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Differential strand separation at critical temperature:
A minimally disruptive enrichment method for
low-abundance unknown DNA mutations
Minakshi Guha
1
, Elena Castellanos-Rizaldos
1
, Pingfang Liu
1
, Harvey Mamon
2
and
G. Mike Makrigiorgos
1,2,
*
1
Division of DNA Repair and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer
Institute, Harvard Medical School, Boston, MA and
2
Department of Radiation Oncology, Dana-Farber Cancer
Institute and Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
Received October 4, 2012; Revised October 31, 2012; Accepted November 3, 2012
ABSTRACT
Detection of low-level DNA variations in the
presence of wild-type DNA is important in several
fields of medicine, including cancer, prenatal diag-
nosis and infectious diseases. PCR-based methods
to enrich mutations during amplification have
limited multiplexing capability, are mostly restricted
to known mutations and are prone to polymerase or
mis-priming errors. Here, we present Di
fferential
S
trand Separation at Critical Temperature
(DISSECT), a method that enriches unknown muta-
tions of targeted DNA sequences purely based on
thermal denaturation of DNA heteroduplexes
without the need for enzymatic reactions. Target
DNA is pre-amplified in a multiplex reaction and
hybridized onto complementary probes immobilized
on magnetic beads that correspond to wild-type
DNA sequences. Presence of any mutation on the
target DNA forms heteroduplexes that are subse-
quently denatured from the beads at a critical tem-
perature and selectively separated from wild-type
DNA. We demonstrate multiplexed enrichment by
100- to 400-fold for KRAS and TP53 mutations at
multiple positions of the targeted sequence using
two to four successive cycles of DISSECT.
Cancer and plasma-circulating DNA samples
containing traces of mutations undergo mutation
enrichment allowing detection via Sanger
sequencing or high-resolution melting. The simpli-
city, scalability and reliability of DISSECT make it a
powerful method for mutation enrichment that inte-
grates well with existing downstream detection
methods.
INTRODUCTION
Cancer treatment is gradually moving toward
personalized therapy, allowing patients to avoid unneces-
sary or ineffective treatments (1). Tailoring therapies to
the genetic profile of tumors requires effective diagnostic
tools that would accurately detect clinically relevant
genetic alterations. The detection of mutated DNA is
often masked by an abundance of wild-type DNA
present in stromal tissue or in bodily fluids such as
plasma, urine or sputum (2–4). These low abundance mu-
tations are particularly important for early cancer detec-
tion, assessment of residual disease post treatment, disease
staging and monitoring of therapy following remission/
relapse ( 2 ). To date, several PCR-based technologies
have been developed for enrichment of low-abundance
mutations at known sequence positions including
methods that modify the targeted sequence (e.g. restriction
fragment length polymorphism or PCR-RFLP, restriction
endonuclease-mediated selective PCR or REMS-PCR
and allele-specific PCR), peptide/locked nucleic acid
approaches and co-amplification at lower denaturation
temperature-PCR or COLD-PCR (2,5–7). COLD-PCR
enables enrichment of any mutation on the sequence,
without prior knowledge of the mutation type or
position within the PCR amplicon. Accordingly, COLD-
PCR can be used in conjunction with downstream
sequencing methods to reveal low-level unknown muta-
tions (5,7,8). This is achieved by using preferential de-
naturation of mismatch-forming mutations at critical
denaturation temperature. Upon two consecutive
COLD-PCRs, mutations can be enriched by 100-fold or
more (7–11). Further mutation enrichment can be a chal-
lenge since polymerase errors and mis-priming events
increase upon additional PCR cycling.
