Supporting data for online publication
SINGLE MOLECULE PCR PROTOCOL
Estimation of the number of template copies
DNA concentration was measured using a NanoDrop® ND-1000 spectrophotometer and was
diluted to a concentration of approximately 103 mitochondria /µl. Assuming that 1 million base
pairs weighs 1pg (1x10-3ng), 1 mitochondrial genome of approximately 17,000 base pairs weighs
1.7x10-3ng. Fifty microtitre aliquots (single use aliquots) at a concentration of 103
mitochondria/µl (hereafter described as stock DNA) were stored at -20oC. Stock DNA was
serially diluted and dispensed in a 96-well microtitre plate in order to obtain 5 different DNA
concentrations (16 wells per concentration and 16 negative controls). In order to assess which
DNA concentration conforms to expectations from smPCR, DNA was subjected to the hemi
nested smPCR protocol (see below) and the proportion of positive wells for each DNA
concentration was estimated. According to a Poisson distribution, 36.8% of the amplifications
from DNA at a concentration of a single molecule are not expected to contain a molecule of the
desired template, another 36.8% are expected to contain a single molecule, and the remainder are
expected to contain multiple molecules (Stephens et al. 1990). In order to decrease the number of
false positives (positive amplifications resulting from multiple molecules), template with a final
concentration of 0.3 amplifiable molecules should be used, meaning that approximately one third
of the amplifications will yield a product derived from a single molecule template and less than
5% of positive amplifications will be derived from multiple molecules (Kraytsberg et al. 2004).
Therefore, only amplifications derived from DNA dilutions that resulted in a proportion of
positive amplifications conforming to a DNA concentration of 0.3 amplifiable molecules are
sequenced.
Selection of loci and design of PCR primers
In order to increase PCR specificity a hemi nested PCR approach was used, which involves the
use of two sets of primers employed in two successive PCR reactions (hereafter described as
Phase I and Phase II PCR). During Phase I PCR the first set of primers, that includes the
forward-external (FEXT) and the reverse primer (REXT), are used to generate a DNA product
that is longer than the final target sequence (Fig. 1.). The product from Phase I PCR is then used
to start a second PCR (Phase II) using a set of primers that involves the same reverse primer used
in Phase I and a new forward primer (forward-internal, FINT). The forward-internal primer
binding site is located within the first amplified sequence, in a region nearby the forwardexternal primer binding site.
To assure that the low numbers of positive amplifications (which are expected in a smPCR
approach) are the result of single molecule amplifications and not due to PCR inefficiency, two
fragments (amplimers) from different regions of the mitochondrial genome were amplified. Cosegregation of the two markers would re-assure PCR effectiveness; therefore if an aliquot gives a
positive amplification for one of the markers, then the same aliquot should give a positive
amplification for the other marker. If the two markers segregate independently then low number
of amplifications might be due to PCR inefficiency. The markers chosen were a fragment of the
cytochrome b gene (CYTB amplimer) and a fragment that spans part of the 12S and 16S
ribosomal genes (12S amplimer) (Fig. 2).
To design specific Lacerta lepida primers to amplify the CYTB amplimer the entire cytb gene
was first amplified with modified versions of primer L14919 (TRNAGLU, 5’- AAC CAC CGT
TGT ATT TCA ACT - 3’) and L16064 (TRNATHR, 5’- CTT TGG TTT ACA AGA ACA ATG
CTT TA - 3’) (Burbrink et al. 2000) using the conditions described in the manuscript.. After
aligning the entire cytb sequences, specific primers for Lacerta lepida were designed (CYTBFEXT, 5’-TTA CAA AAT TAT TAA CTC CTC CT - 3’; CYTB-FINT, 5’ - GCC TAT GTC
TTA TTA TTC AAG - 3’ and CYTB-REXT, 5’ - GGT TTA CAA GAA CAA TGC TTT A 3’). The final CYTB amplimer is 1143 bp long. For the 12S amplimer, published (Paulo et al.
