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An improved fluorescence polarization assay in 5'-nuclease reaction for gene promoter methylation detection
Journal of Biotechnology 211 (2015) 81–86
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An improved fluorescence polarization assay in 5’-nuclease reaction for gene promoter methylation detection Haichuan Su b , Zhongliang Wu c , Yukun Wang a , Yinghao Jiang a , Shaoying Qiang d , Hong Cheng e , Wenchao Liu d,∗ , Ju Zhang a,∗ a Institute of Gene Diagnosis, State Key Laboratory of Cancer Biology, School of Pharmacology,The Fourth Military Medical University, Xian, Shaanxi 710032, China b Department of Oncology, Tangdu Hospital, The Fourth Military Medical University, Xian, Shaanxi 710038, China c Department of Neurology, Xijing Hospital, The Fourth Military Medical University, Xian, Shaanxi 710033, China d Department of Oncology, Xijing Hospital, The Fourth Military Medical University, Xian, Shaanxi 710032, China e Department of Pathology, Xijing Hospital, The Fourth Military Medical University, Xian, Shaanxi 710032, China
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Article history: Received 10 May 2015 Received in revised form 28 June 2015 Accepted 10 July 2015 Available online 18 July 2015 Keywords: Gene promoter Methylation Homogeneous Fluorescence polarization 5’-nuclease reaction
a b s t r a c t The detection of gene promoter methylation plays increasing roles in personalized medicine. In this study, an improved gene promoter methylation assay based on fluorescence polarization in 5’-nuclease reaction was developed. The novel assay offered a homogeneous annealing and cleavage reaction fully integrated with PCR which used a probe labeled with fluorescence without quencher to obtain the decreased fluorescence polarization values. In this platform, gene promoter methylated and unmethylated alleles were detected simultaneously in a tube. O6 -methylguanine-DNA methyltransferase gene promoter methylation in 103 glioma tissue samples and epidermal growth factor receptor gene promoter methylation in 116 primary non-small-cell lung carcinoma tissue samples were detected by the novel assay and sequencing, absolute quantitative analysis of methylated allele in parallel. The accuracy of the results measured by the improved fluorescence polarization assay was evaluated using the paired-samples t test. No significant difference was found ( P > 0.05). Therefore, the improved fluorescence polarization assay in 5’-nuclease reaction demonstrated a homogeneous, reliable and cost-effective method for gene promoter methylation analysis in clinic. That would provide a scientific basis for applying a reasonable therapeutic regimen in future treatment. © 2015 Elsevier B.V. All rights reserved.
1. Introduction DNA methylation only takes place at position 5’ of the cytosine ring in cytosine-guanine dinucleotides (CpG) and 15% of the total CpG is located in CpG islands. In 60% of human genes, the CpG islands reside in promoters, first exon and 5 untranslated region. CpG islands are usually maintained free of methylation. Gene promoter methylation is a critical signal defining heritable epigenetic states of transcription and is associated with transcriptional repression (Taby and Issa, 2010). Moreover, gene promoter methylation is reversible. Gene promoter methylation plays an important role in normal development as well as disease processes. Transcriptional inactivation by cytosine-5 methylation at promoter CpG islands of
∗ Corresponding authors. Fax: +86 29 84779726. E-mail addresses: [email protected] (W. Liu), [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.jbiotec.2015.07.003 0168-1656/© 2015 Elsevier B.V. All rights reserved.
