A tailored LNA clamping design
principle: efficient, economized, specific and
ultrasensitive for the detection
of point mutations
Hao Yang‡ a,
Mengqiu Yan‡ a ,
Gaolian Xu a, Xiaohua Qian a,
Ruiying Zhao b,
Yuchen Han b,
Lin Zhang c,
Hongchen Gu*a,
Hong Xu*a
aSchool
of Biomedical Engineering/Med-X Research Institute, Shanghai Jiao Tong
University, Shanghai, 200030, PR China
bShanghai Chest
Hospital, Shanghai Jiao Tong University, Shanghai, 200030, PR China
cShanghai Mag-Gene Nano Tech Co., Ltd, Shanghai,
200030, PR China
‡ To whom contribute equally to the article.
* To whom correspondence should be
addressed.
Hong Xu, Add.: School of
Biomedical Engineering/Med-X Research Institute, Shanghai Jiao Tong
University,1954 Huashan Road, Xuhui District, Shanghai, China; Tel:
62933743; Fax: 62932907; Email: xuhong@sjtu.edu.cn
Abstract
In the development of personalized medicine, the ultrasensitive
detection of point mutations that correlate with diseases is important
to improve the efficacy of treatment and guide clinical medication. In
this study, locked nucleic acid (LNA) was introduced as an amplification
suppressor of a massive number of wild-type alleles in an
amplification refractory mutation
system (ARMS) to achieve the detection of low-abundance mutations with
high specificity and sensitivity of at least
0.1%. By integrating the length
of clamp, base type, number and position of LNA modifications, we have
established a “shortest length with the fewest LNA bases” principle
from which each LNA base would play a key role in the affinity and the
ability of single base discrimination could be improve. Finally, based
on this LNA design guideline, a series of the most important single
point mutation sites of epidermal growth factor receptor (EGFR) was
verified to achieve the optimal amplification state which as low as
0.1% mutation gene amplification was not affected under the wild gene
amplification was completely inhibited, demonstrating that the proposed
design principle has good applicability and versatility and is of great
significance for the detection of circulating tumor
DNA.
Key words: locked nucleic acid (LNA), LNA clamping modification
principle, point mutant, ultrasensitive detection with high specificity,
cell-free tumor DNA.
Introduction
Point mutants are a class of somatic mutants that serve as significant
biomarkers for heritable human diseases, drug development, cancer
detection, companion diagnostics and
prognosis[1, 2] . The greatest challenge of
point mutant detection is the identification of very low-abundance
mutant targets against a background of large amounts of wild-type
alleles. Since the ratio of mutant targets to wild-type genes is
1%-0.1% or even lower, detection methods with high sensitivity and
specificity are urgently required[3, 4] . To
meet these requirements, numerous methods have been developed, including
amplification refractory mutation system
(ARMS)[5] , next-generation sequencing
(NGS)[6, 7] , high-resolution
melting[8] and
microarrays[9] . High-resolution melting and
microarrays are quick and high throughput whereas usually lack
sensitivity and specificity.
Next-generation sequencing (NGS)
can reach the limit of detection below 0.1% by enhancing the sequencing
depth to more than 10000X[10] but is
expensive and time-consuming.
ARMS is based on the application of allele-specific (AS) PCR primers
that are perfectly matched to the mutant targets at 3’
end[11, 12] . However, imperfect
hybridization between the AS primer and wild-type gene may lead to
false-positive results with relatively low efficiency[13, 14] . To further improve the
specificity, peptide nucleic acid (PNA)[15] , a high-affinity DNA analogue with a
polypeptide-like skeleton, was introduced to bind the wild-type gene
during the PCR annealing stage, which impedes the extension of the AS
primer and thus inhibits the amplification of wild-type
genes[16] . Simultaneously, the PNA-DNA
duplex provides excellent mismatch discriminatory power, and has already
been used as a clamping agent for the detection of many mutant
alleles[17-19] . But PNA also has some
shortcomings, including poor solubility, high cost, a long synthesis
period, and an inflexible design pattern (changing the length only)[20] . In particular, PNA could also inhibit
the amplification of mutant templates with ultra-low concentrations and
potentially producing false negatives[18] .
Locked nucleic acid (LNA), another DNA analogue with excellent water
solubility[21, 22] , can be synthesized
using the same process as conventional oligonucleotides with relative
low cost. Moreover, LNA-modified oligonucleotides have great flexibility
in terms of different numbers, positions and base types, thus they have
been widely used as probes[23, 24] or
primers for nucleic acid detection[25, 26] .
