Isolation of Nucleic Acids and Microorganisms from Ice and Permafrost
Eske Willerslev
Anders J. Hansen
Department of Evolutionary Biology,
Zoological Institute,
University of Copenhagen,
2100 Copenhagen Ø,
Denmark.
Hendrik N. Poinar*
Max Planck Institute for Evolutionary Anthropology,
Deutsche Platz 6
D-04103 Leipzig
Germany
Present address: MacMaster University, Department of Anthropology and Geology, 1280
Main Street Hamilton Ontario, Canada, L8S 4M4
Keywords: glacial ice, permafrost, ancient DNA, DNA damage, viable cells, exobiology
Teaser: Ancient DNA, RNA and microbes from fossil ice and permafrost.
DNA and RNA are unstable molecules relative to many of their cellular counterparts. It
has been predicted that DNA will persist some 104 years in temperate- and 105 years in
colder environments. Thus, ice and permafrost may be the best place for long-term
nucleic acid preservation. Despite this, recent claims of putative viable microbes and
nucleic acids from ice- and permafrost hundred-of-thousand to million-of-years-old are
not properly authenticated and may simply result from contamination. Here, we discuss
processes that restrict long-term survival of DNA/RNA molecules in ice and permafrost
and various sources of contamination. Additionally, we present a set of precautions,
controls and criteria to ensure to the greatest extent possible authentic cultures or
sequences from ancient ice and permafrost.
Main text (without ref. Fig. Tables, boxes) max 2500 words… it’s currently slightly to
long about 300 words.
Numerous ice and permafrost cores (permanently frozen soil) have been drilled from many
countries including Greenland, Canada, Alaska, Russia and the Antarctic (Fig. 1). Ice cores
can be more than 3 km in length and constitute a record of continual snow accumulation as
old as 800 thousand years (kyr) (1-2). Permafrost cores are up to 400 m depth and are
presumed to contain frozen soils that are putatively up to 8.1 million years (Ma) (3-4).
In recent years both glacial ice- and permafrost soils have been the focus of great
attention, as they are believed to be ideal for long-term preservation of microorganisms as
well as bio-molecules, due to their constant low temperatures. Several papers claim the
isolation of viable microbes, as well as the recovery of DNA and RNA molecules, from up to
100 kyr old ice cores and viable microbes from up to 2-3 Ma old permafrost cores (5-17). If
true, these discoveries could fundamentally alter views about microbial physiology, ecology,
evolution and have recently become the model for future missions to recover extraterrestrial
life forms on distant planets, such as Mars and Jupiter’s moon Europa (Box 3). In addition,
Siberian permafrost (2g) has recently been shown to contain DNA sequences from various
mammals including wooly mammoth, horse and bison up to 30 kyr and plant DNA as old as
300-400 kyr even in the absence of obvious macrofossils (18) making permafrost cores
potential important in plaeobiological reconstruction (Fig. 2).
However, both the culturing of microbes and the amplification of ancient DNA/RNA
molecules from ice and permafrost are beset with difficulties. Firstly, theoretical and
empirical studies have shown that short fragments of DNA (100-500bp) do not survive in the
geosphere for more than 10 kyr in temperate environments and 100 kyr in colder ones due to
spontaneous hydrolytic and oxidative damage which may accrue post mortem (19-22) (Fig. 3,
Box 1). RNA has an even shorter half-life than DNA (19) and an absolute upper limit of 1 Ma
has recently been suggested for any amplifiable nucleic acids (23). DNA from some resting
cells such as bacterial endospores may survive longer than theory predicts for naked
molecules due to special adaptations (19, 24). Nevertheless, endospores and other resting cells
have no active repair during dormancy and damages will accumulate in their genomes in a
time and environment dependent manner, finally becoming lethal (25). Some have suggested
that continual metabolic activity and DNA repair can explain the long-term survival of both
spore-forming and non-spore-forming microbial cells in glacial ice and permafrost (26).
Although some indications of this exists in permafrost settings (27-28) direct evidence is
lacking and currently such assumptions must be considered speculative. The revival of
dormant microbes and amplification of DNA and RNA sequences from remains hundreds of
thousands to millions of years old is, from a theoretical point, expected to be difficult, if not
impossible. The culturing of microbes from ice and permafrost using non-specific media
combined with the extreme sensitivity of PCR, places doubt on the claims of isolation to date.