tumor suppressor genes is thought to be an important mechanism in human carcinogenesis. Aberrant cytosine-5 methylation at gene promoter CpG islands results in cancer progression and chemotherapy resistance (How Kit et al., 2012; Normanno et al., 2013; Ren et al.,2011). The detection of gene promoter methylation plays increasing roles in personalized medicine (Mikeska et al., 2012). O6 -methylguanine-DNA methyltransferase gene (MGMT) and epidermal growth factor receptor gene (EGFR) promoter methylation are prognosis and prediction molecular biomarkers in the monitoring of carcinogenesis and the personalized cancer treatment. The DNA repair protein MGMT removes alkyl adducts from the O6 position of guanine and MGMT promoter methylation has been associated with loss of MGMT protein. The MGMT promoter methylation has relation not only with glioma progression-free survival but also with outcome treated with alkylating agents (Cankovic et al., 2013; Hegi et al., 2008; Jacinto and Esteller, 2007). EGFR promoter methylation results in transcriptional silencing of EGFR in non-small-cell lung carcinoma (NSCLC). It is of clinical interest that
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NSCLC cells with EGFR promoter hypermethylation are resistant to EGFR inhibitor (Anglim et al., 2008; Jakopovic et al., 2013; Li et al., 2013; Montero et al., 2006). The detection methods for DNA methylation, such as methylation-specific polymerase chain reaction (MS-PCR), methylation-specific multiplex ligation-dependent probe amplification (MLPA), combined bisulfite restriction assay (COBRA), sequencing, pyrosequencing, MethyLight and absolute quantitative analysis of methylated allele (AQAMA), have been developed (Eads et al., 2000; Herman et al., 1996; Hernández et al., 2013; Nygren et al., 2005; Wu et al., 2011; Shaw et al., 2006; Xiong and Laird, 1997; Zeschnigk et al., 2004). A detection method for gene promoter methylation in clinical diagnosis should be reliable, cost-effective, and a one step format is preferred to prevent crossover contamination. Enhancement and innovation for DNA methylation detection technologies are currently in progress. The fluorescence polarization (FP) technique can be used to detect any significant change in molecular weight of a fluorescent molecule (Ohiso et al., 2000; Bao et al., 2010). FP is a good detection method for 5’-nuclease reaction, where a fluorescent probe is cleaved by 5’-nuclease activity of Taq DNA polymerase during PCR only when it is annealed to a perfectly complementary template. The FP assay in 5’-nuclease reaction has been used as a homogeneous and reliable method for genotype in one step (Sherif et al., 2001). The detection format of FP assay in 5’-nuclease reaction avoids crossover contamination resulting from sample separation and is adaptable to automated analysis. FP plate reader can detect more than one kind of fluorophore in one tube. In our study, the inclusion of a pair of methylated and unmethylated allele-specific probes labeled respectively with reporter fluorophores 6-carboxyfluorescein (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) at the 5 -end without quenchers allowed the simultaneous, homogeneous detection of methylated and unmethylated alleles in one step. In this study, the successful adaptation of FP assay in 5’-nuclease reaction to a reliable, cost-effective gene promotor methylation detection were reported. To evaluate the feasibility of the improved FP assay, MGMT promoter methylation in 103 glioma tissue samples and EGFR promoter methylation in 116 primary NSCLC tissue samples were detected by the novel assay and sequencing, AQAMA in parallel. The research confirmed the improved FP assay in 5’nuclease reaction would be applicable in clinical diagnosis.
2. Material and methods 2.1. Primers and probes Two pairs of universal primers ( MGMT -forward and MGMT reverse, EGFR -forward and EGFR -reverse) and the methylated and unmethylated allele-specific probes were designed based on GenBank accession nos. AL355531 and X17054. The universal primers amplified the bisulfite-modified DNA target but did not discriminate between methylated and unmethylated alleles, as they did not cover any potential CpG dinucleotide sites. It was possible to amplify both methylated and unmethylated alleles simultaneously in one tube. Most current researches for the detection of methylation are based on the conversion of unmethylated cytosine into uracil after sodium bisulphite treatment, and the uracil is converted to thymidine during subsequent PCR. In this study, FP technique and PCR-annealing-cleavage were combined to develop gene promoter methylation assay. The methylated and unmethylated allele specific probes recognized their target sequences in the same promoter as the binding sites of the probes covered the four target CpG and TpG dinucleotide sites (Table 1). DNA methylation was discriminated when the two differently-labeled methylated
Table 1 Sequences of primers and probes. Name Sequence MGMT-forward primer 5’-ttggatatgttgggatagtt-3’ MGMT-reverse primer 5’-cccaaacactcaccaaatc-3’ EGFR-forward primer 5’-ggttttttgatttygtttagta-3’ EGFR-reverse primer 5’-ccttacctttcttttcctcc-3’ FAM-unmMGMT-probe FAM-5’-taggttttt gtggtg tgtattg-3’ TAMRA-mMGMT-probe TAMRA-5’-aggtttt cgcggtg cgtat cg- 3’ FAM-unmEGFR-probe FAM-5’-atg tgattttt tggga tggt tg-3’ TAMRA-mEGFR-probe TAMRA-5’-tg cgattttt cggga cggt cg-3’ MGB-unmMGMT-probe FAM-5’- tgtggtg tgtat tg-BHQ-3’ MGB-mMGMT-probe VIC-5’- cgcggtg cgtat cg-BHQ-3’ MGB-unmEGFR-probe FAM-5’- tgattttt tggga tggt tg-BHQ-3’ MGB-mEGFR-probe VIC-5’- cgattttt cggga cggt cg-BHQ-3’ The CpG dinucleotide sites were highlighted in bold and underlining.