Nevertheless, the principle of LNA-modified oligonucleotide design used
as clamping systems remains rare [27-29] .
The great challenge for the clamping system is to completely inhibit the
amplification of massive numbers of wild-type gene, while with great
sensitivity to mutation. Faced with this arduous task, there are few
systematic studies on the thermodynamic parameters of LNA
clamping[30, 31] . In the absence of
effective design theory guidelines, people either designed LNA-modified
clamping based on their own experience or obtained optimized
LNA-modified clamping through many tedious repeated optimization
experiments. In this study, based
on the LNA-mediated PCR clamping system (LMPC), we proposed for the
first time how to realize an optimized LNA clamping design strategy
using AS primers for the detection
of point mutations to achieve high sensitivity and specificity. First,
LNA clamping are systemically investigated via different parameters,
including the number of, base type, and position of LNA modifications,
ensuring that LNA clamping can completely inhibit the wild-type gene
amplification signal but amplify mutant targets with high efficiency.
And then, LNA clamping acquired by the new design guideline was
successfully used as an amplification suppressor in ARMS PCR to achieve
the ultrasensitive detection of the five most important mutant of
epidermal growth factor receptor (EGFR) alleles with sensitivity of
0.1% against a background of 10,000 wild-type alleles.
Materials & methods
2.1 Oligonucleotides, templates and reagents
LNA-modified oligonucleotides (labeled with NH2-C6 at
the 3’ end), primers, plasmids and TaqMan probes were all obtained from
Sangon Biotech (Shanghai) Co. Ltd.
with the sequences shown in Supplemental Table S1 and S3. All the TaqMan
probes were designed using PrimerExpress® software (Applied Biosystems,
Foster City, CA). Champagne TaqTM DNA polymerase and EvaGreen® Dye (20x
stock solution in water) were purchased from Vazyme Biotech Co. Ltd. and
Biotium, respectively. The EGFR Multiplex Cell Free Reference Standard
Set was purchased from Horizon Discovery (HDx).
AmoyDx®
DNA and RNA Extraction Kits, the
Silica-based Spin Column and the Human EGFR Gene Mutation Detection Kit
(Multiplex real-time PCR) were purchased from Amoy Diagnostics Co., Ltd.
2.2 Preparation of PCR templates
The genomic DNA of A549 cells was extracted using a Genomic DNA Kit
(TianGen Biotech Co. Ltd) and served as a wild-type background with the
concentration quantified using digital PCR (QX200™ Droplet Digital™ PCR,
Bio-Rad). Plasmids containing
different EGFR point mutation fragments (L858R, T790M, S768I, L861Q and
G719A) were constructed and quantified by digital PCR.
2.3 Real-time PCR
Standard real-time PCR was carried out in a total
volume of 20 μl containing 0.15 μM
each primer, 0.1 μM TaqMan MGB
probe, 1.5 μM LNA oligonucleotides
and 1x buffer (10 mM Tris-HCl, pH
8.3, 10 mM KCI, 0.3 M dNTP and 1.5 mM MgCl2). Thermal
cycling was performed using a
LightCycler® 96 System (Roche) with
two amplification steps: initial
denaturation at 95°C for 2 min, followed by 15 cycles
of 95°C for 1 s, 70°C for 20 s and
65°C for 20 s, then 30 cycles of 95°C for 1 s, 70°C for 20 s and 60°C
for 20 s. The fluorescence signal was collected in the second step.
2.4 Melting curve experiments
The composition of the 20 μl melting reaction included 1.5 μM LNA
clamping, 1.5 μM complementary segments (Supplemental Table S2), 1x Eva
Green, and 1x PCR buffer. Melting curve assays were carried out on the
LightCycler® 96 System (Roche)
with following program: 55°C for 10 min, denaturation at 95°C for 10 s,
and cooling to 55°C for 50 s, followed by finally increasing the
temperature from 55°C to 97°C at a heating rate of 0.2°C/s, and the
fluorescence was acquired at 0.1°C intervals. The melting curve was
analyzed using LightCycler® software.
2.5 Agarose gel electrophoresis of
PCR products
10 μl of PCR product was mixed with 1 μl of loading buffer, and
electrophoretic analysis was carried out in 2% agarose gel stained with
Tanon™ Nucleic Acid Dye (10000×). The sample was electrophoresed at a
voltage of 150 mV for 30 min and then imaged on a Vilber Lourmat.