These factors combined with high density and global distribution of microbes makes the risk
of contamination with contemporary ubiquitous microbial cells and exogenous DNA/RNA
molecules extremely high (17). There is increasing evidence that microbes are found
everywhere i.e. in the laboratories, reagents, tools and on people handling the cores or
performing the experiments. As only about 1-5% of the potential modern contaminants, i.e.
extant microbial diversity is estimated to be currently known (29) finding an unpublished
microbial sequence or culture does not imply it is ancient making it extremely difficult to
verify the results of any ancient microbial study. The high risk of contamination is likely to be
the main reason for the many conflicting results within the field of ice core and permafrost
culturing and genetics (Box 2) and seriously calls for standardized procedures within the field.
DNA and RNA longevity in ice and permafrost
The DNA molecule is a relatively unstable molecule in comparison to other cellular
counterparts such as carbohydrates, plant lignin and cutin. In metabolically active tissues,
damage to the genome is rapidly and efficiently repaired via a host of repair pathways (19).
However, in inactive cells (dead or dormant) DNA and RNA molecules accrue in an
environment and largely temperature-dependent manner. In addition to cleavage via
endogenous nucleases and microbial onslaught the two main processes responsible for DNA
and RNA’s half life in the geosphere are spontaneous hydrolysis and oxidation that are
dependent upon the availability of free water and free-oxygen radicals (19-22, 30) (Fig. 3,
Box 1).
High altitude polar ice caps with temperatures as low as –30°C to –50°C and no
surface melting can be considered dry environments. Nevertheless, they do contain acidic
liquid veins, which reportedly runs along triple boundaries of the crystals (31, 32) potentially
exposing the DNA/RNA from dead and dormant cells to hydrolysis and acidic pH.
Furthermore, low altitude icecaps and bedrock samples (T~ 0°C) contain up to 1‰ of free
water and thus despite being dry, nucleic acids will still be subjected to some water and hence
hydrolytic damage, even in ice cores.
Although, there is little if any oxygen diffusion through glacial ice below the first 7080 m when the snow becomes solid, DNA and RNA molecules will be exposed to oxidation
before this occurs, which can take several hundred to several thousand years depending on the
rate of snow accumulation.
In permafrost, ice makes up 92-97% of the total water volume while the remaining 38% of the water is in an unfrozen state, which depends upon the temperature and sediment
texture (16). Therefore, nucleic acids in permafrost will be exposed to spontaneous
hydrolysis. The rate of hydrolysis is likely to take place at a higher rate in permafrost than in
high altitude polar ice due to warmer temperatures (T =-9 to -12C, north east Siberian
permafrost, -22°C, Antarctic permafrost compared to -30 C to -50C polar ice caps).
The high methane values (up to 40 ml/kg) and redox potentials, Eh ~ +40 to –250
mV, in Siberian permafrost suggest largely anaerobic condition (3, 33) and hence oxidative
damage to the DNA/RNA molecules may be minor. This is in contrast to Antarctic
permafrost that reportedly contains redox potentials, Eh ~ +260 to +480, suggesting largely
aerobic conditions (33). This factor, combined with slightly alkaline pH values in Antarctic
permafrost (Miers Valley Antarctica, pH=7.95-8.45, Taylor Valley Antarctica, pH= 8.95-9.5)
as opposed to those close to neutral in Siberian permafrost should make Siberian permafrost
a better place for long term DNA preservation, and Antarctic permafrost more prone to
oxidative and alkylation DNA damage.
The effect of temperature on spontaneous chemical decay is described by the
Arrhenius equation: k=Ae-Ea/RT, where k is the rate constant, A is the pre-exponential
factor that depends on the reaction, Ea is the activation energy, R is the gas constant (8.31 KJ
mol-1 at 1 atm) and T is the temperature (Temperature Kelvin). Accordingly, any decrease in
temperature will induce an exponential decrease in the reaction rate (34). Rough calculations
on the influence of depurination (Fig. 3, Box 1) on DNA survival show that a bacterial
genome of 3.0x106 bp (puines and pyrimidines in ratio 1:1) will be fragmented to roughly
100bp stretcheswithin 500 years at 15C, 81 kyr at -10C and 1.7 Ma at -20C (Fig. 4). Thus
polar ice and permafrost should, from a theoretical standpoint, be the best places for the
longterm storage of nucleic acids.