and unmethylated allele specific probes annealed to their target sequences and were cleaved in the same tube (Fig. 1). FAM-unm MGMT-probe and TAMRA-mMGMT-probe were respectively specific for the unmethylated and methylated alleles of the MGMT promoter. FAM-unmEGFR-probe and TAMRA-mEGFR-probe were respectively specific for the unmethylated and methylated alleles of the EGFR promoter. In AQAMA, the methylated and unmethylated allele discrimination occurred using the specific minor-groove-binding (MGB) molecule containing probes covered the same CpG dinucleotide sites of the methylated and unmethylated alleles (Table 1) (Zeschnigk et al., 2004). The AQAMA was based on real-time PCR using fluorescence quenching detection. The MGB probe was fitted with a minor groove binder to keep the probe length to a minimum and thus maximize the effect of the one-base mismatch (Afonina et al., 1997). DNA methylation was discriminated when two differently-labeled MGB probes hybridized with their target sequences in the same tube (de Kok et al., 2002). All primers and probes were synthesized and labeled by Invitrogen (Shanghai, China). 2.2. Standard plasmid construction In order to develop the improved FP assay, standard controls with methylated and unmethylated target DNA sequences should be detected first, and standard plasmids should be constructed. Bisulfite-modified DNA from peripheral blood leukocytes of healthy volunteers was amplified using the two pairs of primers to create standards for the unmethylated allele; to create standards for the methylated allele, bisulfite-modified DNA from peripheral blood leukocytes of healthy volunteers was treated in vitro with SssI methyltransferase (New England Biolabs, M0226S, Boston, USA) before amplification. The PCR products of methylated and unmethylated alleles ( MGMT promoter, 100 bp; EGFR promoter, 156 bp) were introduced into pGEM-T-easy Vector (Promega, A1360, USA) to construct recombinant plasmids, according to the manufacturer’s instructions. The recombinant plasmids were identified by sequencing. The plasmids p-methylated- MGMT and p-methylated- p16INK4a contained the PCR products of methylated alleles. The plasmids p-unmethylated- MGMT, p-unmethylatedEGFR contained the PCR products of unmethylated alleles. The plasmids were purified using a Quick Plasmid Miniprep kit (Tiangen, DP105, Beijing, China), the concentration of DNA was determined by measuring the optical density at 260 nm. 2.3. Template preparation One hundred and three glioma tissue samples were obtained from patients undergoing surgery in the Department of Neurology, Xijing Hospital and the Tangdu Hospital of The Fourth Military
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Template DNA Bisulphite treatment Conversion template DNA Forward Primer
Amplification
---g(a)c-g(a)c—g(a)c---
---c(t)g-c(t)g--c(t)g---
Reverse Primer Methylated probe
Annealing-cleavage
n----cg-cg--cg-cg---------gc-gc--gc- gc------
Unmethylated probe
Methylated amplicon
n ---- tg- tg--tg-tg----
Small Fluorophores
------ac-ac-ac-ac-----n
Unmethylated amplicon Small Fluorophores
Decreased FP Values of TAMRA n
Decreased FP Values of FAM
=TAMRA
=FAM
Fig. 1. Schematic of the improved FP assay in 5’-nuclease reaction. Template DNA was treated with sodium bisulphate. Unmethylated cytosine residues were conversed to uracil, which were converted to thymidine during subsequent PCR. Thus, after bisulphite treatment, alleles that were originally methylated have different DNA sequences as compared with their corresponding unmethylated alleles. A pair of universal primers and a pair of TAMRA-m-probe, FAM-unm-probe (methylated, unmethylated) were added to each reaction. The universal primers amplified the bisulfite-modified DNA target to generate amplicons. The results between methylated and unmethylated targets were distinguished by TAMRA-m-probe and FAM-unmprobe. TAMRA-m-probe and FAM-unm-probe annealed respectively to methylated, unmethylated amplicons respectively and the probes were cleaved by the 5’-nuclease activity of Taq DNA polymerase. Subsequently, an intact probe bearing a fluorescent molecule, which has a molecular weight of ∼10,000 Da, were conversed to one base nucleotide molecular bearing a fluorescent molecule, which has a molecular weight of ∼1000 Da. That resulted in the decreased FP values.
