ABSTRACT
Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only
a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii)
that PCR-generated alleles or `false alleles' will arise. A mathematical model has
been developed to account for stochastic events when pipetting template DNA in
a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results
correlate with the mathematical model. The results of 150 typing experiments
are consistent with the proposed model. Based on this model and the level of
observed false alleles, an experimental procedure using the multiple tubes
approach is proposed to obtain reliable genotypes with a confidence level of
99%. This multiple tubes procedure should be systematically used when
genotyping nuclear loci of ancient or forensic samples, museum specimens and
hair or feces of free ranging animals.
The polymerase chain reaction (PCR) (
1
-
3
) is an enzymatic process by which a specific region of DNA is replicated
repeatedly to yield several million copies of a particular sequence. Typically,
nanogram to microgram quantities of genomic DNA are used as a template for the PCR. This corresponds to ~300-300 000 copies of a unique target sequence. Refinements of the
standard technique have even enabled the amplification of DNA extracted from a
single spermatozoid (see for example
4
-
6
), demonstrating that a single molecule can be reliably amplified (
7
). In order to improve the sensitivity of the reaction when the template DNA
contains very few copies of the target sequence, a two-step PCR can be performed (
8
). Because of this extreme sensitivity, there is a high risk of contamination,
against which strict measures must be taken (see for example
9
,
10
).
In molecular studies using ancient samples, forensic samples, museum specimens
and hair or feces of free ranging animals, the amount of DNA available for
genetic typing can be very low and is often in the picogram range. Under these
circumstances, it is possible for one allele of a heterozygous individual not
to be detected, leading to an incorrect genotyping of this individual as a
homozygote. This kind of problem has been encountered in a microsatellite study
using Bonobo (
Pan pygmeus
) feces (
11
). A theoretical study by Navidi
et al.
(
12
) suggested that the use of a multiple tubes approach, distributing the extract
DNA among several tubes, provides more reliable genotyping of dilute DNA
samples than a single tube approach. To our knowledge, this has not been tested
experimentally.
In order to identify each individual of the endangered Pyrenean brown bear
population (
Ursus arctos
), we are currently studying microsatellite polymorphism of DNA extracted from
hairs and feces collected in the field (
13
,
14
). Our preliminary results clearly demonstrated that the amount of extracted DNA
varies greatly among samples and that an incorrect conclusion of homozygosity
may be reached for some extracts. We therefore decided to use the multiple
tubes approach (
12
). Our basic purpose was to determine how many times an experiment would have to
be repeated in order to obtain a reliable genetic typing of a homozygous locus,
with the constraint that only a limited amount of template DNA is available.
So, the greater the number of PCRs, the fewer the number of loci that can be
analysed.
Furthermore, we initially obtained many more genotypes than expected, with each
additional genotype corresponding to a previously observed genotype but with a
single difference: one extra allele at an otherwise homozygous locus. We tried
repeating experiments in order to confirm the presence of these alleles, but
they were not reproduced, even with six or seven repetitions. The problem
became even more complex when three alleles were obtained at the same locus for
some heterozygous individuals. One possible explanation for these unusual
results is sporadic contamination, either by cross-contamination between extracts or by PCR product carry-over. However, in our case, the contamination hypothesis can be ruled out. First, the alleles concerned were
always obtained for the first time in our laboratory, therefore, PCR product
carry-over cannot be responsible. Second, all of the bear DNA samples currently
in the laboratory have been analysed and do not carry the alleles concerned,
ruling out the possibility of cross-contamination. As an alternative hypothesis, we suggest that slippage
artifacts generated during the first cycles of the PCR may explain these false
alleles.
In this paper, we present an experimental procedure which provides reliable
genotyping using very low amounts of template DNA, taking into account (i) the
possibility of not detecting an allele in heterozygous individuals and (ii) the
problem of PCR-generated false alleles. Such procedures should be applied when a few
picograms of DNA are to be amplified to a detectable level, as is the case in
studies of forensic samples, museum specimens and fossils.
Our strategy consisted of developing a mathematical model which takes into account stochastic events when pipetting template DNA in a very
dilute DNA extract. Laboratory experiments were carried out in order to: (i)
determine if experimental results correlate with the mathematical model; (ii) estimate the frequency of PCR-generated artifacts. Based on the results obtained, an appropriate
experimental procedure was designed.
The model is restricted to the genotyping of an individual bearing alleles A and
B at an autosomal locus. The main purpose of the model is to take into account
the stochastic sampling of DNA molecules when pipetting the template DNA for
each PCR. The following assumptions have been made: (i) the DNA extract
contains equal numbers of the alleles A and B; (ii) a single target molecule
can be amplified and detected; (iii) each single target molecule has the same
probability of being amplified; (iv) 100 PCRs can be performed using the DNA
extract and the target DNA molecules are distributed randomly among the 100 PCR
tubes; (v) if the initial proportion between alleles A and B (A/B or B/A) in
the PCR tube is >= 5, then only the most common allele will be detected.
