A 115-bp MethyLight assay for detection of p16 (CDKN2A) methylation as a diagnostic biomarker in human tissues.

Abstract

p16 Methylation is a potential biomarker for prediction of malignant transformation of epithelial dysplasia. A probe-based, quantitative, methylation-specific PCR (MSP) called MethyLight may become an eligible method for detecting this marker clinically. We studied oral mucosa biopsies with epithelial dysplasia from 78 patients enrolled in a published 4-years' followup cohort, in which cancer risk for patients with p16 methylation-positive dysplasia was significantly higher than those without p16 methylation (by 150-bp MSP and bisulfite sequencing; +133 ~ +283, transcription starting site, +1). The p16 methylation status in samples (N = 102) containing sufficient DNA was analyzed by the 70-bp classic (+238 ~ +307) and 115-bp novel (+157 ~ +272) MethyLight assays, respectively.
p16 Methylation was detectable in 75 samples using the classic MethyLight assay. The methylated-p16 positive rate and proportion of methylated-p16 by the MethyLight in MSP-positive samples were higher than those in MSP-negative samples (positive rate: 37/44 vs. 38/58, P=0.035, two-sided; proportion [median]: 0.78 vs. 0.02, P <0.007). Using the published results of MSP as a golden standard, we found sensitivity, specificity, and accuracy for this MethyLight assay to be 70.5%, 84.5%, and 55.0%, respectively. Because amplicon of the classic MethyLight procedure only partially overlapped with the MSP amplicon, we further designed a 115-bp novel MethyLight assay in which the amplicon on the sense-strand fully overlapped with the MSP amplicon on the antisense-strand. Using the 115-bp MethyLight assay, we observed methylated-p16 in 26 of 44 MSP-positive samples and 2 of 58 MSP-negative ones (P = 0.000). These results were confirmed with clone sequencing. Sensitivity, specificity, and accuracy using the 115-bp MethyLight assay were 59.1%, 98.3%, and 57.4%, respectively. Significant differences in the oral cancer rate were observed during the followup between patients (≥60 years) with and without methylated-p16 as detected by the 115-bp MethyLight assay (6/8 vs. 6/22, P = 0.034, two-sided).
The 115-bp MethyLight assay is a useful and practical assay with very high specificity for the detection of p16 methylation clinically.

Full text

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RESEARCH ARTICLEOpen Access
A 115-bp MethyLight assay for detection of p16
(CDKN2A) methylation as a diagnostic biomarker
in human tissues
Jing Zhou1, Jie Cao2, Zheming Lu1, Hongwei Liu2and Dajun Deng1*
Abstract
Background: p16 Methylation is a potential biomarker for prediction of malignant transformation of epithelial
dysplasia. A probe-based, quantitative, methylation-specific PCR (MSP) called MethyLight may become an eligible
method for detecting this marker clinically. We studied oral mucosa biopsies with epithelial dysplasia from 78
patients enrolled in a published 4-years’ followup cohort, in which cancer risk for patients with p16 methylation-
positive dysplasia was significantly higher than those without p16 methylation (by 150-bp MSP and bisulfite
sequencing; +133 ~ +283, transcription starting site, +1). The p16 methylation status in samples (N = 102)
containing sufficient DNA was analyzed by the 70-bp classic (+238 ~ +307) and 115-bp novel (+157 ~ +272)
MethyLight assays, respectively.