Medical University between March 2009 and July 2013. One hundred and sixteen primary NSCLC tissue samples were obtained from patients undergoing surgery in the Department of Oncology, the Department of Thoracic Surgery, Xijing Hospital and Tangdu Hospital of The Fourth Military Medical University from March 2009 to October 2013. The study was approved by the Human Research Protective Committee of The Fourth Military Medical University, and written informed consent was obtained from each patient. Each cancer tissue specimen was cut into two parts; one part was paraffin-embedded for histological diagnosis, the other for molecular analysis. Genomic DNA was extracted from 20 to 30 mg cancer tissue using a QIAamp DNA Mini kit (QIAGEN, 51,304, Germany), following the manufacturer’s instructions. The extracted genomic DNA was bisulfite-modified using an EZ DNA Methylation-GoldTM kit (Zymo Research, D5005 USA), according to the manufacturer’s instruction. 2.4. Gene promotor methylation detection using the improved FP assay Firstly, the improved FP assay in 5’-nuclease reaction for individually assessing methylated or unmethylated alleles by one step was developed. Serial dilutions from 107 copies to 102 copies of p-methylated- MGMT or p-methylated- EGFR were used as the methylated positive standards and serial dilutions from 107 copies to 102 copies of p-unmethylated- MGMT or p-unmethylated- EGFR were used as the unmethylated positive standards. The pGEM-
T-easy Vector plasmid, which didn’t contain any PCR product of target methylated or unmethylated alleles, was used as the negative control. The 25 l reaction system included 1.5 U TaqDNA polymerase (Tiangen, ET101, China), 1 M of each primer, 100 M of each deoxynucleotide triphosphate (dNTP, Tiangen, CD111, China), 4.0 mM MgCl2 and 100 nM of the methylated or unmethylated allele-specific probe. The reaction was carried out with initial denaturing at 97 ◦ C for 5 min followed by 35 cycles of denaturing at 95 ◦ C for 40 s, annealing at 55 ◦ C for 40 s, extension at 72 ◦ C for 40 s, and cooling to 25 ◦ C. FP value (millipolarization [mP]) was directly proportional to the fluorescent molecular weight. A fluorescent molecule with high molecular weight rotated slowly in space and FP values was high. A one-base nucleotide bearing fluorophore had a molecular weight of ∼1000 Da and a 18–25 base probe bearing fluorophore had a molecular weight of ∼10,000 Da. In the 5’-nuclease reaction, when the fluorescent probes annealed to the complementary amplicons, they were cleaved into one-base nucleotides bearing fluorophore by the 5 -nuclease activity of Taq DNA polymerase and a decreased FP values was detected. Conversely, the fluorescent probe remained intact and FP values remained high. Therefore, when a template with only methylated allele was detected, TAMRA-m-probe annealed to the amplicon and was cleaved, and the FP values of TAMRA decreased. When a template with only unmethylated allele was detected, FAM-unm-probe annealed to the amplicon and was cleaved, and the FP value of FAM decreased (Fig. 1). Reactions were performed in duplicate. And then the FP values of TAMRA and FAM
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were measured using an FP plate reader. FP values were measured for FAM with excitation at 485 nm and emission at 530 nm; for TAMRA with excitation at 535 nm and emission at 590 nm. Secondly, the improved FP assay in 5’-nuclease reaction for simultaneous methylated and unmethylated allele assessment in one step was developed. FP technique can detect two different fluorophores with distinct excitation and emission frequencies. Each reaction of the FP assay was carried out as described above with minor modifications. In reaction buffer, the mixtures of methylated and unmethylated standards were used as templates and 80 nM each of the methylated and unmethylated allele-specific probe were included. As the template with methylated and unmethylated allele was detected, both TAMRA-m-probe and FAM-unm-probe annealed respectively to their target amplicon and were cleaved, and the FP values of both TAMRA and FAM decreased. Therefore, the methylated and unmethylated alleles were homogeneous detected simultaneously. Lastly, MGMT promoter methylation of 103 glioma samples and EGFR promoter methylation of 116 primary NSCLC samples were assessed by the improved FP assay in 5’-nuclease reaction. The unmethylated allele, which should always be detected, served as an internal control to test inhibitors and assess the quality of the template. The gene promoter methylation in each sample was determined by data analysis of FP values.