Additional factors which influence the Arrhenius equation are the pH and heavy
metal ion chelation, via the activation energy, and pressure through the gas constant. The
presence of much higher concentrations of heavy metals, soil humics and microbial cells of
permafrost soils makes the nucleic acids of this environment more prone to additional forms
of damage including microbial degradation, crosslinking reactions, and alkylation types
damages. Contrarily the higher pressure in deep glacial ice (up to 300 bar) as opposed to
permafrost is likely to significantly increase the reaction rates, such as depurination, reducing
the half-life of any DNA/RNA molecules. Nevertheless, the constant low temperatures of
high latitude polar ice caps and permafrost must make them among the best environments on
Earth for long-term microbial and DNA/RNA preservation.
The risk of contamination
Despite preliminary success in the retrieval of short DNA/RNA fragments as well as viable
cells from glacial ice and permafrost, there remains a noticeable lack of reproduction of
results, and in the few cases where reproducibility has be unintentionally performed the
results are highly conflicting (Box 2). This could be attributed to differences in
methodological efficiency, but are more likely a result of the varying degrees of
contamination. In general, the risk of contamination is high for PCR and studies on the
culturing of permafrost from unspecific media (17, 35-37). This combined with the low
amount of cell numbers and DNA molecules in glacial ice and permafrost makes
contamination a serious problem (e.g. Hans Tausen ice core ~103 cells/l; GRIP ice core ~cell
numbers beyond detection (38); Kangerlussuaq glacial ice ~0.7-3 ngDNA/l; Kolyma Lowland
and Laptev Lowland permafrost cores ~107 cells/g and ~12-160 ngDNA/g; Bacon Valley
permafrost ~ DNA amount beyond detection (39). Currently there are two issues which need
to be addressed, the level of microbial and DNA contamination derived from the drilling and
coring procedure, as well as all laboratory manipulations, including DNA extraction and
amplification.
Drilling and core storage
The first source of contamination is the drilling procedure itself. Permafrost cores are
preferentially drilled for the isolation of organisms. Special rotation-column coring methods
to avoid drilling fluids and handling procedures have been developed to minimize
contamination from externally derived cells, but not with DNA/RNA molecules (12, 18). Ice
cores, on the other hand, are often drilled for isotopic studies providing information about past
climatic conditions (1) and no special procedures are used to minimize biological
contamination during drilling and handling.
We have found that different ice core drilling methods vary in respect to the level of
contamination. Mechanical drilling without liquid fluid in the borehole results in numerous,
small cracks; making it difficult to clean the ice core samples post coring and increases the
chance of contamination (Table l in Box 2). Drilling with fluid such as Exxol D60 (lamb oil)
and HCFC (Freon 141B) often results in good quality ice cores with few noticeable cracks.
Although the drilling fluid used is likely to be contaminated (not yet tested systematically), it
only penetrates the ice where cracks are already present. This has been shown experimentally
(40) and would disturb ice core measurements of dust and heavy metal concentrations (38,
41). However, to look at the extent of penetration into the cores (small cracks are invisible),
the drilling equipment and fluids should be spiked with variously sized and easily
recognizable microorganisms e.g. Serratia marcescens bacteria (as has been done in
permafrost coring (12, 18, 42)) and ideally with additionally nonhuman, non- microbial DNA
of various lengths that can be easily amplified and quantitated by real time PCR. During
logging, cutting and storing of the cores in the field sterile gloves, caps and facemasks should
be worn, which will minimizebut not exclude human and bacterial contamination (Table 1).
In the laboratory, various procedures have been proposed to minimize sample
contamination (5, 9-11, 13, 43). To date no studies have compared the efficiencies of these
methods. However, we find that removing ½-1 cm of the surface with a pre-sterilized
microtone knife (treated with 5% Sodium hypochlorite) is highly efficient (11, 18). For ice
cores, scraping off the outer surface must be carried out in a laminar flow hood in a room
maintained at –20ºC to avoid the formation of water film at the surface. The small size of the
permafrost samples needed for biological studies (a few grams) makes it possible to handle
short core samples (e.g. ~10 x 10 cm) in a positive airflow hood or glove box in a regular
clean laboratory (see below). In such a facility, ½-1 cm of the core surface can be removed
with a sterile microtome knife (treated with 5% Sodium hypochlorite) (Table 1).
Laboratory contamination
Microorganisms are ubiquitous in all envioronments and thus presumably contaminating
bacterial DNA must be everywhere as well, and has been found in e.g. the sequences of the
Human Genome Project (44) or inadvertently been PCR amplified from several low biomass
samples (36). It is therefore prudent to assume that all laboratory reagents and tools are
contaminated with microbial cells and nucleic acids. Buying laboratory reagents and
equipment marked “sterile” is not a guarantee as sterility assurance level is 1x10-6(45).