2.6 Cell-free DNA and clinical sample detection
The EGRF cfDNA point mutation reference standard samples were diluted in
wild-type DNA (10000 copies) to obtain a series of mutation samples with
concentrations of 1%, 0.4%, 0.2%, 0.1%, and 0% (negative). The
genomic DNA of clinical formalin-fixed and paraffin-embedded (FFPE)
samples was extracted using DNA Extraction Kits (Amoy Diagnostics) and
tested by the Human EGFR Gene Mutation Detection Kit (Amoy Diagnostics)
and LMCS we developed respectively.
Results and discussion
3.1 Establishment of the LNA-mediated real-time PCR clamping system
The LNA-mediated PCR clamping
system (LMPC) is schematically
presented in Figure 1 Here, the AS
primer is specifically complementary to mutant targets, while the
LNA-modified amplification suppressor (LNA clamping) is perfectly
complementary to wild-type with the mutant site in the middle. The
completely complementary
LNA-wild-type
gene heteroduplex has a higher melting temperature (Tm) than that of AS
primer-wild-type gene. Hence, during amplification, the formation of a
heteroduplex between the LNA-modified amplification suppressor and
wild-type DNA is favored over the formation of the primer-wild-type DNA
duplex, thus leading to the suppression of AS primer hybridization and
extension, which promises high specificity. However, due to the
existence of the single base mismatch, the Tm for LNA-modified
amplification suppressor hybridization with mutant-type DNA was sharply
decreased, leaving the AS primer able to hybridize effectively with
mutant targets and therefore to achieve the highly efficient
amplification of rare mutant
targets in the presence of a large number of wild-type alleles. For
LMPC, the expected result is to completely
inhibit the amplification of the
massive number of wild-type gene (such as 10000 copies) while exerting
as little effect as possible on the amplification efficiency of
low-abundance mutant targets (e.g., lower than 10 copies), as shown in
Figure 1B (optimal state).
Unfortunately, under real circumstances,
the
amplification of mutant targets
would be suppressed in most clamping systems (as shown in Figure 1B,
deficient state) even with the use of LNA oligonucleotides as clamping
agents due to the lack of LMPC design theory. To obtain an LNA-modified
amplification suppressor with excellent performance, the role of the
difference in Tm between the LNA-mutant-type and LNA-wild-type
heteroduplex was explored by investigating the effects of length, number
of LNA bases and modification position. As illustrated in the schematic
amplification curve of q-PCR (Figure 1B), although the wild-type gene
could be inhibited by LNA clamping in both situations, there is a
significant difference in mutant targets. In the optimal state, the
presence or absence of LNA clamping had little effect on the
amplification of rare mutant targets, while inhibition occurred in the
deficient state. In general, to avoid limiting mutant amplification, the
Tm of LNA clamping with a mutant-type DNA heteroduplex should be far
lower than that of the AS primer, which
makes
the LNA
clamping unable to recognize and
hybridize with mutant targets during PCR. The premise is that as the
suppression of mutant targets by LNA clamping weakens, the detection
sensitivity should be gradually improved. Therefore, this research
aims to determine what kind of LNA
clamping is the most effective as well as to establish an LNA clamping
design principle, which will greatly improve the performance of LMPC
amplification for the highly sensitive detection of mutant targets.
3.2 The key factors
in mismatch discrimination of LNA
clamping
As shown in the Figure 1, the structure of the LNA clamping may be
important for the sensitivity and specificity of the detection of the
mutation targets, and our pre-experiments showed that the mismatch
discrimination capability of the fully modified LNA oligonucleotides was
not as good as that of the
interval-modified
LNA oligonucleotides (LNA
modification at every second base, the data were not shown in this
article), and the same result had also been reported in a previous
study[27, 32] . It is precisely this
interesting phenomenon to make us ponder whether there is a more
rational design philosophy from which the clamping system can perform
better with as few as LNA bases in mutant target detection. To
investigate the design rules and obtain the optimal design parameter,
the L861Q of the EGFR point mutant was chosen as the research model.
First, several interval LNA
modification oligomers with different lengths (11-15 bp) were designed
to explore the influence of LNA-modified clamping length on the
detection performance. As the length of LNA increased, the Ct value of
wild-type gene in real-time PCR increased gradually, indicating that the
amplification efficiency of wild-type gene decreased gently (Figure S1).
