patients with non-small-cell lung cancer using a dual pna … · 1 ultrasensitive and quantitative...
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1
Ultrasensitive and quantitative detection of EGFR mutations in plasma samples from
patients with non-small-cell lung cancer using a dual PNA clamping-mediated LNA-
PNA PCR clamp †
Shichao Zhang,a,c,† Zhiyao Chen,a,† Chenrong Huang,a Cheng Ding,b Chang Li,b Jun Chen,b
Jun Zhao*b and Liyan Miao*a,c
aDepartment of Clinical Pharmacology, The First Affiliated Hospital of Soochow University,
Suzhou, 215006, PR China
bDepartment of Thoracic Surgery, The First Affiliated Hospital of Soochow University,
Suzhou, 215006, PR China
cCollege of Pharmaceutical Sciences, Soochow University, Suzhou, 215006, PR China
†These authors contributed equally to this work.
*Correspondence:
Liyan Miao
No. 188, Shi Zi Street, Suzhou, 215006, PR China. Tel.: (+86) 512-6778-0467; Fax: (+86)
512 67780040; E-mail address: [email protected].
Jun Zhao
No. 188, Shi Zi Street, Suzhou, 215006, PR China. Tel.: (+86) 512-6778-0507; Fax: (+86)
512 67780507; E-mail address: [email protected].
Electronic Supplementary Material (ESI) for Analyst.This journal is © The Royal Society of Chemistry 2019
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List of Electronic Supplementary Information (ESI)
Table S1 Sequences of the PCR primers and probes used for the LNA-dPNA PCR clamp.
Table S2 Clinical characteristics of the patients with NSCLC.Figure S1 Efficiency of the LNA-dPNA PCR clamp at various
concentrations of the M-LNA probe (A), cycle numbers (B), concentrations of IPs (C), and concentrations of OPs (D).
Figure S2 Mutation enrichment detected by the LNA-dPNA PCR clamp at various concentrations of the W-PNA probe using 103 M:103 W (A) and 103 M:106 W (B) as templates.
Figure S3 Mutation enrichment detected by the LNA-dPNA PCR clamp at annealing temperatures of 72 °C (A-1, B-1), 70 °C (A-2, B-2), 68 °C (A-3, B-3), and 66 °C (A-4, B-4) using OPs and IPs.
Figure S4 Mutation enrichment detected by the LNA-dPNA PCR clamp at annealing temperatures of 64 °C (A-1, B-1), 60 °C (A-2, B-2), and 56 °C (A-3, B-3) using OPs and IPs.
Figure S5 Evaluation of the effects of one-tube nested PCR (A) and dual PNA clamp (B) on the LNA-dPNA PCR clamp.
Figure S6 Evaluation of the sensitivity (A), specificity (B), and linearity (A) using ddPCR for EGFR L858R detection.
Figure S7 Evaluation of the reproducibility of LNA-dPNA PCR clamp in detection of EGFR L858R.
Figure S8 Typical results for EGFR L858R analysed using the LNA-dPNA PCR clamp (A, C, E, G) and ddPCR (B, D, F, H) from plasma (A, B, E, F) and tissue (C, D, G, H) samples.
Table S3 Comparison of ddPCR and the LNA-dPNA PCR clamp for detecting plasma EGFR mutations and tissue EGFR mutations.
Table S4 Comparison of EGFR mutations in tumour tissues and matched plasma samples for different tumour stages.
Table S5 Comparison of labour and cost between the LNA-dPNA PCR clamp and ddPCR for quantifying plasma mutations.
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Table S1. Sequences of the PCR primers and probes used for the LNA-dPNA PCR clamp.