Results: p16 Methylation was detectable in 75 samples using the classic MethyLight assay. The methylated-p16
positive rate and proportion of methylated-p16 by the MethyLight in MSP-positive samples were higher than those
in MSP-negative samples (positive rate: 37/44 vs. 38/58, P=0.035, two-sided; proportion [median]: 0.78 vs. 0.02,
P <0.007). Using the published results of MSP as a golden standard, we found sensitivity, specificity, and accuracy
for this MethyLight assay to be 70.5%, 84.5%, and 55.0%, respectively. Because amplicon of the classic MethyLight
procedure only partially overlapped with the MSP amplicon, we further designed a 115-bp novel MethyLight assay
in which the amplicon on the sense-strand fully overlapped with the MSP amplicon on the antisense-strand. Using
the 115-bp MethyLight assay, we observed methylated-p16 in 26 of 44 MSP-positive samples and 2 of 58 MSP-
negative ones (P = 0.000). These results were confirmed with clone sequencing. Sensitivity, specificity, and accuracy
using the 115-bp MethyLight assay were 59.1%, 98.3%, and 57.4%, respectively. Significant differences in the oral
cancer rate were observed during the followup between patients (≥60 years) with and without methylated-p16
as detected by the 115-bp MethyLight assay (6/8 vs. 6/22, P = 0.034, two-sided).
Conclusions: The 115-bp MethyLight assay is a useful and practical assay with very high specificity for the
detection of p16 methylation clinically.
Background
Aberrant methylation of CpG islands is a very stable modi-
fication of genomic DNA that often inactivates gene
expression pathologically. Methylation of a target CpG
island in even 0.1% of a cell population obtained from
fixed/frozen tissues or body fluids can be detected readily.
The high stability and high sensitivity of detection make
DNA methylation one kind of optimal clinical biomarker
for the prediction of potential malignancy progression of
precancerous lesions, metastasis/recurrence of cancer, and
chemo/radio-therapy sensitivity [1].
It is well recognized that complete methylation of
CpG sites within CpG islands around transcription start
sites represents deep-silencing of gene expression estab-
lished during embryo development and cell differentia-
tion. Well-documented examples include the silencing
of tissue-specific genes, gene imprinting, inactivation of
parasite DNA and X-chromosome. However, the methy-
lation of CpG islands in tumor suppressor genes,
* Correspondence: dengdajun@bjmu.edu.cn
1Key Laboratory of Carcinogenesis and Translational Research (Ministry of
Education), Department of Aetiology, Peking University Cancer Hospital &
Institute, Beijing, 100142, China
Full list of author information is available at the end of the article
Zhou et al. BMC Medical Genetics 2011, 12:67
http://www.biomedcentral.com/1471-2350/12/67
© 2011 Zhou et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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including p16, is a progressive process encountered dur-
ing carcinogenesis [2-4]. De novo methylation often
occurs post gene silencing at a few seeding CpG sites in
initiation and precancerous stages, and ultimately
extends to the full CpG island in advanced cancer. This
complicates the development of an assay to detect the
methylation status of a target CpG island in which com-
plete methylation is not established. For example,
methylation of crucial CpG sites within a CpG island
that correlates with clinical outcomes should first be
identified, and then a proper detection approach with
high specificity for clinical diagnosis should be designed.
Unfortunately, such crucial CpG sites are not well char-
acterized for most CpG islands. This often leads to the
dissimilar detection of methylation at different CpG
sites within a target CpG island between different
laboratories. Contradictory results often arise from dif-
ferent kinds of detection assays, or the same assay with
different detection sensitivity [5].
Tumor suppressor gene p16 (CDKN2A) controls cell
proliferation through the P16-CDK4-RB pathway at the
G1®S checkpoint of the cell cycle [6]. Frequent, aber-
rant methylation of a crucial CpG island is the main
mechanism of inactivation for p16 in the early stages of
carcinogenesis [1]. A number of nested case-control stu-
dies and followup cohorts consistently showed p16
methylation as a potential biomarker for the early pre-
diction of malignant transformation of epithelial dyspla-
sia, one kind of precancerous lesion in many organs/
tissues including the oral/oesophageal/gastric mucosa
[7-13]. Although bisulfite-clone sequencing provides
detailed information about the methylation status of
each CpG site in the cloning molecules, it is often used
as a confirmation assay rather than a regular detection
assay because of its low detection sensitivity (> 20%),
labor, and time costs. A number of assays including
MSP, MethyLight, Pyrosequencing, and DHPLC are
often used to detect p16 methylation in laboratory
research [3,7-16]. Among them, MethyLight, based on
MSP primers, may become one of the most eligible,
convenient, quantitative, and sensitive assays for the
clinical detection of p16 methylation primarily because
it uses a methylation-specific primer set and real-time,
sequence-specific probe validation. In the present study,
we evaluated the sensitivity, specificity, and accuracy of
a 70-bp classic assay in which the amplicon partially
overlapped with the MSP amplicon, and a 115-bp novel
MethyLight assay in which the amplicon fully over-
lapped with the MSP amplicon (Figure 1). The data was
collected from 102 oral epithelial dysplasia samples
obtained from a followup cohort study, in which malig-
nant transformation of this disease correlated with p16
methylation detected by MSP and was confirmed by
clone sequencing [13].