As an alternative to PCR-based approaches, a bead-
based method known as DNA enrichment by allele-
*To whom correspondence should be addressed. Tel: +1 617 525 7122; Fax: +1 617 582 6037; Email: mmakrigiorgos@lroc.harvard.edu
Nucleic Acids Research, 2012, 1–9
doi:10.1093/nar/gks1250
ß The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which
permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
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specific hybridization (DEASH) has been described to
enrich for low-abundance mutations (12). DEASH uses
allele-specific biotinylated oligonucleotides that hybridize
competitively to the location of the targeted base substi-
tution followed by capture on streptavidin-coated
paramagnetic beads. The simplicity of DEASH is
powerful, as it circumvents the use of enzymatic steps
and the potential generation of artifacts in the course of
genotypic selection. Nevertheless, the approach is limited
to the enrichment of known mutations since it requires
two carefully designed probes that match both the
mutant and the wild-type alleles.
Here, we describe a novel method based on Di
fferential
S
trand Separation at Critical Temperature (DISSECT) to
enrich unknown mutations within mixed DNA popula-
tions. Similar to COLD-PCR, DISSECT uses differential
denaturation of DNA heteroduplexes and can therefore
enrich mutations at any position on the sequence,
enabling mutation scanning and discovery via down-
stream sequencing. At the same time DISSECT avoids
polymerase extension or other enzymatic steps, as it is
entirely based on repeated cycles of hybridization and
preferential denaturation on solid support (streptavidin-
coated magnetic beads). Since the target sequence
remains unmodified during DISSECT, the resulting
mutation-enriched DNA pool can be combined with any
existing downstream detection method. Thus, DISSECT
can generate DNA template enriched for mutations by
200-fold or more, resulting in radically enhanced ability
to identify low-level nucleic acid alterations. Here, we
validate DISSECT for a variety of mutated DNA
targets of clinical interest (KRAS and TP53) and demon-
strate its simplicity, scalability and application to low-level
mutation enrichment in clinical cancer samples.
MATERIALS AND METHODS
Cell lines and tumor specimens
Human cancer cell lines SW480 and PFSK-1 were
purchased from American Type Culture Collection
(ATCC), and genomic DNA was extracted from
cultured cells using a DNeasy
TM
Blood and Tissue kit
(Qiagen, Valencia, CA, USA) as per the manufacturer’s
protocol. Genomic DNA for A549, NCI-H69, SNU-182
and HCC1008 were purchased directly from ATCC. To
demonstrate mutation enrichment, genomic DNA from
human cancer cell lines SW480, NCI-H69, SNU-182,
HCC1008, A549 and PFSK-1 were serially diluted into
WT DNA as a test panel for various mutations
(Supplementary Table S1). Human male genomic DNA
(Promega Corporation, Madison, WI, USA) served as
the wild-type control for all experiments. DNA mutation
abundances examined were 10, 5, 3, 1, 0.1 and 0.05%
mutant-to-WT ratios. For the capture of multiple muta-
tions in a single-tube, genomic DNA of SW480, NCI-H69
and SNU-182 were combined to generate a 5 and 1%
mixed mutant DNA dilution. All experiments were
carried out in parallel with wild-type controls and
mutant mixtures and were replicated at least three times
for validation.
Clinical colorectal tumor and lung tumor
samples containing low-level mutations previously
identified using COLD-PCR-sequencing, dHPLC and
REMS-PCR-sequencing (7,9,13,14) were also used to val-
idate DISSECT. Additionally, plasma-circulating
DNA donated by a radiation therapy patient following
informed consent and institutional review board
approval was used to demonstrate mutation enrichment
using DISSECT. Plasma-circulating DNA was previously
screened and validated to contain a TP53 mutation (15).
DNA obtained from both plasma and clinical samples
were pre-amplified using 20 cycles of multiplex PCR
prior to DISSECT, as described below.
Multiplex pre-amplification
Multiplex PCR primers (Supplementary Table S2) were
designed for 50 most commonly mutated exonic regions
identified in lung and esophageal cancers according to the
COSMIC database (16). These regions contain a mutant
count of greater than 1 for any single point mutation.
