones, teeth, or
coprolites).
4. I now employ an isopropanol precipitation (versus ethanol). This form of DNA
precipitation greatly removes inhibitors from samples (Hänni et al., 1995).
5. In PCR I now use very little Taq polymerase. I figure that if the inhibitors are
removed and the template DNA is few in copy number, then one needs not employ large
amounts of Taq. Using large amounts of Taq makes PCR a hyper-hyper system for the
amplification of DNA (contamination included). I have seen much less contamination
since reducing the amount of Taq in PCR reactions.
6. To remove the presence of co-extracted PCR inhibitors, I now repeat the silica
extraction a number of times, if needed. For example, in the study I performed on
coprolites, some samples needed to be re-silica extracted three or four times before I was
able to PCR amplify the mtDNA (Kemp et al., 2006; Kemp et al., 2005).
7. I have developed (with Cara Monroe) a system for determining whether a sample
contains DNA or is inhibited from amplifying (thus leading to false negatives) (Kemp et
al., 2006).
Contamination Control
As DNA extracted from ancient remains tends to be in low copy number and is highly
degraded (Lindahl, 1993; Pääbo, 1990), aDNA extractions are highly susceptible to
contamination originating from modern sources. Modern contaminating DNA can be in
higher copy number and more fully intact than the endogenous aDNA and, thus, can
compete with aDNA during polymerase chain reaction (PCR) amplification. In fact,
contamination can completely out-compete aDNA in PCR (Kemp and Smith, 2005).
Ancient DNA extractions can become contaminated via two sources: surface
contamination of the bone or tooth from handling the material or later in DNA laboratory,
during DNA extraction and analysis.
The former source of contamination can originate at any step of an aDNA study from the
time of excavation of the remains to the time of DNA extraction. Modern contamination
of the bone or tooth surface can arise from anyone who has had direct contact with the
material, including the archaeologist that excavated the remains, any archaeological
researchers that analyzed (e.g. cataloging, measuring) the remains, as well DNA
laboratory personnel. That a skeletal or tooth surface can become contaminated, it is
particularly important to successfully remove the contamination before DNA extraction
begins. To accomplish this goal, the remains are treated with a bleach solution to remove
surface contamination (Kemp and Smith, 2005).
The latter source can originate from reagents, labware, PCR carryover, or DNA lab
personnel. As such, procedures that reduce contamination are implemented, including:
the use of DNA free lab-ware and reagents, all processing of ancient materials is
performed in a laboratory physically separated from the one in which modern DNA is
examined, and the use of negative controls in both DNA extraction and amplification to
monitor contamination, if present (following the advice of Kelman and Kelman, 1999).
DNA Extraction
NOTE: If available, all reagents should be bought from manufacturers that guarantee
them to be contamination free (they usually provide the highest quality and are
consistent). If not available, for example 5M Ammonium Acetate, make the reagents
with DNA-free water (Gibco) and filter them through 0.2 m filters (Nalgene, or others).
Remove  0.5 g from the whole (NOTE: there is no need to grind the material to
powdered form. Some prefer to do so as it increases surface area. However, I argue that
it increases the opportunity to introduce contamination). Submerge the sample in 6%
sodium hypochlorite (full strength household bleach) for 15 min (Kemp and Smith,
2005). Rinse the sample with DNA free water (Gibco) to remove the bleach (1-2 times).
In a 15mL conical tube (make sure to use polypropylene, as polystyrene will melt if
exposed to phenol:chlorofrom), submerge the sample in 2 mL of molecular grade (DNA
free) 0.5 M EDTA, pH 8.0 (Gibco) for > 48 hours. An extraction control, to which no
bone is added, should accompany the extraction and be subjected to all of the steps that
follow.
To the sample add 3 mg of Proteinase K (Invitrogen, Fungal Proteinase K) and incubate
at 60-65O C for ~3 hours (NOTE: the activity of this Proteinase K is highest at 65O C, but
will rapidly denature at higher temperatures.
Extract DNA from the digested sample using a three-step phenol/chloroform method: two
extractions adding an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) to
the EDTA, followed by one extraction with an equal volume of chloroform:isoamyl
alcohol (24:1).
To aid in the removal of co-extracted polymerase chain reaction (PCR) inhibitors,
precipitate DNA out of solution with isopropanol (Hänni et al., 1995). This is performed
by adding one half volume of room temperature 5 M ammonium acetate and, to this
combined volume, one volume of room temperature absolute isopropanol, then storing
the solution overnight at room temperature.
