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DNA fragments can be analyzed for discovering their nucleotide sequence; this represents
the more detailed information that can be obtained from a DNA region.1 Walter Gilbert and
Frederick Sanger shared the Nobel Prize in Chemistry in 1980 “for their contributions
concerning the determination of base sequences in nucleic acids.” With this information, it is
possible to explore gene sequences, regulatory elements, make comparisons between
homologous genes across species, and identify mutations. Sanger’s method, known also as
dideoxy sequencing or chain termination, is based on the use of dideoxynucleotides
(ddNTPs) in addition to the normal nucleotides [deoxynucleotides (dNTPs)] found in
DNA.2 ddNTPs are the same as nucleotides except for a hydrogen group on the 3′ carbon
instead of a hydroxyl group (OH). These modified nucleotides, when integrated into a
sequence, prevent the addition of further nucleotides because a phosphodiester bond cannot
form between the ddNTP and the next incoming nucleotide, and thus, the DNA chain is
terminated.3 This process will produce lots of fragments, all with different lengths attributed
to the random integration of the ddNTPs. Automated sequencing has been developed in order
to perform all reactions in a single tube containing the 4 ddNTPs, each labeled with a
different color dye that fluoresces at a specific wavelength. Fluorescence emissions are
read via a machine performing capillary electrophoresis.1,2 In this way, laser determines the
identity of each fragment according to the wavelengths at which it fluoresces. The results are
then depicted in the form of a chromatogram, which is a diagram of colored peaks that
correspond to the nucleotide in a specific location of the sequence.1
In some cases, chromatograms resulting from a Sanger sequencing can present some
problems, concerning the following: 1) the lack of sequence data due to the absence of the
priming site, degraded primers, inefficient primer binding, insufficient amount of DNA
template, degraded DNA, and inhibitory contaminant in the sample (i.e., salts, phenol,
EDTA, and ethanol); 2) low peaks throughout because of an insufficient amount of DNA
template or inhibitory contaminant in the samples (i.e., salts, phenol, EDTA, and ethanol),
insufficient amount of primer, and inefficient primer binding; 3) poor sequence at the start
followed by weak signal attributed to self-complementarity of the primers; 4) overlapping
peaks in the sequence data because of multiple priming sites, residual primers, poor
purification during primer synthesis, mixed plasmid prep, and insertion or a deletion in PCR
product; 5) sequence starting well but signal weakening gradually because of the formation
of secondary structures or too much template; 6) overlapping peaks following a stretch of
mononucleotide sequence attributed to the enzyme slippage giving varying lengths of the
same sequence following this region; and 7) artifacts with large peaks obscuring the real
sequence due to dye blogs caused by unincorporated dye.4
Here, we describe a curious situation that can double in size the sequence depicted in the
chromatogram because of the massive presence of repetitive sequences in the genome of
interest. In our case, the genome in consideration, belonging to Triticum, is composed of
approximately 80% of repetitive sequences.5
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A couple of primers (forward, 5′-ATCTCCCAGCAACCATCTTGAC-3′; reverse, 5′-
GAAATCATCCGGTCTCCGATT-3′) were designed with Primer36 to amplify a specific
DNA fragment of 230 bp from durum wheat. PCRs were performed in a 9700 thermal cycler
(Applied Biosystems, Foster City, CA, USA) in 10 µl reaction mixtures containing 50 ng
template cDNA, 0.02 µM forward and reverse primer, 0.2 mM concentration of each dNTP,
1× buffer, 0.4 U Taq DNA polymerase (Life Technologies, Carlsbad, CA, USA), and 1.5
mM MgCl2. Amplification was achieved using 3 minutes of initial denaturation at 94°C, 38
cycles of 30 seconds at 94°C, 30-second annealing at 58°C, and 30-second extension at
72°C, followed by a final 5-minute incubation at 72°C. The fragment amplified was checked
on 2% agarose gel with a 100-bp molecular size standard (Fermentas, Vilnius, Lithuania).
The band corresponding to the fragment of interest (Fig. 1) was cut and purified using a kit
for nucleic acid purification (MACHEREY NAGEL, Duren, Germany). Finally, sequencing
reactions and purification were carried out with the GenomeLab Dye Terminator Cycle
Sequencing Quick Start Kit using the standard protocol provided by Beckman Coulter (Brea,
CA, USA). The fragment was sequenced with both forward and reverse primers. The
reaction volume was set at 10 µl; the reaction mix was prepared with dye terminator cycle
sequencing (2 µl), sequencing reaction buffer (0.5 µl), H2O (2.54 µl), the appropriate amount
of the target fragment (4.4 µl), and the sequencing primer (0.56 µl of a 40 µM stock
solution). Sequencing reactions were achieved after 40 cycles, each one following a 20-
second denaturation at 94°C, 20-second annealing at 54°C, and a 4-minute extension at
60°C. Finally, 5-minute incubation at 4°C was carried out for stopping the reaction. PCR
products were analyzed on an automated sequencer (CEQ 8800; Beckman Coulter), and
chromatograms were visualized using the proprietary software for sequence analysis.