Multiplex pre-amplification from 20 ng genomic DNA
or 10 ng plasma-circulating DNA was performed in a
total volume of 25 ml using a mixture of 100 primers
(50 paired sets) at a final concentration of 0.3 mM for
each primer with 0.3 mM dNTPs, 3 mM MgCl
2
,1
Kapa HiFi buffer and 0.5 U of Kapa HiFi HotStart
DNA polymerase (Kapa Biosystems, Woburn, MA,
USA) reported to have an error rate of 2.8 10
7
mis-incorporations/bp. Multiplex PCR cycling was per-
formed according to the manufacturer’s recommendations
(Kapa Biosystems) for a total of 15–25 PCR cycles using
63
C as optimal annealing temperature. Amplicon lengths
ranged from 120 to 190 bp in size depending on the
amplicon (Supplementary Table S2). Following multiplex
cycling, 1 ml of exonuclease I (New England Biolabs,
Ipswich, MA, USA) was added to each reaction and
incubated at 37
C for 30 min and 80
C for 15 min to
remove unincorporated primers.
DISSECT procedure and downstream analysis
Probe design and preparation. Streptavidin-coated
Dynabeads (Life Technologies, Grand Island, NY,
USA) were coupled to biotinylated probe sequences of
30–95 nt long matching the wild-type target sequences of
TP53, KRAS and EGFR genes (Supplementary Table S3).
Probes for TP53 exons 5–9 and EGFR exons 19–21 were
synthesized by Integrated DNA Technologies Inc.
(Coralville, IA, USA). The probe for KRAS exon 2 was
synthesized to contain three locked nucleic acid bases,
LNA (Exiqon, Woburn, MA, USA). The LNA design
for KRAS exon 2 was based on previous reports for
LNA-assisted detection of KRAS mutation hotspots
(17,18). ‘Dual-biotin’ was used for the design of capture
probes to strengthen the bond between the streptavidin-
coated beads and the immobilized oligonucleotides during
the thermal cycling procedure (19). Dual-biotinylated
probes were conjugated to streptavidin beads as per the
manufacturer’s protocol (Invitrogen Life Technologies,
Grand Island, NY). Dynabead probes were finally
resuspended in a stock buffer containing 6 SSPE
2 Nucleic Acids Research, 2012
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(0.9 M NaCl, 60 mM Na
2
HPO
4
, 6 mM EDTA) to a final
concentration of 10 mg/ml and kept at 4
C.
DISSECT protocol. 5–10 ml of Dynabead probes were
incubated with a 1:50 dilution of pre-amplified DNA in a
total volume of 50 mlof1 Phusion Buffer (New England
Biolabs). To hybridize DNA onto beads, DNA was
denatured at 95
C for 2 min, then annealed at 60
C for
2 min, 58
C for 2 min, 56
C for 2 min, 54
C for 1 min and
25
C for 5 min using an Eppendorf Mastercycler EP
machine (Eppendorf Inc., Haupage, NY, USA). Beads
were then separated using a Dynamag-PCR magnet
(Invitrogen Life Technologies, Grand Island, NY) and
washed twice with 50 mlof1 Phusion buffer to remove
unbound DNA fragments. Washed beads were resus-
pended in 55 ml1 Phusion buffer, and a 5-ml aliquot
was collected for testing DNA bound to beads.
Single-stranded mutant DNA was preferentially
denatured by heating the bead solution at a critical
denaturation temperature (e.g. 75
C) for 2 min followed
by magnetization, thereby collecting enriched mutated
DNA sequences in the supernatant. Critical denaturation
temperature (T
c
) of the shorter biotinylated probe se-
quences was predicted using IDT’s Oligo Analyzer that
predicts the melting temperature (T
m
) of the matched
(wild-type) sequence at the buffer conditions used for
DISSECT. A range of three to four potential T
c
values
were then experimentally tested to derive the optimal T
c
for the probe tested, ranging from 3
C below the predicted
T
m
and increasing temperature in 0.5
C steps until T
m-1
.