When the length of LNA clamped up to 14 bp, of which the Tm was more
than 10 degrees higher than that of the AS primer, wild-type gene (10000
copies) were completely inhibited within 30 cycles of amplification plus
ten cycles of preamplification. Similarly, the amplification of mutant
targets (the ratio of mutant targets to wild-type gene was 1% and
0.1%, respectively) maintained the same trend, but the inhibition
efficiency was weaker than that of the wild-type gene owing to the
existence of a mutant base (Figure
S1D).
For LMPC, the formation of LNA/wild-type gene duplexes is the key factor
to inhibit wild-type gene amplification. To evaluate the stability of
LNA/DNA duplexes, the melting temperature (Tm) of the LNA clamping was
measured using a melting curve (Figure S1) and predicted by IDT
OligoAnalyzer (Table S3) respectively. Figure 2A illustrates that
Tm (perfect match) is higher than
Tm (mismatch) and both are
increased with the length of LNA-modified clamping. In addition, the
difference in Tm between matched and mismatched duplexes (denoted as
ΔTma, ΔTma =Tm (perfect match)-Tm
(mismatch)) of 861 LNA1-LNA5 is very close(9.6°C-11.2°C). The high Tm
means high binding affinity, so if the Tm is high enough, the extension
of AS primer on wild-type gene can be completely inhibited and the
wild-type gene would be totally bound by the clamping during the
annealing stage of PCR (such as LNA4 and LNA5). On the other hand, in
such a high Tm based on above interval LNA modification clamping, the
Tm (mismatch) between LNA clamping
and mutant targets would be also high, which would reduce the
amplification efficiency of mutant targets. It is clear in principle
that Tm (perfect match) is a key factor in inhibiting the amplification
of wild-type gene, but Tm (mismatch) determines the efficiency of mutant
targets amplification indicating that the simplex LNA clamping
modification pattern, which has no influence on
ΔTma,
would make it difficult to achieve high mismatch discrimination ability.
Therefore, an LNA clamping design model needs to be explored in a much
calmer and more rational manner.
To further reveal the effect of
ΔTma, the
mismatch discrimination ability of LNA-modified (LNA4) or unmodified
oligomers (LNA6) at the mutant
site was designed and studied. The Ct value of mutant targets are 16.56
(1%), 19.86 (0.1%) and 18.25 (1%), 20.91 (0.1%) for LNA4 and LNA6,
respectively, suggesting that the inhibition of LNA4 for mutant targets
is significantly lower (Figure S2). Then, the Tm values of the matched
and mismatched states of both oligomers were analyzed according to the
melting curves shown in Figure 2B. It is noticeable that the
ΔTma of 861 LNA4 and LNA6 are 10.2°C and 6.96°C,
respectively, indicating that the modification of LNA on the mutant
site, which made ΔTma higher, would dramatically
increase the mismatch discrimination ability, leading to higher
amplification efficiency of mutant targets. Therefore, not only is high
Tm of LNA-modified clamping needed to ensure the amplification
suppression of wild-type gene, but more importantly, a
high ΔTma is
required to guarantee both effective suppression of wild-type gene
amplification and the minimum impact on the mutation target
amplification efficiency.
3.2 The influence of the number of LNA bases on the mismatch
discrimination ability of LNA clamping
Although interval modification with LNA enhanced the sensitivity of the
LNA clamping system to 0.1% under the background of 10000 copies of
wild-type gene, the amplification efficiency of mutant targets was still
suppressed unfortunately compared with the LNA-free group, as described
above (the Ct value of real-time PCR was increased by at least 2.7, as
shown in Figure S1). To find the
essential factor of ΔTma in LMPC,
a series of LNA clamps with
different numbers of LNA bases but with similar Tm
were designed (LNA7-10). The Tm of
these clamping segments is high enough to ensure that the wild-type
amplification could be completely restrained (shown in Table S3). In
addition, the mutation location was selected preferentially for
modification according to the result of Figure 2B. As shown in Figure 3,
the signal of wild-type gene could be completely inhibited by all LNA
clamping to avoid the interference on mutant signal. It is worth noting
that the Ct value of mutant targets decreases as the LNA-modified bases
increases (Figure 3A, 3B and 3D). So, at the same Tm, the amplification
efficiency of rare mutant targets could be improved by adding LNA bases.