Primer name Sequence (5’-3’)Tm
(C)
Length of
amplicon
EGFR858-IP-F CAGCATGTCAAGATCACAGATT 61.0
EGFR858-IP-R CCTTACTTTGCCTCCTTCTG 60.087 bp
EGFR858-OP-F CTACTTGGAGGACCGTCGCTTGGTGC 69.5
EGFR858-OP-R CCTGGTCCCTGGTGTCAGGAAAATGCT 69.5200 bp
EGFR858-W-PNA TGGGCTGGCCA ~ 63.9
EGFR858-M-LNA 6-FAM-TTGGGCGGGCCAAA-TAMRA 65.8/
EGFR858-UP HEX-TCTTTCTCTTCCGCACCCAGC-BHQ1 67.3 /
EGFR19-2237-IF ATCCCAGAAGGTGAGAAAGT 60.7
EGFR19-2237-IR GGGCCTGAGGTTCAGAG 60.8121 bp
EGFR19-2237-OF TGGCACCATCTCACAATTGCCAGTT 66.9
EGFR19-2237-OR GCAGCTGCCAGACATGAGAAAAGGT 66.8207 bp
EGFR19-W-PNA AGGAATTAAGAGAAGCAACATCT ~ 64.5 /
EGFR19-2237-LNA 6-FAM-CAAGGCCGAAAGCC-IABkFQ 70.5 /
EGFR19-2240-UP HEX-ACAGCAAAGCAGAAACTCACATCGA-BHQ1 67.7
EGFR-T790M-IF ATCTGCCTCACCTCCAC 60.3
EGFR-T790M-IR GGAGCCAATATTGTCTTTGTGT 61.296 bp
EGFR-T790M-OF AGCCACACTGACGTGCCTCTCC 67.9
EGFR-T790M-OR ATCTGCACACACCAGTTGAGCAGGT 68.1211 bp
EGFR-T790M-PNA GCTCATCACGCAGCTCAT ~ 66.3 /
EGFR-T790M-LNA 6-FAM-TCATCATGCAGC-IABkFQ 65.4 /
EGFR-T790M-UP HEX-TCCCGGACATAGTCCAGGAG-BHQ1 66.3 /
EGFR797-2390-IF ATCTGCCTCACCTCCAC 60.3
EGFR797-2390-IR ACCAGTTGAGCAGGTACT 60.1115 bp
EGFR797-2390-OF CTCTCCCTCCCTCCAGGAAGCCTA 67.3
EGFR797-2390-OR TCCCTGATTACCTTTGCGATCTGCACAC 67.9213 bp
EGFR797-2390-PNA CTTCGGCTGCCTCCTGG ~ 69.7 /
EGFR797-2390-LNA 6-FAM-TTCGGCTCCCTCCT-IABkFQ 71.1 /
EGFR797-2390-UP HEX-AGCTGCGTGATGAGCTGC-BHQ1 66.0 /
Note: The Tm values of the primers and probes were calculated by OligoAnalyzer 3.1
(Integrated DNA Technologies, Inc., Iowa, USA). The LNA bases were marked in italic. The
Tm values of the PNA probes were 2-13 °C higher than the values calculated by
OligoAnalyzer 3.1. Because a base mismatch occurs between the W-PNA and the mutant
sequence, the Tm of W-PNA shifts 2-13 °C.
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Table S2. Clinical characteristics of patients with NSCLC.
Patients with NSCLC
(N=212)
Age, median
(range)63 (32-83)
Sex (n, %)
Male 112 (52.8%)
Female 100 (47.2%)
Smoking history
(n, %)
Never smoked 149 (70.3%)
Smoker 63 (29.7%)
Histological
type
Adenocarcinoma 159(75.0%)
Squamous cell
carcinoma38(17.9%)
Others 15(7.1%)
Stage
Stage I 102 (48.1%)
Stage II 48 (22.7%)
Stage III 49 (23.1%)
Stage IV 13 (6.1%)
5
10 20 30 40
0.0
0.8
1.6
2.4
3.2
CTBlank=UD
CT0.4 µM=21.0
CT0.1 µM=24.1
CT0.2 µM=22.2
CT0.6 µM=20.7CT0.8 µM=20.0
CT1.0 µM=20.2
Del
ta R
n
PCR cycles10 20 30 40
0.0
0.8
1.6
2.4
3.2
CTBlank=UD
CT5 cycles=16.2CT10 cycles=11.4
CT15 cycles=6.3
Del
ta R
n
PCR cycles
10 20 30 40
0.0
0.8
1.6
2.4
3.2
CTBlank=UD
CT0.4 µM=11.6
CT0.2 µM=11.5
CT0.1 µM=11.6
Del
ta R
n
PCR cycles
CT0.6 µM=11.7
10 20 30 40
0.0
0.8
1.6
2.4
3.2
CTBlank=UD
CT0.2 µM=6.3
CT0.1 µM=6.9
CT0.02 µM=13.9
CT0.05 µM=8.6
Del
ta R
n
PCR cycles
A B
C D
Figure S1. Efficiency of the LNA-dPNA PCR clamp at various concentrations of M-LNA
probe (A), cycle numbers (B), concentrations of IPs (C), and concentrations of OPs (D). All
experiments were performed at the same concentration of template (106 mutant plasmid), and
each assessment was performed twice. The selected conditions are marked with red arrows.