We found that adequate mutation enrichments are
obtained for denaturation temperature within 2
C for
most 30–45 nt probes, and optimal values for T
c
can be
arrived at using the approximation T
c
= T
m
(wild-type)-2
(data not shown). For longer 75–95 nt probes, the publicly
available UMelt software developed by the Wittwer la-
boratory (http://dna.utah.edu/umelt/umelt.html) was ini-
tially used to predict T
m
. The optimal T
c
was
experimentally derived using high-resolution melting
(HRM) analysis on the LightScanner HR96 system
(Idaho Technologies Inc.). Following elution from
beads, a 5-ml aliquot of the supernatant enriched for
mutant DNA was saved for analysis, while the remaining
supernatant was used for additional rounds of DISSECT
to increase the mutation enrichment as described in
Figure 1. These additional rounds of DISSECT required
a single 50 ml1 Phusion buffer replacement following the
step-down anneal procedure to eliminate unbound DNA
material in the supernatant. The same T
c
was used for
each denaturation step following capture of target DNA
from supernatant. For the multiplexed DISSECT
(combined bead capture), the same DISSECT procedure
was carried out, but with 2–4 ml of each kind of Dynabead
probe in a total bead volume of 8–16 ml per sample. The
capture probes used for multiplexed DISSECT were
designed to have similar T
m
so that a common critical
denaturation temperature could be used for multiplexed
mutation enrichment.
Downstream analysis . DNA eluted from beads following
DISSECT was amplified by conventional PCR or
COLD-PCR (5,14) followed by HRM analysis (9) and
Sanger Sequencing (Eton Bioscience, Cambridge, MA,
USA) (15). For post DISSECT amplification, nested
primers were used for each target region amplified in the
original pre-amplification reaction (Supplementary
Table S4).
RESULTS
The principle of DISSECT
The DISSECT procedure presented in Figure 1 illustrates
a bead-based method by which low-level mutations can be
enriched using repeated cycles of annealing and denatur-
ation at a critical temperature. Following multiplex
pre-amplification (Step 1) from genomic DNA, amplicons
are captured on magnetic beads using sequence-specific
binding to bead-immobilized DNA probes (Step 2).
Target DNA sequences are captured on beads by denatur-
ation of the double-stranded DNA at 95
C, followed by
step-down annealing procedure that allows single-
stranded DNA to anneal to complementary oligonucleo-
tides (probes) attached to streptavidin-coated magnetic
beads. These probes contain the wild-type sequence of
the targeted DNA regions (Supplementary Table S3),
and therefore the presence of a mutation at any
sequence position of the target DNA leads to a
mismatch that lowers the melting temperature (T
m
)of
the duplex. In Step 3 of Figure 1, mutation-containing
DNA is preferentially released from beads by denatur-
ation at a critical denaturation temperature (T
c
) corres-
ponding to the probe-target duplex. T
c
was defined as
detailed in the ‘Materials and Methods’ section. The
critical denaturation step preferentially elutes mutant
DNA strands into the supernatant, while a fairly large
fraction of the wild-type sequences remains substantially
bound to the beads and are subsequently removed by
magnetization. Steps 2 and 3 can be repeated multiple
times to enable further enrichment of mutation-containing
DNA regions. In the final step, the released mutation-
enriched sequences are PCR-amplified and analyzed.
HRM and Sanger sequencing were used for downstream
analysis. It should also be noted that although the
amplicons generated from the pre-amplification reaction
range from 120 to 190 bp, only the region of the amplicons
that corresponds to the immobilized probes actually
becomes enriched for mutations during DISSECT.