Results and Discussion
Detection of p16 methylation by a classic 70-bp
MethyLight assay
An eligible PCR-based molecular assay for diagnosis
should meet several essential requirements including high
specificity, real-time validation using a sequence-specific
probe, positive confirmation with direct sequencing, and
refractory to carry-over contamination. Combination of
MethyLight using methylation-specific primers with
probes containing an anti-contamination system, com-
posed replacing dTTP with dUTP and the addition of a
uracil glycosylase UNG in the PCR reaction mixture, may
become an ideal method for the clinical detection of
methylation in a specific CpG island. In a 4-year followup
cohort, we reported that methylated-p16 was a potential
biomarker for early prediction of malignant transforma-
tion of oral epithelial dysplasia [13]. Among patients of at
least 60 years of age, the sensitivity and specificity of
methylated-p16 were 77% and 78%, respectively. Hall et al.
reported similar results [14]. Therefore, the using Methy-
Light as a clinical assay to detect methylated-p16 was
feasible.
The 70-bp classic MethyLight for methylated-p16 was
evaluated using either genomic DNA of baseline or fol-
lowup samples (n = 102) from patients enrolled in the
mentioned cohort (n = 78). After genomic DNA was
converted to SafeBis templates as described in the meth-
ods section, the methylated-p16 was analyzed with the
classic MethyLight. Methylated-p16 was detected in 75
of 102 tested samples. The methylated-p16 MethyLight-
positive rate and proportion of methylated-p16 in 44
methylated-p16 MSP-positive samples were higher than
those in 58 MSP-negative samples, respectively (positive
rate: 37/44 vs. 38/58, P = 0.035, two-sided; proportion
[median]: 0.78 vs. 0.02, P <0.007). Using the prognosis-
related MSP-results of methylated-p16 as a golden stan-
dard, we found sensitivity, specificity, and accuracy for
the classic MethyLight were 70.5%, 84.5%, and 55.0%
with a cut-off point of RCN set at 0.073, respectively
(Figure 2A).
Development of a 115-bp novel MethyLight assay
After conversion of unmethylated cytosine residues to
uracil (or thymine in PCR products; C ® U/T) residues,
a double stranded DNA molecule is transformed into
two non-complementary single-stranded DNA mole-
cules (C≡G ® U/T≠G), as illustrated in Figure 1. Inter-
estingly, all current methylation detection assays for the
p16 CpG islands are designed according to the anti-
sense-strand sequence of the p16 exon-1, while none
target the sense-strand. The main reasons may include
the good performance of first 150/151-bp MSP-m/u for
methylated/unmethylated-p16 in cell line and tissue
samples, and the very high content (111/175) of thymine
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Sequences of primers and probe matched to the sense-strand of methylated-p16 exon-1
? ?’???????????????????????????????????? ?????????? ?? ??????????????????????????????????????? ?? ??? ?? ??? ?? ??????????????????????????????????????????????? ?? ?????? ?? ??? ?? ???? ?? ?????????????????????????????? ?’????????
?’????? ?? ?????? ?? ??? ?? ???? ?? ?????’