Note: The efficiency of the LNA-dPNA PCR clamp was affected by the concentrations of the
M-LNA probe, IPs, and OPs as well as the cycle numbers. Therefore, we optimized these key
factors and found that a high efficiency was achieved when performing the LNA-dPNA PCR
clamp with 0.8 μM M-LNA probe (Figure S1A), 0.2 μM IPs (Figure S1B), and 15 cycles of
pre-amplification (Figure S1C). We also found that a higher efficiency was achieved at a
higher concentration of OPs (0.2 μM), but a smoother S-curve was achieved at a lower
concentration of OPs (0.05 μM). Therefore, we selected 0.05 μM as the optimized
concentration (Figure S1D).
6
10 20 30 40
0.0
0.8
1.6
2.4
0.2
CTW+10 µM PNA=UDCTW+8 µM PNA=UD
CTM+8 µM PNA=17.8
CTW+0 µM PNA=18.5
CTM+0 µM PNA=18.0
CTM+10 µM PNA=17.6D
elta
Rn
PCR cycles
CTW+4 µM PNA=UD
CTM+4 µM PNA=17.6
10 20 30 40
0.0
0.8
1.6
2.4
0.2
CTW+0 µM PNA=7.9
CTM+8 µM PNA=26.7
CTW+10 µM PNA=UD
CTM+0 µM PNA=UD
CTW+4 µM PNA=28.6CTM+10 µM PNA=30.0D
elta
Rn
PCR cycles
CTW+8 µM PNA=UD
CTM+4 µM PNA=29.7
A B
Figure S2. Mutation enrichment detected by the LNA-dPNA PCR clamp at various
concentrations of the W-PNA probe using 103 M:103 W (A) and 103 M:106 W (B) as
templates. “M” means mutant DNA; “W” means wild-type DNA; “UD” means “Undetected”.
Each assessment was performed twice, and the selected conditions are marked with red
arrows.
Note: The mutation enrichment detected by the LNA-dPNA PCR clamp was affected by the
W-PNA probe concentration and annealing temperature. Therefore, we first optimized the
PNA concentration and found that mutant DNA amplification was unaffected, and wild-type
DNA amplification was completely blocked when the concentration of W-PNA was changed
from 4 μM to 10 μM at a lower concentration of wild-type template (103 copies) (Figure
S2A). We selected 8 μM W-PNA as the optimal concentration because more efficient
blockade of wild-type DNA (UD vs. 29.7) and more efficient mutant DNA amplification
(26.7 vs. UD) were obtained when the W-PNA concentration was 8 μM vs. 4 μM at a higher
concentration of wild-type DNA (106 copies) (Figure S2B).
7
10 20 30 400.0
0.4
0.8
1.2
CTW+PNA=UDCTW=UD CTBlank=UDCTM+PNA=UD
Delta
Rn
PCR cycles
CTM=UD
B-1 PCR with IPs at 72 °C
10 20 30 400.0
0.4
0.8
1.2
CTW=19.8
CTBlank=UD
CTW+PNA=22.6
CTM+PNA=19.3
Delta
Rn
PCR cycles
CTM=19.1
1.8
A-1 PCR with OPs at 72 °C
10 20 30 400.0
0.4
0.8
1.2
CTW=23.4
CTBlank=UD
CTW+PNA=33.0
CTM+PNA=24.8
Delta
Rn
PCR cycles
CTM=24.3
9.6
B-3 PCR with IPs at 68 °C
10 20 30 400.0
0.4
0.8
1.2
CTW=19.3
CTBlank=UD
CTW+PNA=24.1
CTM+PNA=18.5
Delta
Rn
PCR cycles
CTM=19.24.8
A-4 PCR with OPs at 66 °C
10 20 30 400.0
0.4
0.8
1.2
CTW=19.4
CTBlank=UD
CTW+PNA=22.6
CTM+PNA=18.6
Delta
Rn
PCR cycles
CTM=19.0
3.1
A-2 PCR with OPs at 70 °C
10 20 30 400.0
0.4
0.8
1.2
CTW+PNA=UDCTW=UD CTBlank=UDCTM+PNA=UD
Delta
Rn
PCR cycles
CTM=UD
B-2 PCR with IPs at 70 °C
10 20 30 400.0
0.4
0.8
1.2
CTW=19.3
CTBlank=UD
CTW+PNA=23.7
CTM+PNA=18.9
Delta
Rn
PCR cycles
CTM=18.9
4.6
A-3 PCR with OPs at 68 °C
CT OPs blocking + IPs blocking=
1.8+0=1.8
CT OPs blocking + IPs blocking=
3.1+0=3.1
CT OPs blocking + IPs blocking=
4.6+9.6=14.2
CT OPs blocking + IPs blocking=
4.8+6.2=11.0
10 20 30 400.0
0.4
0.8
1.2
CTW=18.7
CTBlank=UD
CTW+PNA=24.9
CTM+PNA=19.1
Delta
Rn
PCR cycles
CTM=19.1
6.2
B-4 PCR with IPs at 66 °C
Figure S3. Mutation enrichment detected by the LNA-dPNA PCR clamp at annealing
temperatures of 72 °C (A-1, B-1), 70 °C (A-2, B-2), 68 °C (A-3, B-3), and 66 °C (A-4, B-4)
using OPs and IPs. “M” means mutant DNA; “W” means wild-type DNA; “PNA” means W-
PNA probe; “UD” means “Undetected”. Each assessment was performed twice, and the
selected conditions are marked with red arrows.