KRAS and TP53 mutation enrichment
As a large portion of lung cancer mutations occur in
KRAS (20) and TP53 genes (21,22), we implemented
DISSECT to analyze and enrich point mutations within
these two genes. Three successive rounds of DISSECT
were performed from a 1% mutation abundance of both
SW480 and A549 cell lines using a KRAS exon 2 probe at
a critical denaturation temperature of 75
C. Figure 2A
shows a 40- to 50-fold enrichment of two KRAS mutations
(c.35C>A, p.G12V and c.C>T, p.G12S) after DNA
elution following three rounds of DISSECT. Similarly,
tumor sample TL119 containing a double KRAS
mutation (c.34_35CC>AA, p.G12F) showed the same
degree of enrichment (Figure 2A). PCR-amplified
DISSECT products were also analyzed by HRM
Nucleic Acids Research, 2012 3
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analysis showing incremental mutant enrichment for every
additional round of DISSECT for both SW480 and A549
mutation-containing DNA (Figure 2B). Beads containing
a non-LNA probe designed for TP53 exon 8 were equally
effective in enriching for low-level mutations from an
original 1 and 0.1% mutant abundance (Supplementary
Figures S1–S4). DISSECT was able to successfully
enrich low-level TP53 exon 8 T
m
-reducing mutations
(c.818C>T, Supplementary Figure S1), T
m
-retaining mu-
tations (c.841C>G, Supplementary Figure S2) and T
m
-
increasing mutations (c.823A>C, Supplementary Figure
S3). All three mutations were enriched with a single
bead-bound oligonucleotide probe, indicating the ability
to enrich different types of single-base substitutions within
the length of the DNA sequence covered by the probe.
These experiments yielded up to 100- to 200-fold
mutation enrichment after three rounds of DISSECT,
equivalent to mutant enrichments of 5-fold/cycle, as
inferred by comparing the apparent mutation abundance
in the Sanger chromatographs to the initial abundance of
0.1%. The gradual increase in mutation enrichment of
TP53 exon 8 after each round of DISSECT was
demonstrated via HRM analysis following DISSECT,
using 5, 1 or 0.1% original mutation abundances
(Supplementary Figure S4).
Next, to evaluate longer probes for use with DISSECT,
a 90-nt long TP53 exon 8 probe was used. In
Supplementary Figure S5A, serial dilutions of SW480
DNA into wild-type DNA (0.3, 1, 5 and 10% abundance)
were used to enrich for a c.818G>A mutation using two
rounds of DISSECT. Furthermore, a 1% mutation abun-
dance using DNA with a C>G mutation (HCC1008
DNA) within the same exon 8 target region resulted in
similar mutation enrichment (Supplementary Figure
S5B). Finally, a single-tube, multiplexed mutation enrich-
ment tested using 76, 95 and 90 nt probes for exons 5, 7
and 8, respectively, are depicted in Supplementary Figure
S5C. The data demonstrated that two rounds of
DISSECT in conjunction with the 76- to 95-nt probes
result in 20- to 60-fold mutation enrichments,
depending also on the mutation type and position.
The longer probes provide a broader ‘footprint’ for the
use of DISSECT in the enrichment of unknown
mutations.
Limits of mutation detection via Sanger sequencing and
HRM following DISSECT
Using an initial mutation abundance of 0.1%, conven-
tional PCR or single-round COLD-PCR-Sanger
sequencing failed to detect the mutation (Figure 3A).
Figure 1. DISSECT flowchart. Following multiplexed pre-amplification from genomic DNA (Step 1), enriched DNA target regions are captured on
complementary probes immobilized to magnetic beads resembling the wild-type form of the target sequence (Step 2). Mutation-containing target
DNA forms heteroduplexes that are subsequently denatured at a critical temperature leaving preferentially wild-type DNA bound to beads (Step 3).
Eluted DNA is separated and re-bound to a fresh aliquot of beads. Steps 2 and 3 are repeated to increase the mutation enrichment. Aliquots of
eluted DNA from each round are analyzed by downstream assays (Sanger sequencing and HRM were used in this work).