ML-Primer-F2 ML-Probe-2 ML-Primer-R2
(115-bp MethyLight)
Sequences of primers and probe matched to the antisense-strand of methylated-p16 exon-1
? ?’??????????????????????????? ?????? ?? ????? ?????????? ?? ?????????????????????????????????????????????????????????????????????????????????????????????????????????????? ????????? ?? ???????????????????? ?’???????????
?’?? ?????? ?? ????? ????
?????? ?? ??????’
Primer-R Primer-F
(150-bp MSP)
? ?’??????? ?? ???? ?? ??????????????? ?? ?????? ?? ????? ???????????????????????????? ?? ?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ?’???????????
?’??????? ?? ???? ?? ???????????’
ML-Primer-R1 ML-Probe-1 ML-Primer-F1
(70-bp MethyLight)
Sequence of the p16 exon-1 double-stranded DNA
? ?’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ?’???????
? ?’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? ?’???????????
Bisulfite modification
Figure 1 The sequence of the p16 exon-1 before and after bisulfite conversion. Locations of amplicons, primers, and probes used in the
150/151-bp MSP-m/u, 70-bp, and 115-bp MethyLight assays within the sense-strand or antisense-strand were underlined and labelled.
A: 70-bp classic MethyLight B: 115-bp novel MethyLight
Figure 2 ROC curves of detection of methylated-p16 by two MethyLight assays. The sensitivity and specificity of the MethyLight assay at
various points of relative copy number in 102 tested samples were calculated according to the result of the 150-bp MSP. The cut-off points of
RCN were marked by the deep-red circlets. A: For the 70-bp classic MethyLight, the area under the curve is 0.776 (95% CI: 0.677-0.874), P =
0.000. When the cut-off point of RCN was 0.0725, the sensitivity and specificity were 0.705 and 0.845, respectively. B: For the 115-bp novel
MethyLight assay, the area under the curve is 0.787 (95% CI: 0.689-0.884), P = 0.000. When the cut-off point of RCN was 0.0002, the sensitivity
and specificity were 0.591 and 0.983, respectively.
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residues in the unmethylated sense-strand present after
bisulfite modification, which makes it difficult to design
a proper unmethylation-specific primer set that can be
used as control MSP-u in the case that p-16 is not
methylated (Figure 1). However, in the MethyLight
assay, instead of using the template corresponding to
unmethylated p-16, the COL2A1 gene, without a CpG
island, is recommended as an optimal common refer-
ence for all tested CpG islands for quantification of
modified genomic DNA in the tested samples [17].
Using this strategy, the sense-strand of the methylated-
p16 can be used to design a MethyLight assay.
The amplicon (+238 ~ +307; transcription starting site,
+1) of the 70-bp MethyLight partially overlapped with
the 150-bp MSP amplicon (+133 ~ +283) (Figure 1). To
investigate the feasibility of using the p16 exon-1 sense-
strand for detection of methylated-p16, we designed a
115-bp novel MethyLight assay according to the sense-
strand believing it might correlate with the 150-bp MSP
better than the 70-bp MethyLight assay. This theory was
based on the fact that the 115-bp MethyLight amplicon
matched to the 150-bp MSP amplicon better than the
70-bp MethyLight amplicon (Figure 1). Using the novel
MethyLight assay, we observed methylated-p16 in 26 of
44 MSP-positive samples and 2 of 58 MSP-negative ones
(P=0.000). This result was confirmed by clone sequencing
two representative samples (Figure 3). When the RCN
cut-off point was set at 0.0002, the sensitivity, specificity,
and accuracy of the 115-bp MethyLight were 59.1%,
98.3%, and 57.4%, respectively (Figure 2B).