Autoclaving does not destroy all presence of DNA, only denatures it, and thus certainly will
not remove solutions of short DNA fragments (≤100bp). Ethanol is a better bacteriostatic
agent than it is a sterilant, but certainly does not destroy DNA (46). Therefore, to efficiently
reduce the risk of contamination, it may be helpful to treat all reagents, with ultra-filtration
(primer solutions: through 50K NMWL filters, other reagents for extraction and PCR through
30K NMWL filters), tubes and water with UV-radiation (45W for 72 h), all glassware via
baking (>180°C over night), and/or 5% Sodium hypochlorite for 48 h (11, 18, 35) (Table 1).
Handling of the cores, culturing experiments, DNA extraction and PCR setups must be
carried out in positive air hoods or glove boxes in fully equipped laboratories dedicated to
culturing of ancient microorganisms or the recovery of ancient DNA/RNA molecules (clean
laboratory). These laboratories should be physically separated from each other and from other
laboratories and must have separate ventilation systems, nightly UV-radiation of surfaces and
have surfaces cleaned frequently with household bleach. In the clean laboratories sterile
gloves, caps and facemasks should always be worn (11, 18, 35, 47) (Table 1).
At least one control should be applied for every experimental step i.e. a control to
monitor possible contamination from the air within the hood or glove box (air control), a
clean filter used for concentration of melt water (filter control, ice cor e studies), Petri plates
that contain only media (culture controls), DNA extractions without template added extraction
control, performed in ratio of 1:5) and PCR controls without DNA extract added (PCR
controls, performed in a ratio of 1:1) (11, 18, 35, 47) (Table 1). It is noteworthy that blank
controls may appear clean after PCR while low level contaminants in sample extracts may
still be amplified due to “product carryover” (35, 47). Likewise, empty blank controls are not
a test for sample contamination. Therefore, some specific additional criteria are needed in
order to authenticate ancient DNA/RNA and cultures from ice and permafrost.
Box 3. Criteria of authenticity
Below are a set of criteria, currently appropriate for authenticating DNA/RNA sequences and
cultures recovered from ice and permafrost cores (Table 1).
There should be an inverse relationship between amplification efficiency and fragment length
i.e. the concentration of short templates should be relatively higher than longer ones due to
sequence fragmentation. Presumably this should be the case even in the presence of viable
cells as each viable cell should be accompanied by a relatively large number of degraded ones
of the same type (11, 35, 37, 47).
Amplification products should be cloned and sequenced. Using specific primers for PCR this
can determine damaged-induced errors (37, 48) (Box 1) while “universal” primers may reveal
diversity and possible contamination (11, 18).
Results should be independently replicated by another laboratory (both cultures and
DNA/RNA molecules) using either the same core sample or, ideally, a different sample of the
same age drilled in close proximity. This may be complicated by an uneven distribution of
cells in the sample. Therefore, it is wise to pool several independent DNA extracts prior to
PCR and to pool several PCR reactions prior to cloning to increase homogenization. For
studies with high sequence diversity statistical methods have been developed to test for
reproducibility (18). Many spectacular DNA claims have failed this criterion of authentication
(49) and the need for independent replication of results in ice and permafrost studies is
strengthened by the considerable variation in results obtained by different groups (Box 2).
In theory, a single viable cell should be followed by a relatively larger number of amplifiable
template sequences due to larger numbers of dead than viable cells and to the presents of
multi copy genes (17). Thus, viable cultures should be verified by recovering fragments of its
DNA directly from the sample. This could be done in an independent laboratory.
The discovery of previously unknown cultures or novel nucleotide sequences cannot be used
as criteria for their authenticity in microbial studies. However, finding clear age related
patterns in DNA/RNA damage may be a way forward but has yet to be tested The
identification of sequences from less contamination prone organisms such as extinct
mammals and plants are stronger proof of their authenticity from that specific core sample,
although their age is questionable (18).
Quantifying the amount of DNA molecules by e.g. Picogreen fluorescence assay or starting
templates by e.g. quantitative or “Real Time” PCR can provide information concerning
possible contamination. It is difficult to reproduce results when PCR begins with less than
approximately 1000 template molecules (50).
Assessing the total amount, composition, and relative extent of diagenetic change in amino
acids can provide indirect evidence of DNA or RNA survival (22). Direct evidence of the
state of the DNA may be addressed by enzymatic assays (51) or by mass/spectrometry (21).