2.7. Gene promotor methylation detection using sequencing and AQAMA To evaluate the accuracy of the improved FP assay, MGMT and EGFR promotor methylation of the samples were detected by sequencing and AQAMA in parallel. In sequencing assay, 50 l reaction system included 2.5 M of each universal primer. The bisulfite-modified template of each glioma tissue sample was subjected to the primers of MGMT forward and MGMT -reverse, the bisulfite-modified template of each NSCLC tissue sample was subjected to the primers of EGFR -forward and EGFR -reverse. PCR products stained with ethidium bromide were detected visually on gel under UV, and they were purified with the Wizard DNA Clean-up System (Promega, USA) followed by sequencing on an ABI 377 instrument by the AmpliCycle sequencing kit (PerkinElmer, San Jose, California). The gene promotor methylation of each sample was assayed in duplicate. In AQAMA, 25 l reaction system included 2.5 M of each universal primer and 150 nM each of methylated and unmethylated allele-specific MGB-probes. The bisulfite-modified template of each glioma tissue sample was subjected to MGMT promoter methylation detection, and the bisulfite-modified DNA of each NSCLC tissue sample was subjected to EGFR promoter methylation detection. Samples were assayed in duplicate. 3. Results
2.5. Data analysis of the improved FP assay 3.1. Cut off Value of the improved FP assay The negative controls had high FP values for TAMRA and FAM, as no probe was cleaved. The methylated positive standards had decreased FP values for TAMRA, as the TAMRA-m-probes were cleaved. The unmethylated positive standards had decreased FP values for FAM, as the FAM-unm-probes were cleaved. By performing a t distribution test, the FP values of the negative and the positive data sets of TAMRA and FAM were found to be significantly different at a 95% level of confidence. The net change of FP values between the positive and the negative standards for TAMRA and FAM was analyzed. Cut off values for the net change of the positive reactions were determined as described (Ohiso et al., 2000; Bao et al., 2010; Sherif et al., 2001).
2.6. Specificity, sensitivity and reproducibility of the improved FP assay The specificity of the improved FP assay for methylated and unmethylated alleles was affected mainly by the specificity of the 5’-nuclease reaction. Cross annealing between the probes and amplicons would interfere with the specificity of the 5’-nuclease reaction. In the specificity determination reaction for unmethylated alleles, TAMRA-m-probe and FAM-unm-probe were used in a cocktail, 106 –108 copies of methylated plasmid were used as template. In the specificity determination reaction for methylated allele, TAMRA-m-probe and FAM-unm-probe were used in a cocktail, 106 –108 copies of unmethylated plasmid were used as template. FP values of TAMRA and FAM were monitored for 24 h. To determine the sensitivity of the improved FP assay, the mixtures of methylated and unmethylated allele plasmid were used as template in the ratios 1:99, 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91 and 10:90. The methylation allele of each mixture was determined based on the FP values of TAMRA and FAM. To determine the reproducibility of the FP assay, the mixtures with the following ratios of methylated to unmethylated allele plasmid were analyzed three times on different days: 10:90, 50:50, 70:30 and 90:10. The effect of varying amounts of template on the results in samples was also investigated.