8
10 20 30 400.0
0.4
0.8
1.2
CTW=19.2
CTBlank=UD
CTW+PNA=24.9
CTM+PNA=18.8
Delta
Rn
PCR cycles
CTM=18.85.7
A-1 PCR with OPs at 64 °C10 20 30 40
0.0
0.4
0.8
1.2
CTW=18.2
CTBlank=UD
CTW+PNA=24.2
CTM+PNA=18.6
Delta
Rn
PCR cycles
CTM=18.66.0
B-1 PCR with IPs at 64°C
10 20 30 400.0
0.4
0.8
1.2
CTW=17.7
CTBlank=UD
CTPNA+W=24.3
CTPNA+M=18.1
Delta
Rn
PCR cycles
CTM=18.2
6.6
CT OPs blocking + IPs blocking=
5.7+6.0=11.7
CT OPs blocking + IPs blocking=
6.2+6.6=12.8
CT OPs blocking + IPs blocking=
6.5+7.1=13.6
10 20 30 400.0
0.4
0.8
1.2
CTW=19.0
CTBlank=UD
CTPNA+W=25.2
CTPNA+M=18.6
Delta
Rn
PCR cycles
CTM=18.76.2
A-2 PCR with OPs at 60 °C B-2 PCR with IPs at 60 °C
10 20 30 400.0
0.4
0.8
1.2
CTBlank=UD
CTPNA+W=24.7
CTW=18.2
CTPNA+M=18.3
Delta
Rn
PCR cycles
CTM=18.3
6.5
A-3 PCR with OPs at 56 °C
10 20 30 400.0
0.4
0.8
1.2
CTW=17.2
CTBlank=UD
CTPNA+W=24.3
CTPNA+M=17.9
Delta
Rn
PCR cycles
CTM=17.77.1
B-3 PCR with IPs at 56 °C
Figure S4. Mutation enrichment detected by the LNA-dPNA PCR clamp at annealing
temperatures of 64 °C (A-1, B-1), 60 °C (A-2, B-2), and 56 °C (A-3, B-3) using OPs or IPs.
“M” means mutant DNA; “W” means wild-type DNA; “PNA” means W-PNA probe; “UD”
means “Undetected”. Each assessment was performed twice, and the selected conditions are
marked with red arrows.
Note: We optimized the annealing temperatures, and 68 °C for the OPs and 56 °C for the IPs
were selected because we found that more efficient blockade was achieved when the OPs
were amplified at 68 °C (14.2) rather than 72 °C (1.8), 70 °C (3.2), or 66 °C (11.0) (Figure
S3). We further observed that more efficient blockade was achieved when the IPs were
amplified at 56 °C (13.6) rather than 60 °C (12.8) or 62 °C (11.7) (Figure S4). A better
specificity was achieved when the IPs were amplified at 60 °C rather than 56 °C when the
clinical samples were amplified (data not shown).