Conclusion and prospects
The culturing of ancient, “viable” microorganisms as well as the recovery of the putatively
oldest strands of DNA and RNA from glacial ice and permafrost holds tremendous promise
yet is at its infancy. At the lowest temperatures of any geological setting and their relative
constancy over long periods of time, ice and permafrost may indeed be the greatest
environmental setting for the long-term survival of endogenous nucleic acids on Earth.
Progress in this new field may be of great importance not only in microbial ecology and
evolutionary biology but also in the search for extraterrestrial chemistry such as the simplest
chemical forms of “life” such as amino acids or simple ribonucleotides, on planet Mars and
Jupiter’s moon Europa that are covered with thick permafrost and ice (Box 3). Yet before this
is to become a full fledge area of biological research much work on the level of diversity and
the survival of nucleic acids and microorganisms in these environments is needed. Strict
adherence to the above-mentioned precautions, controls, and criteria (Table 1) even though
expensive and time consuming are essential to establish reputable claims. Failure to rule out
issues of contamination will leave all ancient DNA results the topic of speculation. It is
therefore essential that journal editors, reviewers and researchers alike subscribe to “all”
criteria of authenticity not just a subset of these.
Acknowledgments
We thank J. Bada, A. Cooper, D. Gilichinsky, S. Bulat, D. Fisher, D. Dahl, J. P. Steffensen I.
Barns, T. B. Brand, S. Mathiasen, T. Quin, B. Schlaf, R. Rønn, T. Mourier, S. O’Rogers and
J. Castello for help and discussion. EW and AJH were financially supported by the
VILLUMKANN RASMUSSEN Foundation, Denmark and HNP by the Max Planck Society.
EW and AJH have contributed equally to the work.
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Fig. l. Some main areas for permafrost and ice core drillings on
the northern hemisphere that are discussed in the text (photos by
D. Gilichinsky and H. Højmark).
Fig. 2. DNA/RNA sequences and viable cultures reported from glacial ice and permafrost of various
age. The figure does not include the many bone and soft tissue remains reported from permafrost
settings. Kyr= thousand of years before present. Ma= million of years before present.
Fig. 3. A) Spontaneous hydrolytic damage of DNA (left) and RNA (right). Different sized arrows indicate
principle sites of damage and their relative frequency. B) Base products produced by oxidative damage. C)
Damage pathways of DNA exemplified by deamination of Cytosine, cleavage of the phosphor backbone and
cleavage by depurination and subsequent ß-elimination (Figure A and B are redrawn from ). For details see Box
1.
Fig. 4. Long-term survival of 100bp of DNA as a function of the rate of
depurination and temperature. The calculations are based upon a genome
size of 3.0x106bp, Arrhenius equation and depurination kinetics of Lindahl
and Nyberg (52) i.e. depurination rate of 4x10 -9 sites sec-1 at 70 C, pH 7.4,
an activation energy of 31 kcal/mol. It is assumed that the positions of
damage are distributed equally over the genome.
Table l. Precautions, controls, and criteria for ice and permafrost genetics and culturing
Procedure
Precaution
Controls
Criteria
Drilling
Fluid based drill (ice) / mechanical drill Recognisable
Empty controls
Storing
(permafrost)
contaminant
IRg
Sterile gloves, face mask, cap
a
added
Independent
Sterilized tools
reproducibility
Cloning
Inspection for cracks Freeze laboratory (ice)c
Time dependent
Airf
(ice)b
d
patternsi
Dedicated clean laboratory (permafrost)
Dividing
for Positive flow bench or glow box
Quantificationj
replication
Sterile gloves, face mask, cap
Preservationj
Removal of surface
Sterilized microtome knife
Melting (ice)
Concentration (ice)
DNA/RNA extractions
PCR set up
Culturing
Dedicated clean laboratoriesd
Positive flow bench or glow box
Sterile glows, face mask, cap
Cleaned reagents, tools, tubesh
Airf
Filter (ice)
Extraction (ratio
1:5)
PCR (ratio 1:1)
Multiple media
aIf possible spike drill equipment and fluids with known, easily recognizable, cultures and variable length nonhuman, non-microbial DNA, that can be easily amplified by PCR to look at the effects of penetration.
bUse light table.
cWith positive flow bench.
dIsolated from other laboratories, fully equipped with a positive flow hood or glove box, separate ventilation
system, nightly UV-radiation. Due not use the same clean laboratories for DNA/RNA work and culturing.
eTreated with 5% Sodium hypochlorite.
fAir filter detecting possible contamination from the air within the positive flow hood or glove box.
gInverse relationship between amplification strength and fragment length.
hUV-radiation, baking silica, acid, bleach and ultra-filtration.
iTime dependent patterns in e.g. diversity and DNA/RNA damage if comparing samples of different ages
preserved at similar conditions.