In the improved FP assay, the FP values of the positive standards and negative controls were significantly different. For TAMRA, the 95% confidence interval was: (49, 180; 120 ± 43) from the methylated-MGMT standards population; (56, 185; 126 ± 40) from the methylated- EGFR standards population; and (237, 254; 243 ± 11) from the negative control population. For FAM, the 95% confidence interval was (42, 168; 105 ± 39) from the unmethylated-MGMT standards population; (46, 163; 116 ± 41) from the unmethylated- EGFR standards population; and (221, 246; 235 ± 10) from the negative control population. These intervals do not overlap, demonstrating a clear separation between positive and negative samples. For MGMT gene promoter methylation detection, cut off values for the net decreasing of the FP value in TAMRA was >56 mP of the negative controls and in FAM was >47 mP of the negative controls. For EGFR gene promoter methylation detection, cut off values for the net decreasing of the FP value in TAMRA was >51 mP of the negative controls and in FAM was >41 mP of the negative controls. The FP values curves for 102 –107 copies of methylated and unmethylated allele was shown in Fig. 2. 3.2. Specificity, sensitivity and reproducibility In the specificity determination for unmethylated or methylated allele, no net change of FP values more than the cut off values was found, indicating that TAMRA-m-probes and FAM-unm-probes exhibited strong specificity for their targets, with no cross reaction. Therefore, few possibilities for misidentification were present. In the sensitivity analysis, the improved FP assay could detect methylated allele for content as low as 5%. In the reproducibility analysis, all the different ratios of methylated to unmethylated allele plasmid were defined as positive, and varying amounts of template in samples had no effect on the results. 3.3. Gene promoter methylation in samples Each sample was detected in parallel using the improved FP assay and sequencing, AQAMA. In the improved FP assay, the gene
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FP assay, sequencing and AQAMA. One sample was identified as EGFR promoter methylation positive by the FP assay and AQAMA but EGFR promoter methylation negative by sequencing. The accuracy of the results measured by the improved FP assay, sequencing or AQAMA was evaluated using the paired-samples t test. There was no significant difference ( P > 0.05), suggesting that the FP assay is as reliable as the conventional detection method.
4. Discussion
Fig. 2. The FP values curves for 102 –107 copies of methylated and unmethylated allele. (A) The FP values curves for 102 –107 copies of MGMT promotor methylated and unmethylated allele. The quantity of p-methylated- MGMT, p-unmethylated- MGMT was determined by measuring the optical density at 260 nm. 102 –107 copies of p-methylatedMGMT were used as the methylated positive standards and 102 –107 copies of punmethylated- MGMT were used as the unmethylated positive standards. FP assay in TAMRA was for methylated positive targets. FP assay in FAM was for unmethylated positive targets. This figure indicated that the FP values fell down with the increasing amount of target positive standards. For TAMRA, FP values decreased from 180 to 49 when copies of p-methylated- MGMT increased from 102 to 107 . For FAM, FP values decreased from 168 to 40 when copies of p-unmethylated- MGMT increased from 102 to 107 . The R 2 values of the trend line of the FP values curves for MGMT promotor methylated and unmethylated allele was no less than 0.9709. (b) The FP values curves for 102 –107 copies of EGFR promotor methylated and unmethylated allele. The quantity of p-methylated- EGFR, p-unmethylated- EGFR was determined by measuring the optical density at 260 nm. 102 –107 copies of p-methylatedEGFR were used as the methylated positive standards and 102 –107 copies of punmethylated- EGFR were used as the unmethylated positive standards. FP assay in TAMRA was for methylated positive targets. FP assay in FAM was for unmethylated positive targets. This figure indicated that FP values fell down with the increasing amount of target positive standards. For TAMRA, FP values decreased from 185 to 56 when copies of p-methylated- EGFR increased from 102 to 107 . For FAM, FP values decreased from 163 to 46 when copies of p-unmethylated- EGFR increased from 102 to 107. The R 2 values of the trend line of the FP values curves for EGFR methylated and unmethylated allele was no less than 0.9714.