And, more remarkable, the Ct value of LNA 10 is almost the same as that
of LNA free (no LNA clamping in PCR mix), indicating that it is possible
to obtain the optimized LNA clamping system as we predicted earlier.
To further reveal the factors that cause differentiated property of
these LNA clamps, melting curve analysis was adopted to measure the Tm
at perfect match and mismatch state respectively. As shown in Figure 3E
and 3F, the melting curves of all matched duplexes for wild-type gene
almost overlap, which is consistent with the predicted results. However,
the Tm of mismatched duplexes for
mutant targets decreased as the number of LNAs increased, improving
ΔTma and
ultimately the enhancement amplification efficiency of mutant targets.
According to the result of LNA7-LNA10 (Figure 3B and Figure 3C), it was
obvious that with the addition of LNA-modified bases to a certain
extent, the ΔTma and detection efficiency of LNA
clamping systems was assuredly increased. However, it should be noted
that it is not enough to focus
only on ΔTma. For example, the ΔTmavalues of LNA4 and LNA5 are close to LNA10 (10.02°C, 9.74°C and 9.02°C,
respectively), but the amplification efficiency of mutant targets was
suppressed in the systems with LNA4 and LNA5 (Figure 2A). This might be
because even when ΔTma is high enough, if the number
of LNA bases is too much, as in LNA4 and LNA5, the Tm of the mismatch
state would also increase further, promoting the formation of the LNA
clamping-mutant target heteroduplex and finally reducing the
amplification efficiency. Hence, it was indicated that the values of Tm
and ΔTma should be kept within a reasonable range.
Here, to highlight the effect of
single base mismatch and the addition of LNA bases more deeply, the Tm
of LNA-modified clamping (LNA 7-10) must be tailored and designed to
maintain a consistent value. It can be seen from the results of LNA7-10,
with the addition of the LNA bases, that although the length of the
clamping segments was shortened from 21 bp to 18 bp, the high Tm could
still be maintained, indicating that the binding affinity from LNA bases
increased. As shown in Table S4 (LNA 7-10), as the number of LNA bases
increased, the Tm contributed by each LNA base
(denoted as
ΔTmb’,
ΔTmb’= (Tm (with LNA)-Tm(without LNA))/number of LNA
bases) also increased, which means that each LNA base plays a key role
in the stability of the LNA-DNA heteroduplex. Hence, the affinity with
the mismatched templates might be dramatically decreased, further
effectively improving the
ΔTma. However,
if there were too many LNA bases in the clamping segments, such as LNA4
or LNA5, even if the length of the clamping segments was only 14 bp or
15 bp, the ΔTmb’
decreased (Table S3), finally reducing the
mismatch discrimination property.
Therefore, it is worth noting that a small amount of LNA modification
not only improves the performance of LNA clamping systems but also
reduces the cost.
3.4 Optimization of LNA Clamping system
To obtain the best performance of the LNA clamping system, the LNA
modification characteristics have been further studied and optimized.
Combining the previous results, there are several key factors for LNA
clamping design: one is high Tm for perfectly matched wild-type gene,
the second is high
ΔTmawhen the single base mismatch occurs, and the third is high increased
affinity per LNA
(ΔTmb’). Thus,
by adjusting the base type,
location and amount of LNA bases, an optimal design model would be
obtained. As mentioned before, we think that the most effective and
economical LNA clamping design strategy should have the
shortest length with the fewest
LNA-modified bases; meanwhile, the Tm is high enough (approximately 75°C
according to our results).
Based on the high Tm, which can inhibit wild-type gene amplification, a
simulation analysis of various LNA modification patterns was evaluated
systematically by applying different lengths from 11 bp-16 bp and
different numbers of LNA-modified bases from 1-7. It is worth noting
that when the length of the LNA clamp is less than 14 bp, the number of
LNA-modified bases must be at least 6 to achieve a relatively high Tm to
inhibit wild-type gene amplification. Therefore, the length of LNA
clamping should be more than 14 bp to minimize the number of LNA bases
(according to the simulation analysis data). On the basis of the rule,
we chose the shortest length with the fewest LNA bases, and
three optimized LNA oligomers were
screened out from a large number of candidate sequences that we
simulated and calculated (LNA11-LNA13). In Figure 4, the sensitivity and
specificity of the obtained three LNA clamps plus LNA 10 were tested
again. As shown in Figure 4E and Figure 4F, ΔTma(LNA11: 11.4°C, LNA12: 11.3°C, LNA13: 8.3°C) and ΔTmb’
(LNA11: 2.57°C, LNA12: 3.20°C, LNA13: 3.70°C) (shown in Table S3 and S4)
of the four LNA clamps are extremely high, indicating that the optimal
LNA clamps with high ΔTma and ΔTmb’
could be obtained from the design principle. Furthermore, the detection
performance at low abundance mutations is verified by real-time PCR. In
addition, the amplification of wild-type gene by primers other than LNA
11, whose Tm is relatively low, can be inhibited (Figure 4A).