JDNA/RNA quantification and indirect/direct evidence of DNA/RNA preservation by. e.g. aminoacid
rasmisation or mass/gas spectrometry should be added to the list of criteria for surprising results largely
contradicting
theoretical
expectations
for
DNA/RNA
survival.
Box 1. DNA and RNA damage
The DNA and RNA molecules are particularly prone to spontaneous hydrolytic damage.
Cleavage of the phosphodiester bond in the sugar backbone by hydrolytic attact, results in
single stranded breaks. In a fully hydrated system phosphodiester cleavage in DNA happens
about once every 2.5 hours at 37°C (a). Single stranded nicks can also be generated by
hydrolytic cleavage of the glycosidic bonds connecting the bases to the sugars causing base
loss (depurination and depyrimidation) and subsequent strand cleavage by ß-elimination (Fig.
3,a,c). In hydrated DNA, both depurination and ß-elimination takes place once per 10h at
37°C. Depyrimidation is estimated to occur with a rate 5% of that for depurination (b). The
lack of the 2’OH group in DNA compared to RNA increases the strength of the
phosphordiester bonds of the sugar backbone but weakens the bond that joins the bases to the
sugars. Thus, RNA has a slower rate of depurination than DNA, yet direct cleavage of its
phosphodiester bonds will be rapid (c) and, therefore, RNA molecules does not survive as
long in the geophere as DNA (Fig. 3a). Single strand nicks are largely responsible for the
reduction in number of amplifiable template molecules in fossil remains, the short
amplification products (usually100-500bp) and lesions blocking the action of the DNA
polymerase (Table 1).
The miscoding bases hypoxanthine, uracil, thymine, and xanthine can be generated by
hydrolytic deamination (hydrolytic cleavage of their amino groups) of adenine, cytosine, 5methylcytosine and guanine respectively (Fig. 3c). Cytosine is particularly prone to this
reaction with about one event every 700 hours in a fully hydrated system at 37°C (a).
Miscoding lesions does not block the DNA polymerase enzyme but cause misincorportation
of erroneous bases during PCR (Table 1).
Free radicals e.g. peroxide (.O2) and hydroxy (.OH) as well as hydrogen peroxide
(H2O2) cause oxidative damages to the DNA and RNA molecules. These radicals are likely to
play an important role in limiting the half-life of DNA and RNA molecules in the geosphere
(a-b, d). Many oxidative lesions block the extension of the polymerase enzyme during PCR
preventing amplification and eventually causing chimera sequences via “jumping PCR” (Fig.
3b, Table l).
The DNA molecule is also prone to condensation type reactions where the exocyclic
amine groups can react with the carbonyl groups of reducing sugars (Maillard products)
causing DNA-protein crosslinks (e). In addition, base-less sites can result in the formation of
interstrand DNA crosslinks (f). Crosslinks will prevent DNA amplification (Table 1).
Table l. Damage in ancient DNA and their effects on PCR amplification
Processes
Modification
Effects
Hydrolysis
Strand break
Few template molecules → Contamination
Short fragment length → Short PCR products
Hydrolysis
oxidation
Oxidation
and Base modifications
Blocking lesions → “Jumping PCR”
Miscoding lesions → base-misincorporations
Sugar
residue Helical distortion → effect on PCR unknown
modifications
Oxidation
and Cross links
Maillard reaction
DNA-DNA cross links → nonamplification
DNA-protein cross links → loss of DNA
References
a. Shapiro, (1981) Damage to DNA coursed by hydrolysis. In Chromosome damage and
repair (Seeberg, E. and Kleppe, K. eds) pp. 3-18, Plenum, New York
b. Friedberg, E.C. et al. (1995). DNA Repair and Mutagenesis 698 pp., Washington,
D.C., ASM Press
c. Lindahl, T. (1993) Instability and decay of the primary structure of DNA Nature 362,
709-715
d. Höss, M. et al. (1996) DNA damage and DNA sequence retrieval from ancient tissues.
