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Tumor Discovery Somatic mutations in POLE and POLD1 in colorectal cancer

Table 1. Comparison of the clinicopathological features of 2.4. Quick-multiplex consensus-PCR protocols
patient cohorts
2.4.1. Thermal cycling conditions
Variable Classification Cohort, n=102; Cohort, n=83; The QMC-PCR was performed as previously described.
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frequency, frequency,
n (%) n (%) The pre-diagnostic multiplex (PDM) reaction was
Sex Male 57 (56) 49 (59) performed in a final volume of 25 µL, containing 1×
HotShot master mix (Cadama Medical Ltd., UK), 250 nM
Female 37 (36) 34 (41) of each primer, and 20 ng template DNA. The PDM
Unknown 8 (8) reactions were carried out separately for the POLE and
Age Median 70 (43 – 88) 71 (43 – 80) POLD1 targets. For the POLE primers, PCR was performed
Dukes’ stage A 6 (6) 4 (5) using a two-step cycling protocol: One cycle at 95°C for
B 29 (28) 29 (35) 5 min, followed by 25 cycles of 95°C for 1 s and 55.5°C for
C 41 (40) 39 (47) 1 s; whereas for the POLD1 targets, the PCR cycling was
3-step: 1 cycle at 95°C for 5 min, followed by 40 cycles of
D 2 (2) 11 (13)
Unknown 24 (23) 0 95°C for 10 s, 55°C for 30 s, and 72°C for 10 s. The single
specific diagnostic (SSD) reaction was performed in a
Vascular invasion V0 49 (48) 42 (51) final volume of 10 µL, containing 1× HotShot master mix,
V1 31 (30) 29 (35) 1 primer pair (each primer at 250 nM final concentration),
V2 1 (1) 1 (1) 1× LC Green PLUS (Idaho Technology, United States),
Unknown 21 (21) 11 (13) and water to complete the total volume. The template
Tumor stage pT1 3 (3) 3 (4) consisted of 1 µL of a 1:100 dilution of the PCR product
pT2 6 (6) 3 (5) from the PDM reaction. The PCR was performed using
pT3 47 (46) 43 (52) a two-step protocol: 1 cycle of denaturation at 95°C for
pT4 24 (23) 21 (25) 5 min, followed by 45 cycles of denaturation at 95°C for 1
s, and annealing at 55°C for 1 s.
Unknown 22 (22) 12 (14)
2.4.2. Co-amplification at lower denaturation
temperature (COLD)-PCR
used to design outer and inner primers for all the targets of Full co-amplification at a lower denaturation temperature
interest, including exons 9 – 14 of POLE and exons 8 – 13 of PCR followed by HRM (full COLD-PCR-HRM) analysis
POLD1. All the inner primers were designed to amplify PCR was used to enrich for minor alleles that were below the
products that are <250 bp. In total, six sets of outer and detection limits of Sanger sequencing. First, the critical
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eight sets of inner primer pairs were designed for POLE temperatures (Tc) for selected POLE and POLD1 targets
and POLD1, respectively. Primer specificities were assessed were determined using gradient PCR with the following
using UCSC in silico PCR (http://genome.ucsc.edu/cgi-bin/ parameters: 1 cycle of initial denaturation at 95°C for 5 min;
hgPcr?command=start), as well as on MFEprimer-2.0 (http:// 10 cycles of denaturation at 95°C for 1 s and annealing
biocompute.bmi.ac.cn/CZlab/MFEprimer-2.0/index.cgi/ at 55.5°C for 1 s; followed by 25 cycles of denaturation at
check_dimer), which also excluded possible primer-dimer Tm ± 4°C for 10 s, annealing at 55°C for 15 s, and extension at
formations. The optimal annealing temperatures for all 72°C for 10 s. Following HR-1 HRM, the lowest denaturation
primers were determined through gradient PCR and gel temperature that produced amplification was chosen as
electrophoresis on a Primus PCR machine. The annealing the Tc for each target. The cycling parameters for the full
temperature for all eight POLD1 outer primer pairs was COLD-PCR were as follows: 1 cycle of initial denaturation
set at 60°C, whereas all eight POLD1 inner primers and at 95°C for 5 min; 45 cycles of denaturation (95°C/30 s),
all the POLE primers (six sets of outer and inner primer hybridization (70°C/30 s), denaturation (Tc/30 s), annealing
pairs) had an annealing temperature of 55°C. The gradient (55.5°C/1 min), and extension (72°C/1 min); with 1 cycle of
PCR cycling conditions were as follows: one cycle at 95°C final extension at 72°C for 10 min.
for 5 min; 40 cycles of denaturation at 95°C for 10 s,
annealing at 55 ± 5°C for 30 s, and extension at 72°C 2.5. HRM analysis
for 20 s (refer Tables A1 and A2 for the outer and inner The PCR products of the SSD reactions were transferred
primer sequences). The specificities of the individual inner to light cycler capillaries (20 µL) (Roche, Switzerland). The
primers were further verified using standard PCR followed products were loaded in the HR-1 HRM instrument (Idaho
by gel electrophoresis (Figures A1 and A2). Technology) and melted by raising the temperature at a

Volume 3 Issue 3 (2024) 3 doi: 10.36922/td.3659