Assumption (iii) is related to previous studies concerning PCR on single
spermatozoids that have shown that only ~60-70% of each DNA molecule can be amplified (
4
-
6
), so only amplifiable DNA is considered later. Assumption (v) is included
because it is difficult to score an allele when the band intensity on the
autoradiograph is five times weaker than the other allele (
12
). Other proportions were also tested, but the results were not substantially
different.
The computer simulations were set up to randomly distribute the given amount of
extracted DNA (i.e. the given number of alleles A and B) among 100 PCR tubes
and then to analyse the results of the DNA amplification in each tube using the
above assumptions. One thousand replications of random sampling of alleles
without replacement were performed for 48 extracts of different DNA
concentrations. The amount of amplifiable DNA in the extracts ranged from one
to 1000 diploid genomes, corresponding to 0.01-10 diploid genomes/PCR tube.
As a precaution against contamination, the extraction procedure was performed in
a special room dedicated only to DNA extraction from ancient samples, hair and
feces. In order to produce enough template DNA for the subsequent
amplification, five DNA extractions were carried out at the same time for a
single feces sample of a brown bear. This sample was collected in the field by
J.-J.Camarra in the Pyrenees mountains. Extraction was performed using the
silica method (
15
-
17
). The bear feces sample was preserved dry until DNA extraction.
For each extraction, ~50 mg dry bear feces was added to 1 ml L6 extraction buffer (10 M GuSCN, 0.1 M Tris-HCl, pH 6.4, 0.02 M EDTA, pH 8.0, 1.3% Triton X-100). After incubation overnight at 60oC with constant agitation, 500 [mu]l of the liquid phase was added to 500 [mu]l fresh L6 extraction buffer and 40[mu]l silica suspension prepared as previously
described (
17
). The mixture was incubated at room temperature for 10 min with constant
agitation. After centrifugation (1 min, 7000
g
), the silica pellet was washed three times with 500 [mu]l L2 buffer (10 M GuSCN, 0.1 M Tris-HCl, pH 6.4), once with 1 ml 100% ethanol and once with 1 ml acetone.
The pellet was then dried at 60oC for 10 min and nucleic acids were eluted at 60oC for 5 min with 200 [mu]l TE (10 mM Tris-HCl, 10 mM EDTA, pH 8.3). The tube was centrifuged (3 min,
10 000
g
) and 160 [mu]l supernatant was carefully removed to avoid pipetting silica particles and transferred to a new Eppendorf tube. The tube was centrifuged again (3 min, 10 000
g
) and only 120 [mu]l was removed to ensure that no silica particles remained in the extract.
The products of the five extractions were then pooled, constituting the 1:1
extract. Two aliquots of this 1:1 extract were used to make two extract
dilutions of 1:2 and 1:4. Five microlitres of each extract was used as template
for each PCR. To detect whether contamination of samples with exogenous DNA had
occurred during the extraction procedure, a tube without bear feces (extraction
negative control) was treated identically through both the extraction procedure
and the amplification.
Fifty PCRs were carried out for each of the 1:1, 1:2 and 1:4 extracts. The DNA
amplifications were performed in a two-step PCR. The first step used diluted external microsatellite primers to
reduce the formation of primer-dimer artifacts (
8
). In the second step, a nested primer was introduced. Three primers were
designed from the sequence of locus G10B (
18
): G10BF (5'-AAGCCTTTTAATGTTCTGTTG-3'); G10BFI (5'-TGCTAATATTTTCTTGAGGACT-3'); G10BR (5'-AGGACAAATCACAGAAACCT-3'.
The first step was performed in a total volume of 25 [mu]l [750 mM Tris-HCl, pH 9.0, 200 mM (NH
4
)
2
SO
4
, 50 [mu]M each dNTP, 1.5 mM MgCl
2
, 5 ng BSA, 0.1 U Red GoldStar DNA polymerase (Eurogentec), 0.01 [mu]M primer G10BF, 0.01 [mu]M primer G10BR, 5 [mu]l extract] and a PCR amplification of 20 cycles was carried out (93oC for 30 s, 55oC for 30 s, 72oC for 1 min, using a Perkin Elmer Gene Amp PCR System
9600). Between the first and second steps, a volume of 25 [mu]l [750 mM Tris-HCl, pH 9.0, 200 mM (NH
4
)
2
SO
4
, 50 [mu]M each dNTP, 1.5 mM MgCl
2
, 5 ng BSA, 0.1 U Red GoldStar DNA polymerase, 1 [mu]M primer G10BFI and 1 [mu]M primer G10BR] was added to the same tube. The second step consisted of
35 cycles of amplification (93oC for 30 s, 55oC for 30 s, 72oC for 1 min). The PCR products were purified on a low melting
point agarose gel, diluted in 200 [mu]l ddH
2
O and 10 [mu]l were used as template for an additional amplification of two cycles (93oC 10 s, 55oC 30 s, 72oC 1 min) performed in a volume of 25 [mu]l [750 mM Tris-HCl, pH 9.0, 200 mM (NH
4
)
2
SO
4
, 50 [mu]M each dNTP, 1.5 mM MgCl
2
, 0.1 U Red GoldStar DNA polymerase, 0.2 [mu]M primer G10BF and 0.02 [mu]M [gamma]-
33
P-labelled primer G10RI]. Amplification products were separated by
electrophoresis on a 6% polyacrylamide gel (sequencing gel) for 2 h. This gel
was dried and exposed to autoradiography film.