4 Nucleic Acids Research, 2012
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In contrast, four rounds of DISSECT followed by conven-
tional PCR resulted to an apparent mutation abundance
of 26% for TP53 exon 8 and 16% for KRAS exon 2,
corresponding to 260- and 160-fold mutation enrichment,
respectively, or 4- to 5-fold mutation enrichment
per DISSECT cycle (Figure 3B, left). The final mutation
enrichment was higher when COLD-PCR was applied to
the eluted DNA following four rounds of DISSECT,
resulting in a mutation enrichment of 350- to 400-fold
(Figure 3B, right). The increased overall enrichment
reflects the additional enrichment provided by
COLD-PCR itself. Three or four rounds of DISSECT
followed by conventional PCR altered the HRM profiles
for both 0.1 and 0.05% mutant KRAS compared with
wild-type DNA (Figure 3C, middle), and these HRM
differences were greater when COLD-PCR was adopted
following DISSECT (Figure 3C, bottom). Similar results
were shown for mutations in TP53 exon 8 and KRAS exon
2 after four rounds of DISSECT from an initial mutation
of 0.05% (Supplementary Figure S6). In summary, three
or four rounds of DISSECT enrich low-level mutations,
originally present at 0.05–0.1% mutation abundance, to
levels of about 15–25%, allowing their subsequent
identification via COLD-PCR, HRM and Sanger
sequencing.
Multiplexing DISSECT
Bead-based processes are inherently scalable and
amenable to multiplexing. For a proof of principle in
multiplexing DISSECT, we prepared a 5 and 1%
mixtures of genomic DNA from cell lines SW480,
SNU-182 and NCI-H69, containing mutations in four dif-
ferent exon regions (Supplementary Table S1) diluted in a
wild-type DNA background. A 50-plex pre-amplification
was performed using this DNA mixture and processed for
the simultaneous mutation enrichment of four mutated
DNA targets using a combination of beads in a single
tube. The oligonucleotide probes attached to the beads
were designed to have similar melting temperatures,
within a ±0.5
C temperature range. During the single-
tube multiplexed DISSECT, a single denaturation
Figure 2. KRAS mutation enrichment. KRAS exon 2 enrichment following three rounds of DISSECT at a critical denaturation temperature of 75
C,
using immobilized KRAS oligonucleotide (30 nt, wild-type sequence). (A) Sanger sequencing chromatographs following conventional PCR amplifi-
cation of DISSECT products for three different KRAS mutations from an initial mutation abundance of 1% (c.35C>A, p.G12V and c.C>T, p.G12S
from SW480 and A549, respectively, and c.34_35CC>AA, p.G12F from a lung tumor sample, TL119). The DNA remaining on beads (left) is
compared with DNA eluted from beads following three rounds of DISSECT. (B) HRM analysis showing increasing mutation enrichment after every
round of DISSECT (R1-R3) for SW480 and A549 samples with 1% input mutation abundance.
Nucleic Acids Research, 2012 5
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temperature of 75
C was used for the enrichment of all
four mutated DNA targets.
Figure 4 shows a combined mutation enrichment of
60-fold following three rounds of DISSECT for KRAS
exon 2, TP53 exon 5, TP53 exon 6 and TP53 exon 8
amplicons, using initial mutation abundances of 1–5%.
HRM analysis also depicts melting curves with increasing
differences for all four mutated amplicons (KRAS exon 2
and TP53 exons 5, 6 and 8) following each round of
DISSECT (Supplementary Figure S7). These results illus-
trate that DISSECT can be readily multiplexed provided
the T
m
of the bead-bound probes are matched.
Analysis of clinical tumor samples and circulating
DNA from plasma
To validate the use of DISSECT with clinical specimens,
we used previously tested DNA from lung and colorectal
tumor samples containing mid- to low-abundance TP53
and KRAS mutations (7, 14). Figure 5A shows strong
mutation enrichment following three rounds of
DISSECT for low-abundance TP53 exon 8 mutations in
tumor samples CT20, TL6 and TL121. HRM analysis also
demonstrates differences between the original DNA
captured on TP53 exon 8 beads versus DNA eluted after
three rounds of DISSECT followed by conventional
PCR-HRM (Figure 5B). Additional tumor specimens
were tested for enrichment of mutations from other
hotspot regions including TP53 exons 7 and 9 and
KRAS exon 2 (Supplementary Figure S8 ). A list of the
clinical samples, mutation positions and enrichment
following DISSECT is listed in Supplementary Table S5.