The Ct values of these LNA
clamping systems (Figure 4B, 4C and 4D) were almost the same as that of
the LNA-free system when 1% and 0.1% mutant targets were detected
respectively, demonstrating that the optimized amplification efficiency
of mutant targets can be achieved successfully in these systems.
Hence, combining the experimental
observations with previous suggestions, we propose the following
principles to obtain the optimal LNA clamping system for the
ultrasensitive detection of single point mutant targets with low
abundance:
- The length of the LNA clamp should be short (14-16 bp), and the number
of LNA-modified bases should be 4-6. LNA clamping with the shortest
length and the fewest LNA bases should be selected as the most
valuable.
- The ΔTma of perfectly matched and mismatched LNA/DNA
duplexes should be more than 10°C, and the increased affinity per LNA
base (ΔTmb’)
should be as high as possible.
- The Tm of the LNA oligonucleotides should be at least 10 degrees
higher than that of the AS primer to prevent the binding of the AS
primer to wild-type templates.
- The mutant site of the oligonucleotides must be modified, and the
mutant site should be placed in the middle of the clamping chain.
In addition, based on the design strategy proposed above,
to realize the precise regulation
of the LNA-modified base
automatically,
a homebrew analysis software for
obtaining the optimal LNA clamping sequence based on the OligoAnalyzer
interface has been developed in the Python language. Through a
combination of local traversal and cloud computing, we first enter the
sequence containing mutation information into the software and annotate
the mutant site. Then, several optimal LNA clamps can be obtained
automatically and quickly, avoiding tedious labor operations and
significantly saving time (Figure S4).
3.5 Validation and application of the LNA clamping design model
To further verify the reliability
and universality of the LNA-modified design rules we proposed,
other point mutation types in
EGFR, such as L858R, T790M, S768I, and G719A, which are the most common
mutation sites of EGFR and are closely related to the guidance of
targeted drug treatment of cancer,
were chosen to validate the LNA
clamping design principle. Wild-type gene (10000 copies) and plasmids
containing different ratios of EGFR point mutation templates were used
to explore the sensitivity and specificity of LMPC. The optimal LNA
clamping of different mutant sites were designed via homebrew design
software, and the detection performance was evaluated using real-time
PCR and agarose gel electrophoresis. Because the amplification of
wild-type gene cannot be completely inhibited in the LNA-free group, the
amplification signal of ultra-low-concentration mutation targets (mutant
ratio of 0.1%) would be overlapped by the amplification of wild-type
gene, which would finally interfere with the assessment of LMPC (Figure
S3). Therefore, to highlight whether LNA clamp would affect the
amplification of mutant targets before and after addition, wild-type
genes were detected separately rather than added to the mutant targets
in each group (LNA and LNA free). As shown in Figure 5, according to the
amplification curves for all point mutant sites, the wild-type
amplification in each mutant site can be totally suppressed by LNA
clamp, and further, the amplification efficiency on mutant targets (100
and 10 copies) of LNA and LNA-free groups are almost identical except
L858R, because the binding affinity of FP in this site was decreased by
the second structure. Then, the amplicons were analyzed by agarose gel
electrophoresis (Figure 5E). There is no target band of wild-type gene
in the LNA clamping group, indicating that LNA clamping can totally
inhibit the amplification of wild-type gene. Moreover, the brightness of
the mutant band in the electrophoretogram was almost the same between
the LNA and LNA-free groups, which is consistent with the real-time PCR
results. Hence, the results indicated that our LNA clamping design
principles are universal for point mutation at different sites.