2008) partial sequences of 12S and 16S genes from Lacerta lepida were aligned and specific
primers were designed in order to amplify a fragment of similar size to that of the CYTB
amplimer. As there are no published sequences for the entire 12S and 16S genes from Lacerta
lepida the published mitochondrial genome of Lacerta viridis (Böhme et al. 2007) was used to
estimate the approximate final size of the amplimer. The primers designed for the amplification
of the 12S amplimer were: 12S-FEXT (5’ - GCA AAT GTT AGG GAA GAG AT - 3’), 12SFINT (5’ - CTA TTT TAA CAA CGC TCT GGG - 3’) and 16S-REXT (5’ - GAG TCA CTG
GGC AGG CAA GA - 3’). The final 12S amplimer is 933 bp long.
PCR amplifications, scoring and sequencing
Phase I PCR consisted of the amplification of both markers using a multiplex approach in a mix
containing 1x PCR Gold buffer (Perkin-Elmer), 4 mM MgCl2, 200 mM each of dATP, dCTP,
dGTP and dTTP (Applied Biosystems), 0.2 mM of forward-external and reverse external primers
(Operon Technologies) for both markers (see above primer selection for each loci) and 0.5U Taq
Gold DNA polymerase (Perkin-Elmer). Five microlitres of this mix were added to 5µl of
template DNA which was previously prepared by performing serial dilutions from stock DNA
and dispensed in a 96 well plate under 1 drop of oil. Negative controls (no DNA) were included
for all amplifications. In order to avoid contamination, Phase I PCR was prepared in a room
separated from any PCR products and DNA solutions at high concentration. Amplifications were
conducted in the normal lab as follows: 93oC for 9min, then 28 cycles of 94oC for 20s, 50oC for
30s, 72oC for 90s. Phase I PCR products were diluted to 1000µl with bi-distilled water (ddH20)
and 5µl aliquots of it were used to perform the Phase II PCR. Phase II PCR consists of
amplifying the two markers independently in two monoplex PCRs. The 5µl aliquots of diluted
Phase I PCR products are supplemented to give a 10µl final volume containing 1 mM each of the
relevant forward-internal and reverse primers, 1x PCR Gold buffer, 4 mM MgCl2, 200 mM each
dNTP and 0.2U Taq Gold DNA polymerase. Amplifications were conducted as follows: 93oC for
9min, then 33 cycles of 94oC for 20 s, 54oC for 30 s, 72oC for 90 s.
PCR products were analyzed by agarose gel electrophoresis (2%), scoring presence and absence
of the expected PCR product. Positive products that could be identified as obtained from a single
molecule were purified by filtration through QIAquick® columns (Qiagen) following
manufacturer’s recommendations and the CYTB amplimer was sequenced in both directions
using Phase II PCR primers. Sequencing reaction mixes consisted of 6.35l of ddH2O, 1.5l of
primer at 3.5M, 1l of BigDye Terminator v3.1TM (Applied Biosystems) and 1l of PCR
product. Sequence reactions were performed as follows: initial incubation at 96ºC for 1min; 25
cycles of incubation at 90ºC for 10s, 50ºC for 5s and 60ºC for 4min.
Fig. 1 Schematic representation of smPCR nested design.
Bp 2540
12S
943bp
Fext_12S
(starts at bp702)
65bp
tRNA-val
Bp 0
16S
1532bp
Fint_12S
(starts at bp811)
Rext_16S
(starts at bp1873)
1062 bp
Bp 1143
Bp 0
CytB
1143 bp
Fint_CytB
(starts at bp116)
Rext_CytB
(starts at bp1049)
Fext_CytB
(starts at bp33)
933 bp
Lacerta lepida sequences available at Genebank
Fragment to be amplified by smPCR (amplimers)
Fig. 2. Schematic representation of two amplimers to be amplified by smPCR and the position of
the primers used in the nested PCR. Base pair numbers in the first scheme are set according to
Lacerta viridis publised 12S and 16S genes, and in the second scheme are set according to
Lacerta lepida cytb gene.
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