Figure 1
Electrophoresis gel and chromatograms. (A) Electrophoresis agarose gel shows the band of the
expected size running with a 100-bp DNA ladder. (B) The aligned chromatograms result from the
sequencing reactions of the target fragment using the forward (S-forward) and reverse primer (S-
reverse). S-reverse chromatogram was reversed and complemented (rev-com) for clearness.

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An amplification experiment was carried out on a target DNA fragment from Triticum
durum using specific primers. We obtained a band of the expected size (230 bp), which was
visualized on an agarose gel (Fig. 1A). After the excision of the band from the gel, the
fragment was purified and sequenced with forward and reverse primers. When analyzing the
data output, the chromatogram resulting from the sequencing reaction with forward primer
(S-forward) was almost double in length (412 bp) compared to the one expected. On the
contrary, the sequencing reaction with the reverse primer (S-reverse) returned a sequence
sizing around the expected length (193 bp). The S-forward sequence had a peak
corresponding to an adenine in position 214, which was about 5 times higher compared to the
average peak height (Fig. 1B).
The S-forward sequence was used as a query for an alignment against the S-reverse sequence
reversed and complemented (Fig. 1B), to verify the nucleotide composition of the expected
fragment. It was also aligned with a 230-bp reference sequence from
another Triticum species. In these alignments, the S-forward aligned with its first half in +/+
orientation, whereas its second half aligned in +/− orientation against both the subjects.
For investigating this unexpected event, the S-forward was aligned with itself. The alignment
output was interesting: in +/+ orientation, clearly the alignment was 100% matching on each
position, whereas in the opposite orientation (+/−), the sequence aligned perfectly, except for
11 bp in the middle of the sequence and 24 bp at the 3′ end of the sequence. When analyzing
the unaligned 24 bp, we found that they corresponded to a “CT” dinucleotide and to the
forward primer sequence (22 bp) in a reverse complement orientation (Fig. 1B). Therefore, in
this Sanger product, there were also present an extra sequence representing the first half of
the same fragment and the sequencing primer both in a reverse complement orientation (Fig.
1B). These 2 findings let us hypothesize that 2 halves of the sequence were merged in a point
composed of a 23 bp partially overlapping sequence. Upon careful observation at the
alignment, we realized that the last 23 bp of the expected S-forward were composed of 2
small inverted repeats of 6 bp separated by 11 bp (Fig. 2A). During sequencing reaction,
these inverted repeats would anneal each other and form a stem-loop structure (11 bp in
length) at the 3′ end of the expected sequence (Fig. 2B); thus, only for S-forward Sanger
product was the sequencing reaction not stopped at the expected nucleotide but continued
and went back to the 5′ sequencing again the target sequence and the primer in reverse
complementary orientation (Fig. 2C). According to this occurrence, the total length of the S-
forward was 412 bp.

Figure 2
Stem-loop formation during Sanger sequencing reaction due to the presence of small inverted repeats
(GAAATC-GATTTC). (A) The two 6-bp inverted repeats (in green) located at the end of the 230 bp
expected fragment spaced by 11 bp. (B) Possible stem-loop formation during the Sanger sequencing
because of 6-bp inverted repeats. (C) Drawing of the DNA hypothesized folding of the total
sequenced fragment.

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Sanger sequencing has been the most diffused sequencing method in the world since it was
released. For this reason, a remarkable importance is given to the problems occurring in all
standard situations in which there is the need to sequence a specific PCR product or a
plasmid insert. Many sequencing problems have been already described, and answers have
been proposed for each of them, including the lack of sequence data, low peaks, poor
sequence, weak signal, overlapping peaks, signal weakening, and artifacts with large peaks
obscuring the real sequence. Here, we have described an event that can falsify sequencing
results providing almost the double length of the expected sequence. This event is likely
caused by a stem-loop formation by means of 2 inverted repeats at the 3′ end of the target
fragment. Because of that, the sequencing reaction is not stopped at the expected nucleotide
but continues until the sequencing primer is itself sequenced. This kind of event contributes
to a misleading double-sized sequencing product. The unusual occurrence of this event does
not exclude its previous observation in these decades of Sanger sequencing, although no
description is reported. Hence, attention should be obviously paid in designing primers for
PCR amplifications, but a special focus should be put also on the expected fragment
composition, particularly when working with complex genomes rich in repetitive sequences
(i.e., those belonging to Triticum genus) because they could alter sequencing results.
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