Recently, many studies have shown the clinical utility of circulating
cell-free DNA (cfDNA) as the most important target of liquid biopsy in
cancer detection. However, ultra-low-abundance mutant target detection
in variable amounts of cfDNA could be extremely challenging due to the
demand for ultra-sensitivity [33] . To test
the performance of the LNA clamping system in cfDNA samples,
the EGRF cfDNA reference standard
samples were diluted in wild-type DNA (10000 copies) to obtain a series
of cfDNA samples with concentrations of 1%, 0.4%, 0.2%, 0.1%, and
0% (In addition, 500 copies of mutant targets as a positive control).
The Ct values gradually increased with decreasing cfDNA concentration,
and as little as 0.1% cfDNA could be detected correctly at all single
mutant sites (Figure 6A). In general, in the field of nucleic acid
detection, the limit of detection of PCR is usually defined as measurand
concentration that produces at least
95% positive replicates.
To make the sensitivity of 0.1%
in these systems more statistically significant, at least 16 replicates
were performed in each site and as shown in Figure S5, 100% positive
replicates could be obtained at S768I, L858R, G719X and T790M, except
for L861Q (93.7%). The results show that our LNA clamping system can
perform well and reach a sensitivity of 0.1% (10 copies of mutant
targets in 10000 wild-type gene) in cfDNA, which is comparable to the
level of digital PCR and achieves the limit sensitivity of real-time
fluorescence quantitative PCR technology. In addition, LMPC we proposed
here has excellent specificity. This is because that the templates in
cfDNA sample contain all mutant sites, but only one site can be
amplified in specific LMPC under such a complex sample, indicating that
this system has the determination of different sites with high
selectivity.
Furthermore, there are still some differences between the real samples
and the standard samples. Therefore, it is crucial to further explore
the properties of our system in clinical samples. The commercial EGFR
Mutation Detection Kit was selected as a reference, and 24
clinical FFPE samples were tested
using commercial kits and our LNA clamping system. As shown in Figure
6B, all the mutant results of EGFR were identified, and the detection
results of the commercial kit test and our clamping system are exactly
the same as the 100% compliance rate in these 24 samples, demonstrating
that the method has definitely excellent performance in actual clinical
sample detection and prospects for clinical application
Conclusions
In this study, we reported novel,
universal and efficient LNA clamping design rules
for ultra-low abundance point
mutant detection. A series of LNA clamps with high Tm,
ΔTma and increased affinity per LNA base
(ΔTmb’) can easily be obtained through this system,
which could completely suppress the amplification of massive wild-type
gene but with almost no influence on a few copies of mutant targets.
Therefore, it can overcome the bottleneck problem in existing clamping
systems that the amplification efficiency of the mutant targets must be
decreased to inhibit the wild-type gene amplification signal. Then, the
sensitivity of rare mutant target detection could be greatly improved,
which is extremely important for the detection of ctDNA. Furthermore,
the design pattern can dramatically decrease the length of
oligonucleotides and the number of LNA-modified bases, which is suitable
for multiplex PCR. In addition, optimal LNA clamping could be obtained
efficiently and easily by homebrew analysis software. Moreover, the LNA
clamping system was applied in the most common point mutation of EGFR
and can achieve a highly sensitive and specific result in both cfDNA
standard samples and clinical samples, providing an efficient strategy
for mutant target detection in ctDNA and good prospects for clinical
application.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work is supported by the National Natural Science Foundation of
China (Grant No. 31927803); Exploratory Projects of Natural Science
Foundation of Shanghai (Grant No. 19ZR1476000); Innovation Research Plan
supported by Shanghai Municipal Education Commission (Grant No.
ZXWF082101) and SJTU funding (ZH2018ZDA35 and 19X150010008); Cross
Research Fund of Biomedical Engineering of Shanghai Jiao Tong University
(Grant No. ZH2018QNA69).
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Figure legend
Figure 1. The LNA-mediated real-time PCR clamping system (LMPC). (A) The
LNA oligomer was designed to perfectly match the wild-type sequence. The
forward primer partially overlapped the LNA binding position, and the 3’
end of the primer was designed to bind the point mutation site. During
PCR, an LNA/DNA hybrid with a perfect match inhibited annealing of the
primer, thus suppressing the amplification of wild-type DNA. However, an
LNA/DNA hybrid with a single base-pair mismatch did not restrain the
amplification of mutant alleles. (B) Various LNA modification modes
based on length, base number, and position were evaluated. LNA
modification has a great impact on clamping performance. The optimal
amplification state of LMPC is that the amplification of wild-type gene
can be inhibited completely with no influence on mutant targets. But in
most clamping system, the amplification of mutant targets would be
suppressed as deficiency state.