The stochastic sampling of target DNA in very dilute extracts is illustrated by
a subset of the simulation results shown in Table
1
. The number of target alleles varies greatly among tubes and some tubes do not
contain any target molecules. Figure
1
shows the probabilities of obtaining a positive PCR (either allele A or B), only allele A or both alleles (correct genotype) when using different quantities of template DNA. One unit (U) of the template DNA corresponds to the amplifiable diploid content of one cell. For 1.5 U template DNA, the probability of obtaining a PCR product is 0.95. For 2.4 U
template DNA, the probability of obtaining a PCR product is 0.99, but the
probability of obtaining a correct genotype is only 0.80. The probabilities of
identifying the correct genotype at the 0.95 and 0.99 levels are obtained for
5.0 and 8.0 U template DNA respectively. The maximum probability of obtaining
only one allele is for 0.7 U template DNA. Figure
2
considers only positive PCRs and presents the probabilities of obtaining a
correct genotype and an incorrect genotype (e.g. only allele A) using different quantities of template DNA. The probability of obtaining only allele A decreases rapidly when using more template DNA.
A mathematical model has been developed to design a procedure to obtain reliable
genotyping based on PCR when using very low quantities of template DNA. This
model takes into account the random sampling of template molecules in the
extract and assumes that a single template molecule can be detected. The results of 150 typing experiments clearly show that the genotyping of very dilute DNA samples follows a stochastic process (Fig.
3
and Table
2
), consistent with the proposed model.
Based on our proposed mathematical model, the probability of obtaining a correct
genotype at the 99% confidence level corresponds to the use of 8 U template
DNA, which is ~56 pg DNA for mammals. However, the probability of obtaining a PCR product
at the 99% level is reached at only 2.4 U template DNA. Therefore, when using
between 2.4 and 8 U template DNA, there is a high risk of not detecting one
allele, despite obtaining positive PCRs in almost all experiments. This problem
is illustrated in Figure
1
by the gap between the two curves corresponding to the probability of obtaining
a positive PCR and of obtaining a correct genotype. In our experience, when
working with bear hairs and feces collected in the field, the amount of
template DNA used per PCR is usually <8 U and therefore the risk of missing one allele is almost always present.
Excluding the possibility of sporadic contamination, as explained in the
Introduction, an alternative explanation can be proposed for the observation of
false alleles. Amplification of microsatellite loci often produces shadow bands
below the main band. These artifacts correspond to smaller numbers of repeats
in the microsatellite array (
19
-
21
) and could result from slippage during the amplification process. If a single
target DNA molecule is used as template and if such a slippage occurred during
the first cycle of the PCR, then this artifact could be amplified in
approximately the same proportion as the true target molecule and would be
visible on the autoradiograph. If this hypothesis is true, then the occurrence
of false alleles should be proportional to the intensity of shadow bands
observed in microsatellite loci and the most common false alleles should
correspond to the most intensive shadow bands. In addition, all intermediates
between the most intensive false alleles (due to slippage during the first
cycle) and very faint false alleles (due to slippage during the following few
cycles) should be observed. At present, we have carried out ~1000 typing experiments involving dinucleotide repeat loci using brown bear
hair or feces as a source of DNA. Despite the difficulty in scoring false
alleles due to large variations in band intensity, the occurrence and the
length of the false alleles observed seem to support all three of the
assumptions above (data not shown).
The guidelines we propose for genotyping very dilute DNA samples are only valid
under the following conditions: (i) a single target molecule can be detected;
(ii) the amount of template DNA is very low, in the picogram range, but is not
accurately known. Our goal was to obtain a reliable genotyping, with a
confidence level of 99%, taking into account the stochastic sampling of
template DNA, the possibility of generating false alleles and the risk of
contamination. One approach would be to concentrate the extract and to perform
a single PCR per locus. The results of the computer simulation demonstrate the
dangers of the one tube approach with `concentrated' extract. The problem is
that the quantity of extracted DNA is unknown and this quantity could be high
enough to give a PCR product, but insufficient for a reliable genotyping (Fig.
1
). Therefore, we propose the multiple tubes approach as a more reliable method
that can monitor the three potential sources of errors: stochastic sampling,
false alleles and sporadic contamination. When using the multiple tubes
approach, it is important to determine the number of experiments that are
necessary to obtain a reliable genotype.
We would like to thank Christiane Dubois-Paganon for her valuable technical help during the genetic analysis. This
work was supported financially by the University Joseph Fourier, the Centre National de la Recherche Scientifique, the French Ministry of the Environment (DRAEI-EGPN and DNP) and a LIFE Programme of the European Union.
REFERENCES
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