Comparison of two MethyLight assays
Furthermore, we compared the results of two Methy-
Light assays and found 23 of the 41 (56.1%) classic
MethyLight positive samples are also 115-bp MethyLight
positive; whereas only 4 of 61 (6.6%) classic MethyLight
negative samples are 115-bp MethyLight positive (P =
0.000). The detailed overlap information for the results
of methylated-p16 in all 102 tested samples was ana-
lyzed (Figure 4). Apparently, when the RCN cut-off
points were set for the two MethyLight assays, the novel
and classic assays had the similar accuracy (57% and
55%). However, the 115-bp MethyLight had a very high
specificity, while the classic MethyLight had a higher
sensitivity. Most importantly, the sensitivity and specifi-
city of the novel MethyLight assay are consistent regard-
less of whether the RCN cut-off point was used during
the calculation process. This indicates the 115-bp assay
could be used as a qualitative assay for clinical detection
of methylated-p16.
We further analyzed the clinical outcome of methy-
lated-p16 as detected by two MethyLight assays. Among
Standard sense-strand sequences of the methylated p16 CpG island
Before bisulfite treatment???’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
After bisulfite treatment?? ?’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
Bisulfite sequences of two representative tissue samples K286 and K285
K286 Clone#9??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#12??’???????????????????????????????????????????????????????????????????????????????????????????????
K286 Clone#13??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#1??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#2??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#3??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#5??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#16??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#11??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#7??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#15??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#8??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#10??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#14??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#4??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K286 Clone#6??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’

K285 Clone#1??’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K285 Clone#?? ?’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K285 Clone???? ?’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K285 Clone?????’???????????????????????????????????????????????????????????????????????????????????????????????
K285 Clone#???’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K285 Clone#???’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K285 Clone????’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K285 Clone#???’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K285 Clone#???’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K285 Clone????’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
K285 Clone#?? ?’????????????????????????????????????????????????????????????????????????????????????????????????????????????????????-3’
?
??????????????????????-3’
??????????????????????-3’
Figure 3 Results of clone sequencing of the 115-bp MethyLight PCR products. The sense-strand sequences of the methylated p16 CpG
island with and without bisulfite treatment were listed. The bisulfite-treated template of two 115-bp MethyLight-positive samples was amplified
with the same primer set. The PCR products of these two representative samples were clone-sequenced, respectively. 99.6% (223/224) and 93.5%
(144/154) cytosines at CpG sites within total 16 and 11 clones (14 CpG sites/clone) from the sample K286 and K285 were maintained and 77.8%
(548/704) and 90.9% (440/484) cytosines at non-CpG sites within these clones (44 non-CpG sites/clone) were converted to thymines, respectively.
These results indicate that these clones are fully methylated at all CpG sites.
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30 patients of at least 60 years of age, methylated-p16
was detected in 8 baseline samples by the 115-bp
MethyLight assay (with or without the cut-off value).
During the followup period, oral cancer developed in 6
of 8 methylated-p16 positive patients (75.0%), but only
6 of 22 patients (27.3%) without methylated-p16 devel-
oped oral cancer [odd ratio 8.00 (95% CI, 0.98~80.93;
P = 0.034, two-sided). Among 34 patients analyzed
using the classic MethyLight assay (with cut-off value
0.073), the odds ratio of methylated-p16 was 3.64 (6/10
vs. 7/24; 95% CI, 0.62~21.91; P = 0.130). These results
suggest that the 115-bp MethyLight assay might be
better suited to detect the methylated-p16 biomarker
than the classic MethyLight assay.
Conclusions
The 115-bp MethyLight assay maybe a practical assay
for the detection of methylated-p16 biomarker for clini-
cal diagnosis.
Methods
Patients and oral biopsies
102 genomic DNA samples (> 500 ng) were extracted
from paraffin-embedded oral mucosa biopsies containing
mild or moderate dysplasia lesions from 78 patients
enrolled in a 4-year follow-up cohort (NCT00835341,
available at http://ClinicalTrials.gov) [7,13]. Briefly, the
fixed tissue block was cut into 10 μm slides, treated
with xylene to remove the paraffin, rehydrated with
graded ethanol, mixed with lysis buffer containing 100
μg proteinase K, digested at 56°C overnight, and incu-
bated 10 min at 95°C to stop the digestion [18]. DNA
present in the digestion solution was precipitated with
ethanol and dissolved in 50 μl TE buffer. DNA concen-
tration was determined spectrophotometrically with
diphenylamine as described [19]. The average recovery
rate of genomic DNA was 77.6%. 61 samples were base-
line biopsies and the remaining 41 samples were taken
during the followup periods. Methylation status of the
antisense-strand of exon-1 within the p16 CpG island
was determined using a 150-bp MSP assay in which
DHPLC was used as the detector; the results were
further confirmed through clone sequencing (Figure 1).