Centrifuge the tubes for 30 min at 3100 rpm to pellet the DNA. Discarded the liquid and
air-dry for 15 minutes. There may or may not be a visible pellet (In my experience a
visible pellet doesn't guarantee that DNA has been preserved in the sample, and vice
versa). Wash the pellet with 1 mL of 80% ethanol by vortexing for about 30 seconds
(making sure to dislodge the pellet, if visible, from the side of the tube). Centrifuge the
tubes for 30 min at 3100 again to pellet the DNA. Pour off the ethanol and allow the
tubes to air-dry for 15 min. To further remove co-extracted PCR inhibitors, the resuspended the DNA in 300 L of DNA-free ddH2O and perform a silica extraction (Höss
and Pääbo, 1993) using the Wizard PCR Preps DNA Purification System (Promega),
following the manufacturer’s instructions (except elute the DNA in the final step with
100 L of ddH2O). Store the final solution at -20OC.
PCR Amplification (Screening for the defining markers of the New World
haplogroups)
PCR amplification reactions contain 8.76 L of DNA-free ddH2O (Gibco), 2.4 L of 2
mM dNTPs, 1.5 L of 10X PCR Buffer, 0.45 L MgCl2 (50mM), 0.18 L of each primer
(20 M), 0.06 L of Platinum Taq (Invitrogen), and 1.5 L DNA template. Negative
controls (PCR reactions to which no DNA template was added) should accompany every
set of PCR reactions to monitor the presence of contaminating DNA. Coordinates,
numbered according to the Cambridge Reference Sequence (Anderson et al., 1981), the
primers I use are found in Table 1.
PCR conditions are as follows: 94O C for 3 min, 40 cycles of 15 second holds at 94O C,
55O C, and 72O C, followed by a final three minute extension period at 74O C. Run 5-6
L of the amplicons on a 6% polyacrylamide gel. Stain the gel with ethidium bromide
and visualize under UV light, to either confirm successful amplification for later
restriction enzyme digest, or to score the presence or absence of the 9-bp deletion.
Sequencing Reactions
The sequencing PCR reaction differs slightly from the one used to amplifying the regions
containing the haplogroup-defining polymorphisms. PCR amplification reactions contain
17.52 L of DNA-free ddH2O (Gibco), 4.8 L of 2 mM dNTPs, 3.0 L of 10X PCR
Buffer, 0.9 L MgCl2, 3.6 L of each primer (20 M), 0.06 L of Platinum Taq
(Invitrogen), and 3.0 L of DNA template (NOTE: here I use the same amount of Taq in
a 30 L PCR reaction as I do in a 15 L reaction). The primers I use are listed in Table
1. For the amplification of sequencing products “touchdown” PCR is utilized (Don et al.,
1991). The PCR conditions as follows: 5 min hold 94O C, 60 cycles of 15 second holds
94O C, the annealing temperature (which was decreased 0.1O C after each successive
round of amplification), and 72O C, followed by a final three minute extension period at
74O C. The starting annealing temperatures are listed in Table 1. About 3-4 L of the
amplicons are run on 6% polyacrylamide gels, stained with ethidium bromide and
visualized with UV, as described above, to confirm success in amplification.
ExoI digests the remaining amplicons by adding to the amplified product 60 L of
ddH2O, 2 L of the ExoI buffer, and 0.2 L of the ExoI enzyme. Incubate this reaction at
37O C for 90 min and then at 80O C for 20 min to denature the ExoI. Filter the ExoI
digested DNA through a Millipore plate, and re-suspended in 25 L ddH2O.
This product was is ready for sequencing. I have been submitting my PCR product to the
Davis Division of Biological Sciences (DBS) Automated DNA Sequencing Facility, at
the University of California, Davis (so I don't have a protocol for cycle sequencing, etc.).
Sequence the amplicons in both directions to preclude sequencing errors.
Testing for PCR Inhibitors
It is important to know whether a sample contains DNA or it has been co-extracted with
PCR inhibitors that preclude DNA amplification. Following DNA extraction if a sample
does not amplify it should be tested for the presence of PCR inhibitors. To do so, set up a
PCR reaction. Add the straight extract to one reaction. To another reaction add the
straight extract and spike the reaction with a “positive ancient control” (an ancient DNA
sample that is known to work, use the same volume of template as the sample in
question). If the positive ancient control fails to amplify, the sample is inhibited. If the
positive ancient control amplifies, then the sample in question most likely contains no
DNA. If the sample indicates that PCR inhibitors are present, repeat the silica extraction
and repeat the procedure above. Repeat this until the sample no longer shows signs of
inhibitors.