9
10 20 30 40
0.0
0.8
1.6
2.4
3.2
CTBlank=UD
CTwithout OPs=20.0Del
ta R
n
PCR cycles10 20 30 40
0.0
0.8
1.6
2.4
3.2
CTBlank=UD
CTwith OPs=8.6 CT without OPs=20.1
Del
ta R
n
PCR cycles
11.5
A-1 A-2
0 10 20 30 400.0
0.4
0.8
1.2
CTW=19.0
CTM=18.7
CTW+PNA=25.2
CTM+PNA=18.6
Del
ta R
n
PCR cycles
6.2
CTBlank=UD
10 20 30 400.0
0.4
0.8
1.2
CTW=17.7
CTM+PNA=18.2
CTW+PNA=24.3
CTM+PNA=18.1
Del
ta R
n
PCR cycles
6.6
CTBlank=UD
10 20 30 400.0
0.4
0.8
1.2
CTW+PNA=23.7
CTW=19.3
CTM=18.9
CTBlank=UD
CTM+PNA=18.9
Del
ta R
n
PCR cycles
4.6
10 20 30 400.0
0.4
0.8
1.2
CTW=23.4
CTW+PNA=33.0
CTM+PNA=24.8
CTM=24.3
Del
ta R
n
PCR cycles
9.6
CTBlank=UD
B-3
B-1 B-2
B-4
Conventional PCR One-tube nested PCR
PCR with OPs at 68 °C PCR with IPs at 68 °C
PCR with OPs at 60 °C PCR with IPs at 60 °C
Figure S5. Evaluation of the effects of one-tubed nested PCR (A) and dual PNA clamp (B) on
the LNA-dPNA PCR clamp. “M” means mutant DNA; “W” means wild-type DNA; “PNA”
means W-PNA probe. Each assessment was performed twice.
10
A-1
A-3
A-2
A-4
A-5
A-6
A-7
Theoretical Value: 0 M (105 W);
Measured Value: 0 M
Theoretical Value: 100 M;
Measured Value: UN
Theoretical Value: 101 M;
Measured Value: 0.6×101 M
Theoretical Value: 102 M;
Measured Value: 0.80×102 M
Theoretical Value: 103 M;
Measured Value: 0.98×103 M
Theoretical Value: 104 M;
Measured Value: 1.07×104 M
Theoretical Value:105 M;
Measured Value: 1.01×105 M
B-1
B-2
B-3
B-4
B-5
B-7
B-6
Theoretical Value: 0.001%(100 M:105 W);
Measured Value: UN
Theoretical Value: 0.01 %(101 M:105 W);
Measured Value: 0.0083%(1.16×101 M:1.40×105 W)
Theoretical Value: 0.05%(5×101 M:105 W);
Measured Value: 0.032%(4.60×101 M:1.42×105 W)
Theoretical Value: 0.1%(102 M:105 W);
Measured Value: 0.076%(1.04×102 M:1.36×105 W)
Theoretical Value: 1%(103 M:105 W);
Measured Value: 0.67%(0.94×103 M:1.40×105 W)
Theoretical Value: 10%(104 M:105 W);
Measured Value: 6.08%(0.90×104 M:1.39×105 W)
Theoretical Value: 50%(105 M:105 W);
Measured Value: 46.60%(0.89×105 M:1.02×105 W)
Figure S6. Evaluation of the sensitivity (A), specificity (B), and linearity (A) using ddPCR for EGFR L858R detection. “M” means mutant DNA; “W” means wild-type DNA; “UN” means undetermined. Each assessment was performed twice.
11
10 20 30 40
0.0
0.7
1.4
2.1
2.8
Delta
Rn
PCR cycles
0% Blank0.01%
0.2
50% 10% 1% 0.1%
10 20 30 40
0.0
0.7
1.4
2.1
2.8
3.5
Delta
Rn
PCR cycles
0.2 Blank105 104
102
103101 100
10 20 30 40
0.0
0.8
1.6
2.4
3.2
Delta
Rn
PCR cycles
0.2Blank
105104
102103 101 100
10 20 30 40
0.0
0.8
1.6
2.4
3.2
Delta
Rn
PCR cycles
0% Blank0.01%0.2
50% 10% 1% 0.1%
10 20 30 40
0.0
0.8
1.6
2.4
3.2
Delta
Rn
PCR cycles
0.2Blank
104 102103 101100
10 20 30 40
0.0
0.8
1.6
2.4
3.2
4.0
Delta
Rn
PCR cycles
0% Blank0.01%
0.250% 10% 1% 0.1%
A-1
A-2
A-3
B-1
B-2
B-3
y = 3.3506x+ 5.3655R² = 0.9942
y = 3.9568x+ 3.6407R² = 0.9867
y = 3.6471x+ 3.6985R² = 0.9967
y = 3.4613x+ 3.0756R² = 0.9947
y = 3.535x+ 5.5968R² = 0.9927
y = 3.3654x+ 5.9421R² = 0.9975
Figure S7. Evaluation of the reproducibility of LNA-dPNA PCR clamp in detection of EGFR
L858R. The amplification sensitivity (A) and limit of detection (LOD) (B) were repeatedly
detected in triplicate from sample dilution to real-time PCR. A-1: The amplification
sensitivity was tested first; A-2: the amplification sensitivity was tested second; A-3: the
amplification sensitivity was tested third. B-1: the LOD was tested first; B-2: the LOD was
tested second; B-3: the LOD was tested third. Each assessment was performed twice.