Methylated-p16 was detected in 44 of these samples.
The study was approved by the Institutional Review
Boards of Peking University School of Stomatology and
School of Oncology, and all patients gave written
informed consent.

75 70-bp ML-positive 41
44 150-bp MSP-positive 44
28 115-bp ML-positive 27
20 All-negative 47
2
25
12
36
6

20
1
23
8
10
10

47
1
3
Without Cutoff Point With Cutoff Point
Figure 4 Overlapping information of methylated-p16 by different assays. 102 oral mucosa biopsy samples were tested using three assays;
red circles indicate methylated-p16 positive sample numbers as determined using the 150-bp MSP; blue circles indicate methylated-p16 positive
sample numbers as determined using the 115-bp novel MethyLight assay; violet circles indicate methylated-p16 positive sample numbers as
determined using the 70-bp classic MethyLight assay; the black dashed line circles represent samples without methylated-p16 detected using
the three assays; the number within each open area covered by different cycles represents the exact number of samples containing methylated-
p16 as detected by the corresponding assays. A: Qualitative results without the use of a cut-off point. B: Summary of the total number of
samples with or without methylated-p16 as determined by each assay (left/right case no., without/with cut-off point). C: Qualitative results using
a cut-off point for the two MethyLight assays (relative copy number; 0.073 for the classic MethyLight assay, and 0.0002 for the novel MethyLight
assay).
Case no. Results Case no.
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Preparation of SafeBis DNA by bisulfite treatment
Genomic DNA samples (2 μg) were treated with bisul-
fite for 16 hrs at 50°C without desulfonation as
described [20], purified with the Wizard DNA Clean-Up
System Kit (Promega, Madison, WI), dissolved in 40 μl
TE preheated to 80°C, and stored in three aliquots at
-20°C before use. The unmethylated cytosine residues in
the DNA were converted to uracil (thymine in PCR pro-
ducts) and the methylated cytosine residues remained
intact after this treatment.
Detection of p16 methylation by the 70-bp classic
MethyLight assay
Methylation of CpG sites across the MSP Primer-R
region in the antisense-strand of the p16 exon-1 was ana-
lyzed by the classic MethyLight assay using modified pri-
mers [15]. Briefly, the ML-Primer-F1 (5’-tggag ttttC ggttg
attgg tt-3’), ML-Primer-R1 (5’-aacaa cGccc Gcacc tcct-
3’), and a methylated-p16-specific ML-Probe-1 (6FAM5’-
accCg acccC gaacC gCg-3’TAMRA, TaqMan) were used
to detect the 70-bp methylated p16 templates in the Safe-
Bis DNA (Figure 1). The reference gene COL2A1 was
also amplified with a forward primer (5’-tctaa caatt ataaa
ctcca accac caa-3’), a reverse primer (5’-gggaa gatgg gatag
aaggg aatat-3’), and a COL2A1-specific probe (6FAM5’-
ccttc attct aaccc aatac ctatc ccacc tctaa a-3’BHQ1) [17].