As an additional test, we used DISSECT to enrich mu-
tations from plasma-circulating DNA obtained from an
esophageal cancer patient undergoing radiation therapy.
The circulating DNA was purified from plasma and
pre-amplified using 20 cycles of multiplex PCR.
A biotinylated probe for DISSECT, p53Ex8-FC39
(Supplementary Table S3), was designed to enrich for a
previously identified mutation at an abundance of 0.5%
on TP53 exon 8 (c.847C>T, p.R823C) (15). Three rounds
of DISSECT resulted in a final mutation abundance of
36% (G>A, antisense) (Figure 5C). Alternatively, a
single round of COLD-PCR following two rounds of
DISSECT was performed, which resulted in mutation
abundance of 65% according to the chromatograph. In
summary, DISSECT can readily be applied to enrich
low-abundance mutations from clinical tumor specimens
and circulating tumor DNA prior to downstream
mutation detection.
Figure 3. Mutation enrichment following four rounds of DISSECT. (A) Sanger sequencing following direct conventional PCR or COLD-PCR in the
absence of DISSECT for TP53 exon 8 (C>T mutation) and KRAS exon 2 (C>A mutation) from an original 0.1% mutation abundance (from
SW480 mutant DNA diluted into wild-type DNA). (B) Sanger sequencing following direct conventional PCR or COLD-PCR after four rounds (R4)
of DISSECT. Resulting mutation abundance of 26% for TP53 and 16% for KRAS (corresponding to a final 160- to 260-fold mutation enrichment)
are depicted. DISSECT in combination with COLD-PCR increased the enrichment to 35% for TP53 and 42% for KRAS (corresponding to a final
400-fold mutation enrichment). (C) HRM analysis shows differential melting curves for KRAS exon 2, before and after DISSECT, from original
input mutation abundances of 0.05–0.1%. Data are representative of at least three independent experiments.
6 Nucleic Acids Research, 2012
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DISCUSSION
DISSECT is a novel method for enriching DNA samples
for low-level unknown mutations that is purely based on
the thermal denaturation properties of DNA, without the
need for enzymatic reactions during the enrichment
process. The process is ‘minimally disruptive’ to the
input DNA material since no enzymatic modification
such as RFLP or sequence alterations by PCR primers
and polymerase extension occur during the mutation
enrichment, thus minimizing the probability for artifacts.
By using selective denaturation of mismatch-forming
target sequences bound to immobilized wild-type
sequence at critical temperature, DISSECT enriches for
most types of mutations, enabling mutation scanning
over the entire length of the probe. This was not
possible using the previously described DEASH competi-
tive hybridization method (12) because (a) special oligo-
nucleotides matching the specific targeted mutation were
needed and (b) selective denaturation confers superior se-
lectivity and faster kinetics over selective hybridization, as
our experience with COLD-PCR has demonstrated (5).
To date, few methods have been able to provide multi-
plexing capability in enriching low-level mutations in
diverse targets (2). Therefore, an advantage of DISSECT
lies in the ability to enrich multiple mutant targets in a
single tube without compromising accuracy or selectivity.
This can inherently enable multiplexed downstream
analysis platforms to be used with DISSECT, such as
matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry, pyrosequencing and next-generation
sequencing, in addition to Sanger sequencing and HRM
implemented here.
In the present work, a pre-amplification step using a
50-plex PCR from genomic DNA was used to generate
target pre-amplification prior to applying DISSECT.
While this step combines conveniently with DISSECT,
the pre-amplification can potentially be omitted by per-
forming direct target capture from genomic DNA, as per-
formed with DEASH (12). This would eliminate potential
artifacts introduced during the initial 15–25 cycles of
pre-amplification. A limitation of DISSECT is that the
original DNA content is gradually reduced following
each round since part of the bead-bound DNA is
removed after the critical denaturation step. Thus, follow-
ing three to four consecutive rounds of DISSECT that
produced up to 100- to 400-fold mutation enrichment,
an amplification was applied prior to downstream analysis
by Sanger sequencing (or, potentially, prior to additional
rounds of DISSECT to increase mutation enrichment
further).