Note: 0.01% (101M:105W) was repeatedly detected in the six parallel tests. A 100
copy/reaction was detected four times in six parallel tests due to its extremely low content.
12
10 20 30 40
0.0
0.8
1.6
2.4
3.2
4.0
0.2
CTC=19.1
CTM=17.5
Del
ta R
n
PCR cycles
10 20 30 40
0.0
0.8
1.6
2.4
0.2
CTC=25.1
CTM=22.7D
elta
Rn
PCR cycles
C D
A B
10 20 30 40
0.0
0.4
0.8
1.2
1.6
0.2
CTC=28.2
CTM=UD
Delta
Rn
PCR cycles
10 20 30 40
0.0
0.4
0.8
1.2
1.6
0.2
CTC=21.3
CTM=UD
Del
ta R
n
PCR cycles
E F
G H
Figure S8. Typical results for EGFR L858R analysed using the LNA-dPNA PCR clamp (A,
C, E, G) and ddPCR (B, D, F, H) from plasma (A, B, E, F) and tissue (C, D, G, H) samples.
“M” means mutant DNA; “C” means universal control; “UD” means “Undetected.” Each
assessment was performed twice.
13
Table S3. Comparison of ddPCR and the LNA-dPNA PCR clamp for detecting plasma EGFR
mutations and tissue EGFR mutations.
LNA-dPNA PCR
clampddPCR
Positive Negative
TotalSensitivity
(%)
Specificity
(%)
Positive 46 0 46 100.0 /Tissues
Negative 0 86 86 / 100.0
Total 46 86 132 / /
Positive 12 5 17 70.6 /Plasmas
Negative 2 113 115 / 98.3
Total 14 118 132 / /
14
Table S4. Comparison of EGFR mutations in tumor tissues and matched plasma samples for
different tumour stages.
Matched plasma samplesTumor
stagesTissues
Positive NegativeSensitivity
I 29 2 27 6.9%
II 11 2 9 18.2%
III 8 1 7 12.5%
IV 3 3 0 100%
15
Table S5. Comparison of labour and cost between the LNA-dPNA PCR clamp and ddPCR
for quantifying plasma mutations.
LNA-dPNA PCR clamp ddPCR
StepsTime
(h)
Cost
($)Steps
Time
(h)Cost ($)
Standard curve
preparation 0.5 0.02
Reagent
preparation0.5 22.04
Reagent
preparation0.5 39.52
Droplet
preparation0.5 28.08
PCR 1.25 0 PCR 2.0 0
Data analysis 0.5 0 Droplet reading 0.5 5.92
Data analysis 0.5 0
Total 2.75 39.68a Total 4.0 56.04 b
a Eight reactions are needed to quantify one mutation site with the LNA-dPNA PCR clamp, including 5 reactions for the
standard curve, 1 reaction for the negative control, 1 reaction for the blank control, and 1 reaction for the sample. The cost of
one reaction is 4.96 $, and the cost for the reaction is as follows: EASY Dilution (for real time PCR): 0.02 $/reaction; PNA
probe: 2.49 $/reaction; TaKaRa Premix Ex TaqTM (probe qPCR): 0.69 $/reaction; PCR tube (Axygen): 0.09 $/reaction; LNA
Probe: 1.54 $/reaction; universal probe: 0.13 $/reaction.
b Four reactions are needed to quantify one mutation site by ddPCR, including 1 reaction for the positive control, 1 reaction
for the negative control, 1 reaction for the blank control, and 1 reaction for the sample. The cost of one reaction is 14.01 $,
and the cost for the reaction is as follows: PrimePCRTM ddPCRTM Mutation Assay (Bio-Rad): 3.52 $/reaction; probes: 1.83
$/reaction; PCR tube and seal foil: 0.16 $/reaction; droplet generation oil: 0.96 $/reaction; droplet generator: 4.76 $/reaction;
DG8 gaskets: 1.3 $/reaction; droplet-reading oil: 1.48 $/reaction.