A uracil DNA glycosylase (UNG) carry-over prevention
system was employed in the MethyLight assay [18]. The
20 μl MethyLight reaction mixture contained 2 μl
10×PCR buffer (Qiagen, Germany), 0.5 units of HotStar
Taq DNA polymerase (Qiagen), 200 μmol/L dATP, 200
μmol/L dCTP, 200 μmol/L dGTP, 800 μmol/L dUTP
(Promaga), 5 mmol/L MgCl2, 75 nmol/L of each primer
(TaKaRa, Beijing), 75 nmol/L probe (TaKaRa), 2 μl
10×UNG Buffer (NEB), 0.4 units UNG (NEB), and 10 ng
template. An ABI7500 thermal cycler was used to
conduct the PCR reactions using the following thermal
conditions: 37°C for 10 min ® 95°C for 30 min ® (95°C
for 15 sec ® 62°C for 1 min) × 45 cycles. The fluores-
cence value was detected at 62°C. Duplicate tubes were
used for each sample, and the average Ct value was used
in the calculations. Relative copy number (RCN) of
methylated-p16 was calculated according to the formula
[2-ΔCt, (ΔCt = Ctmethylated-p16- CtCOL2A1)]. RKO and
MGC803 xenografts from nude mice were also used as
methylated-p16 positive and negative controls in each
experiment, respectively [13]. The calculated RCN of
methylated-p16 in each sample was standardized accord-
ing to the RCN of RKO positive control.
Detection of p16 methylation by the 115-bp MethyLight
assay
The ML-Primer-F2 (5’-CgCgg tCgtg gttag ttagt-3’),
ML-Primer-R2 (5’-tacGc tcGac Gacta Cgaaa-3’), and
ML-Probe-2 (5’-6FAM-gttgt ttttC gtCgt Cggtt-TAMRA-
3’) were used to detect the 115-bp methylated fragment of
the sense-strand of p16 exon-1, which completely over-
lapped the sense-strand sequence corresponding to
the 150-bp MSP amplicon within the antisense-strand
(Figure 1). Other conditions were the same as the classic
MethyLight assay.
Clone sequencing of the 115-bp MethyLight PCR
products of methylated-p16
The SafeBis template from two representative samples
of the 115-bp MethyLight-positive samples was ampli-
fied with the same primer set used in the 115-bp
MethyLight assay (without the ML-Probe-2), and then
clone-sequenced as described [3].
Statistical methods
A ROC curve of the results for each MethyLight assay
was calculated. Results of methylated-p16 in these
tested samples, determined using the 150-bp MSP-m
(and by151-bp MSP-u in the MSP-m negative cases),
were used as the golden standard in the calculation of
sensitivity and specificity for the two MethyLight
assays (Figure 1). These results showed a strong corre-
lation with the malignant transformation of these
lesions in the 4-year followup cohort study [13]. The
accuracy was calculated according to the formula [Sen-
sitivity+Specificity-1]. The Chi-square test and Stu-
dent’s t-test were used to test the significance of
qualitative and quantitative data between different
groups. All tests were two-sided.
Acknowledgements
This work is supported by Capital Program for Development of Health
Science (Grant #434), Beijing Science and Technology Commission (Grant
#Z090507017709016), Key Technologies Research & Development Programs
(863 Grants #2006AA020902 and 2006AA02A402). We thank Dr. Zhaojun Liu
for preparation of ROC curve charts. We also thank Professor Huidong Shi
and Mr. James Wilson (Augusta, Georgia) for language editing.
Author details
1Key Laboratory of Carcinogenesis and Translational Research (Ministry of
Education), Department of Aetiology, Peking University Cancer Hospital &
Institute, Beijing, 100142, China.2Department of Oral Medicine, Peking
University School of Stomatology, Beijing 100081, China.
Authors’ contributions
JZ carried out the molecular epigenetic assays. ZL carried out the clone
sequencing. JC and HL collected the tested samples. DD conceived the
study and drafted the manuscript. All authors read and approved the final
manuscript.
Received: 2 February 2011 Accepted: 13 May 2011
Published: 13 May 2011
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Cite this article as: Zhou et al.: A 115-bp MethyLight assay for detection
of p16 (CDKN2A) methylation as a diagnostic biomarker in human
tissues. BMC Medical Genetics 2011 12:67.
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