Here, we have shown that DISSECT can simultan-
eously enrich diverse targets for multiple mutations in
the same tube using a single denaturation temperature.
The mutation enrichment varies somewhat since the dif-
ferent DNA duplexes may not have the same optimal
critical denaturation temperature and also because
Figure 4. Multiplexed, single-tube DISSECT. Sanger sequencing analysis from a combined bead capture experiment showing simultaneous mutation
enrichment for four independent regions, KRAS exon 2 and TP53 exons 5, 6 and 8 following three rounds of DISSECT, from an original mutation
abundance of 1 and 5% (mixture of SW480, SNU-182 and NCI-H69 mutant DNA diluted into wild-type DNA, MP3). Data are representative of at
least three independent experiments.
Nucleic Acids Research, 2012 7
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mutation enrichment depends on the specific mismatches
and the sequence context.
DISSECT is simple and amenable to scaling and auto-
mation, e.g. using liquid-handling robots, microfluidics or
micro-arrays. Accordingly, we anticipate using DISSECT
in routine genetic screening, especially in cancer-related
applications where it is important to identify low-level
mutations, such as EGFR resistance mutations (23) or pre-
dictive KRAS mutations (24,25). KRAS mutations at an
initial abundance of 0.05–0.1% are capable of being
directly detected via Sanger sequencing following
DISSECT (Figure 3), which illustrates a detection limit
improvement over previously reported methodologies
used to improve KRAS Sanger sequencing limits (13,26).
In the notable reports by Misale et al.(27) and Diaz et al.
(28), BEAM-ing was used to identify KRAS mutations at
the 0.1% level in plasma, as an indicator of developing
resistance to cetuximab treatment in colorectal cancer
patients. The BEAM-ing detection capability is similar
to that provided by DISSECT when followed by
PCR-Sanger sequencing (Figure 3). In addition,
DISSECT coupled with sequencing provides information
on adjacent positions on the same amplicon, thus enabling
‘unknown’ mutation detection and surveying of multiple
‘hotspots’ along the sequence. Accordingly, DISSECT
may also be used for detecting biomarker mutations in
tumor suppressor genes with multiple hotspot mutations
present in bodily fluids such as plasma-circulating DNA,
as was shown for the TP53 gene mutation in Figure 5.
In addition to cancer, DISSECT applications in prenatal
diagnosis or infectious diseases can also be envisioned.
In summary, DISSECT is a novel method for enriching
DNA target sequences for low-level unknown mutations
based on thermal denaturation properties of DNA
heteroduplexes, without the need for excessive amplifica-
tion or other enzymatic reactions. The process is readily
amenable to scaling and automation and can be combined
with existing downstream DNA sequencing platforms.
The simplicity of DISSECT makes it a powerful method
to screen for multiple mutation targets in a single tube
without compromising accuracy or sensitivity.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online:
Supplementary Tables 1–5 and Supplementary Figures
1–8.
Figure 5. Clinical specimen analysis using DISSECT. Conventional PCR-Sanger sequencing (A) and conventional PCR-HRM (B) showing antisense
TP53 exon 8 mutations from lung (c.830C>A, TL6 and c.818C>T, TL121) and colorectal (c.818C>T, CT20) clinical tumor samples, after three
rounds of DISSECT. Results from DNA remaining on beads versus DNA eluted from beads are depicted (C) Plasma-circulating DNA from an
esophageal cancer patient with a low-level mutation (c.847G>A, R283C) was analyzed by DISSECT followed by conventional or
COLD-PCR-Sanger sequencing.
8 Nucleic Acids Research, 2012
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FUNDING
NCI [R21 CA-111994 and R21 CA-155615, in part]. The
contents of this article do not necessarily represent
the official views of the National Cancer Institute or the
National Institutes of Health. Funding for open access
charge: Departmental funds.
Conflict of interest statement. None declared.
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