promotor methylation in samples was determined based on the net change of the FP values in TAMRA and FAM. If the net decreasing of the FP values of a sample was less than the cut off value in TAMRA and more than the cut off value in FAM of the negative controls, the sample was identified as methylation negative. If the net decreasing of the FP values of a sample was both more than the cut off values in TAMRA and FAM of the negative controls, the sample was identified as methylation positive. Of the 103 glioma samples, 44 samples were identified as MGMT methylation positive by the improved FP assay, sequencing and AQAMA. Two samples were identified as MGMT promoter methylation positive by the FP assay, AQAMA but MGMT promoter methylation negative by sequencing. Of the 116 primary NSCLC samples, 30 samples were identified as EGFR promoter methylation positive by the improved
Research on monitoring EGFR promoter methylation with a hybridization-FP assay has been done before. In the hybridizationFP assay for EGFR promoter methylation detection, a pair of EGFR promoter primers was used in an asymmetric PCR and the single-chain PCR product hybridized with the probe labeled with fluorophore at the 3 -end, resulting in an increased FP values. The hybridization-FP assay for EGFR promoter methylation detection used multiple steps in two tubes (Zhang et al., 2013). A homogeneous approach is of particular interest to clinical molecular diagnostics, as it eliminates carryover and cross contamination. In the novel assay, a 5’-nuclease reaction was combined with FP technique to detect methylated and unmethylated alleles simultaneously in one tube. When the probe matched to its target amplicon and was cleaved into one-base nucleotides by the 5 nuclease activity of TaqDNA polymerase, the molecular weight of the fluorophore-labeled molecule decreased. Then the FP values decreased significantly. Therefore, the homogeneous, gel free detection of gene promoter methylation without separation of the PCR products was established. This is more compelling when comparing to other popular assays for DNA methylation detection such as MSP, MLPA, COBRA, the hybridization-FP assay, pyrosequencing and sequencing (Hernández et al., 2013; Herman et al., 1996; Nygren et al., 2005; Xiong and Laird, 1997; Shaw et al., 2006; Zeschnigk et al., 2004). In terms of MSP, MLPA, COBRA, the hybridization-FP assay, pyrosequencing and sequencing, methylated and unmethylated alleles couldn’t be detected simultaneously in one tube, the purified PCR products and gel electrophoresis were required. The improved FP assay obviated the necessity for an open reaction tube after amplification. The improved FP assay was more exact and faster than conventional assays. Furthermore, the unmethylated allele, which could always be detected, served as an internal control to assess the quality of the template DNA in the improved FP assay. This helped simplify handling, as it obviated the need for other controls. A one-step approach is of particular interest for clinical diagnosis, but the labor-intensive and time-consuming steps of the conventional techniques limits their application. A high sensitivity, accuracy and cost-effectiveness are always required in clinical diagnosis. In this study, three samples were identified as methylation positive by the improved FP assay and AQAMA but methylation negative by sequencing. This was likely because of the fact that the sequencing assay was defined by the little amount of methylated PCR product from the specimens with the minor population of gene promoter methylation. This indicated the improved FP assay was more sensitive compared to the gel-based sequencing assay for the detection of gene promoter methylation. The sensitivity of the improved FP assay was 5%, the assay was as reliable as the conventional assays based on fluorescence intensity detection, such as AQAMA and MethyLight. The conventional assays based on fluorescence intensity detection techniques relied on reporter fluorophore intensity released from a fluorescence quencher of a probe (Eads et al., 2000; Zeschnigk et al., 2004; Dimitrakopoulos et al., 2012). However, the improved FP assay relied on the decreased FP values resulted from probe cleaved, the novel assay opened up the possibility of utilizing a less costly