Nucleic Acids Res. 24, 1304-1307
e. Poinar, H.N. et al. (1998) Molecular coproscopy: Dung and diet of the extinct ground
sloth Nothrotheriops shastensis Science 281, 402-406
f. Goffin, C. and Verly, W.G (1983) Inter strand DNA crosslinks due to AP
(apurinic/apyrimidinic) sites FEBS Letters 161, 140-144
Box 2. Conflicting results
Despite preliminary success in the retrieval of DNA/RNA sequences and viable microbes
from polar ice and permafrost (Fig. 2), there remains a solid lack of independent
reproducibility of results. E.g. Neither Christner et al. (a) nor we (Table l) obtained viable
cells or amplification products from the GISP2 or the GRIP ice cores respectively (both
drilled at Summit, Greenland, Fig. 1) using several liters of ice, despite previous reports to the
contrary from just a few milliliters of ice melt (b-c). Others claim the revival of viable
microbial cells and amplification of viral RNA (350bp) (d) from ice core samples up to 100
kyr, from the Vostok drill site in Antarctica (e) or the GISP2 site in Greenland (b-c). Still,
Willerslev et al. (f) could not obtain amplification products as small as 550bp from core
samples 2-4 kyr drilled at the low latitude Hans Tausen ice cap (Fig. 1, Table l) with a
relatively higher microbial cell number (se main text). Furthermore, Priscu et al. (g) and
Christner et al. (h) claim to have found endogenous bacterial DNA (~1.4kb and ~900bp,
respectively) in ice core samples derived from refrozen water of Lake Vostok, an ancient lake
beneath the East Antarctic Ice Sheet. However, according to Tanner et al. (i) one of the
organisms (Afipia) identified by Priscu et al. (g) is a common laboratory contaminant. In
addition, the only sequence identified to the same genera by the two science teams
(Aquabacterium) is by Priscu et al. (g) regarded to be a contaminant as they obtained it from
the blank controls, despite Christner et al. (h) claiming it to be endogenous. Attempts by
Russian scientists to reproduce the results by Priscu et al. (g) have failed (S. Bulat
unpublished). Likewise, recent attempts to obtain microbial DNA sequences from 1.5-2 Ma
permafrost samples from Kolyma Lowland and Laptev Lowland (Fig. 1) have failed (E.
Willerslev et al. unpublished) despite previous claims of viable cultures in the samples (j-k).
These conflicting results could be attributed to differences in methodological efficiency, but
are more likely due to contamination. This call for clear standardized procedures to be
established within the field.
Table 1. Summary of amplification results from Greenland ice cores
Vol.
Age
Ice coresa
Amplification products (bp)b
IRc
(litres)
(yr B.P.) 160 180 340 550 Controls
DNA found
GRIP, Summit, Greenland
72,30ºN, 37,30ºW, 3232 masl.
Dye 3, southern Greenland
65,11ºN, 43, 50ºW, 2480 masl.
Renland, eastern Greenland
71,10ºN, 26,43ºW, 2340 masl.
Hans Tausen, North, Greenland
82,5ºN, 37,5ºW, 1270 masl.
3.5
350
-
-
-
-
-
NA
NA
4.6
250
-
-
-
-
-
NA
NA
5.7
5000
y
y
y
y
-
-
3
3
2000
2500
y
y
y
y
y
y
-
-
Y
Y
Mammalian
(contamination)
Microbial & plants
Microbial
3
4000
y
y
y
-
-
Y
Microbial
a The GRIP, Dye 3 and Hans Tausen ice cores were drilled using fluid and the Renland ice core was drilled without
drilling fluid.
bPresence (y) or absence (-) of visible amplification products of different length.
cPresence (y) or absence (-) of an inverse relationship between amplification efficiency and fragment length. NA= not
applicable.
References
a. Christner, B.C. et al. (2000) Recovery and identification of viable bacteria
immured in glacial ice. Icarus 144, 479-485
b. Catranis, C. and Starmer W.T. (1991) Microorganisms entrapped in glacial ice.
Antarctic Journal of the United States 26, 234-236
c. Ma, L-J. et al. (1999) Detection and characterization of ancient fungi entrapped
in glacial ice. Mycologia 92, 286-295
d. Castello, J.D. et al. (1999) Detection of tomato mosaic tobamovirus RNA in
ancient Glacier ice. Polar Biol. 22, 207-212
e. Abyzov, S.S. (1993) Microorganisms in the Antarctic ice. In Antarctic
Microbiology (Friedmann, E.I., ed), pp. 265-295, Wiley-Liss, New York
f. Willerslev, E. et al. (1999) Diversity of Holocene life forms in fossil glacier ice.