This procedure should not be performed with a positive ancient control, not a positive
modern control. I have found evidence that, in some cases, an inhibited sample will
allow a positive modern control to amplify, while not allowing a positive ancient control
to amplify. While I have not determined exactly why this is the case, it suggests that
there is some threshold involved with DNA concentration and/or quality of the template.
Works Cited
Anderson S, Bankier AT, Barrel BG, DeBulin MHL, Coulson AR, Drouin J, Eperon IC,
Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJH, Staden R, and Young
IG (1981) Sequence and organization of the human mitochondrial genome.
Nature 290:457-465.
Don RH, Cox PT, Wainwright BJ, Baker K, and Mattick JS (1991) Touchdown PCR to
Circumvent Spurious Priming During Gene Amplification. Nucleic Acids
Research 19:4008.
Hänni C, Brousseau T, Laudet V, and Stehelin D (1995) Isopropanol Precipitation
Removes PCR Inhibitors from Ancient Bone Extracts. Nucleic Acids Research
23:881-882.
Höss M, and Pääbo S (1993) DNA Extraction from Pleistocene bones by a silica-based
purification methods. Nucleic Acids Research 21:3913-3914.
Kaestle FA (2000) Report on DNA Analysis of the Remains of "Kennewick Man" from
Columbia Park, Washington. In NP Service (ed.): National Parks Service, website
http://www.cr.nps.gov/aad/kennewick/index.htm.
Kelman LM, and Kelman Z (1999) The use of ancient DNA in paleontological studies.
Journal of Vertebrate Paleontology 19:8-20.
Kemp BM, Monroe C, and Smith DG (2006) Repeat silica extraction: a simple technique
for the removal of PCR inhibitors from DNA extracts. Journal of Archaeological
Science 33:1680-1689.
Kemp BM, and Smith DG (2005) Use of Bleach to Eliminate Contaminating DNA from
the Surfaces of Bones and Teeth. Forensic Science International 154:53-61.
Kemp BM, Smith DG, and Nelson WJ (2005) DNA analysis of ancient coprolites from
Fish Slough Cave. 29th Great Basin Anthropological Conference.
Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709715.
Pääbo S (1990) Amplifying Ancient DNA. In MA Innis (ed.): PCR protocols : a guide to
methods and applications. San Diego: Academic Press, pp. 159-166.
Parr RL, Carlyle SW, and O'Rourke DH (1996) Ancient DNA analysis of Fremont
Amerindians of the Great Salt Lake Wetlands. American Journal of Physical
Anthropology 99:507-518.
Stone AC, and Stoneking M (1993) Ancient DNA from a pre-Columbian Amerindian
population. American Journal of Physical Anthropology 92:463-471.
Wrischnik LA, Higuchi RG, Stoneking M, and Erlich HA (1987) Length mutations in
human mitochondrial DNA: direct sequencing of enzymatically amplified DNA.
Nucleic Acids Research 15:529-542.
Table 1. Primers and Annealing Temperatures.
Target Region
Primer
Coordinates*
Annealing
Temperature
Citation
611F
00591-00611
55O C
(Stone and Stoneking, 1993)
743R
00765-00743
B
8215F
08195-08215
55O C
(Wrischnik et al., 1987)
8297R
08316-08297
C
13256F
13237-13256
55O C
(Parr et al., 1996)
13397R
13419-13397
D
5120F
05099-05120
55O C
(Parr et al., 1996)
5190F
05190-05211
X
14440F
14421-14440
49O C
(Kaestle, 2000)
14591R
14591-14612
HVI-1
15986F
15986-16010
62O C#
Designed by John
McDonough, UC Davis
16153R
16132-16153
O #
HVI-2
16106F
16106-16126
62 C
Designed by John
McDonough, UC Davis
16251R
16230-16251
O #
HVI-3
16190F
16190-16209
58 C
Designed by John
McDonough, UC Davis
16355R
16331-16355
O #
HVI-4
16232F
16232-16249
58 C
Designed by John
McDonough, UC Davis
16404R
16383-16404
HVII-1
00034F
00034-00058
62O C#
Designed by John
McDonough, UC Davis
00185R
00160-00185
HVII-2
00112F
00112-00135
62O C#
Designed by John
McDonough, UC Davis
00275R
00249-00275
HVII-3
00184F
00184-00208
62O C#
Designed by John
McDonough, UC Davis
00356R
00331-00356
* Coordinates, numbered according to the Cambridge Reference Sequence (Anderson et
al., 1981).
#
Touch-down PCR used, decreasing the annealing temperature 0.1O C after each cycle.
A