Proc. Natl. Acad. Sci. USA 96, 8017-8021
g. Priscu J.C. et al. (1999) Geomicrobiology of subglacial ice above Lake Vostok,
Antarctica. Science 28, 2141-2144
h. Christner, B.C. et al. (2001) Isolation of bacteria and 16S rDNAs from Lake
Vostok accretion ice. Environmental Microbiology 3, 570-577
i. Tanner, M.A. et al. (1998) Specific ribosomal DNA sequences from diverse
environmental settings correlate with experimental contaminants Appl.
Environmantal Microbiol. 64, 3110-3113
j. Vorobyova, E. et al. (1997) The deep cold biosphere: facts and hypotheses.
FEMS Microbiol.Rev. 20, 277-290
k. Shi, T. et al. (1997) Characterization of viable bacteria in Siberian permafrost by
16S rDNA sequencing. Microbial Ecol. 33, 169-179
Box 3. The hunt for extraterrestrial life-forms
The interest for detecting extraterrestrial life has been intensified in the past few years due to
the recent success of MARS landings. However, currently there is no solid evidence of past or
present life-forms outside Earth and central issues in exobiology continue to be the search for
“life” on the Planet Mars and one of Jupiter’s moons Europa (Fig. 1a,b). Both are among the
main candidates in the hunt for life beyond Earth due to assumptions of free water in the past
(Mars) or at present (Europa). For both Mars and Europa, ice plays a central role in this
search: Mars has ice caps at each pole that are believed to go approximately 100 Ma back in
time (a) and have a ground floor of permafrost. Europa is believed to contain an ocean
covered by a surface of several kilometers of thick ice. While direct search for life on Europa
may be far in the future, the hunt for life on Mars is already taking place.
There are two routes by which simple biomolecules such as amino acids could have been
shared as the genetic material between Earth and Mars: (i) by convergent evolution and (ii) by
Panspermia. The first idea is supported by evidence suggesting that Earth and Mars were
ecologically rather similar 3.5 to 3.9 billion years ago (the time period when life evolved on
Earth), both having a thick atmosphere of CO2, volcanic activity and surface water (b) and by
“The Miller-Urey primordial soup experiment” which showed simple amino acids and purine
bases amongst its products (c). The Pernspermia Hypothesis (d) that life may have traveled
planets is supported by findings of nucleic acids in meteorites (e) and by the idea that there
has been a significant exchange of material by impact between Earth and Mars over several
millennia (f).
Since the Martian polar ice caps, are believed to sustain temperatures between –50°C and
-110°C (g) they are probably a suitable place to look for remains of amino acids and purine
bases. The same goes for the Martian permafrost. Very rough calculations using the Arrhenius
equation (see main text) suggest that 100bp of DNA can survive 3.4x109- 3.1x1021 years at 50°C and -110°C, respectively (Fig. 4). Although the calculation is highly simplified it does
suggest that any nucleic acids on Mars may be preserved at time scales way beyond what can
be expected on Earth and devices to recover nucleic acids and analogs from the Martian ice
caps has already been suggested (h).
Fig. 1. a) Europa, one of Jupiter’s moons covered with a thick shell of ice. b) Planet Mars. c) The ice cap at
Mars South Pole (From: http://photojournal.jpl.nasa.gov/).
References
a. Fisher, D.A. et al. (2002) Lineations on the ”White Accumulation Areas of the
Residual Northern Ice Caps of mars: Their Relation to the “Accublation and Ice Flow
Hypothesis. Icarusi. 159, 36-56
b. McKay, C.P. (1998) Life on Mars. In The molecular origins of life (Brack, A. ed) pp.
386-406, Cambridge University press, UK
c. Miller, S. (1953) A Production of Amino Acids Under Possible Primitive Earth
Conditions Science 117, 528-529
d. Raulin-Cerceau, F. et al. (1998) From Panspermia to Bioastronomy, the evolution of
the hypothesis of universal life Orig. Life. Evol. Biosph. 28, 597-612
e. Storks, P.G. and Schwatz, A.W. (1981) Geochim. Cosmochim. Acta. 45, 563-568
f. Chyba, C.F. et al. (1990) Commentary delivery of organic molecules to the early
Earth Science 249, 366-373
g. Fisher, D.A. ( 2000) Internal layers in an “Accublation” Ice Cap: A Test for Flow
Icarus 144, 289-294
h. Hansen, A.J. et al. (2003) JAWS: Just add water system – a device for detection of
nucleic acids in the Martian ice caps. Proceedings of the Second European workshop
on Exo/Astrobiology 309-311