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probe labeled with fluorophore at the 5 -end without a fluorescence quencher. Furthermore, the FP assay was not influenced by variations in fluorescence intensities, since it detected the plane polarization of the fluorescence. So the assay was more reliable than conventional assays, such as MethyLight. In addition, the FP assay did not require any real-time PCR equipment; it was performed with a less costly FP plate reader. In this study, the FP values of the negative and positive data sets were significantly distinct. The R2 values of the trend line of the FP values curves for 102 –107 copies of methylated and unmethylated allele was no less than 0.97, demonstrating that FP value of TAMRA and FAM was linearly proportional to the amount of MGMT, EGFR promoter methylated and unmethylated alleles, respectively. This showed a high precision in discriminating the methylated and unmethylated alleles. This high precision is beneficial for high sensitivity, good reproducibility and accuracy. This study reported a homogeneous and cost-effective FP assay in 5 nuclease reaction for detection of MGMT,EGFR promoter methylation. A good compromise was achieved between efficiency, cost, and accuracy and the assay provided a practical tool for clinical molecular diagnostics. MGMT,EGFR promoter methylation serves as biomarker to predict prognosis, progression-free survival, and outcome of treatment. The incidence rate of MGMT promoter methylation was as high as 44.7% in glioma samples and the incidence rate of EGFR promoter methylation was as high as 26.7% in primary NSCLC samples in China. The results indicated that MGMT promoter methylation was common in glioma samples and EGFR promoter methylation was common in primary NSCLC samples. The detection of MGMT promoter methylation before alkylating agent therapy and the detection of EGFR promoter methylation before EGFR inhibitor therapy would provide a scientific basis for applying a reasonable therapeutic regimen in future treatment. Conflicts of interest We have no conflicts of interest to declare. Authors’ contributions Conception and design: Haichuan Su, Ju Zhang, Wenchao Liu. Development of methodology: Ju Zhang, Yinghao Jiang. Acquisition of data (provided and confirm samples, etc.): Haichuan Su, Zhongliang Wu, Shaoying Qiang, Hong Cheng, Wenchao Liu. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Ju Zhang, Wenchao Liu, Haichuan Su. Study supervision, Writing and review of the manuscript: Ju Zhang, Wenchao Liu. Acknowledgements This work was supported by the National High-tech R&D Program (2008AA02Z444), and the National Nature Science Foundation (81071435, 81371891). We thank Professor Sun Jianzhong for manuscript language helping. References Afonina, I., Zivarts, M., Kutyavin, I., Lukhtanov, E., Gamper, H., Meyer, RB, 1997. Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res. 25, 2657–2660.
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A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples. BMC Cancer 12, 486. Eads, C.A., Danenberg, K.D., Kawakami, K., Saltz, L.B., Blake, C., Shibata, D., Danenberg, P.V., Laird, P.W., 2000. MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res. 28, E32. Hegi, M.E., Liu, L., Herman, J.G., Stupp, R., Wick, W., Weller, M., Mehta, M.P., Gilbert, M.R., 2008. Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity. J. Clin. Oncol. 26, 4189–4199. Herman, J.G., Graff, J.R., Myöhänen, S., Nelkin, B.D., Baylin, S.B., 1996. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. U. S. A. 93, 9821–9826. Hernández, H.G., Tse, M.Y., Pang, S.C., Arboleda, H., Forero, D.A., 2013. Optimizing methodologies for PCR-based DNA methylation analysis. Biotechniques 55, 181–197. How Kit, A., Nielsen, H.M., Tost, J., 2012. DNA methylation based biomarkers: practical considerations and applications. Biochimie 94, 2314–2337. Jacinto, F.V., Esteller, M., 2007. MGMT hypermethylation: a prognostic foe, a predictive friend. DNA Repair 6, 1155–1160. Jakopovic, M., Thomas, A., Balasubramaniam, S., Schrump, D., Giaccone, G., Bates, S.E., 2013. Targeting the epigenome in lung cancer: expanding approaches to epigenetic therapy. Front. Oncol., 3261. Li, X.Y., Wu, J.Z., Cao, H.X., Ma, R., Wu, J.Q., Zhong, Y.J., Feng, J.F., 2013. Blockade of DNA methylation enhances the therapeutic effect of gefitinib in non-small cell lung cancer cells. Oncol. Rep. 29, 1975–1982. Mikeska, T., Bock, C., Do, H., Dobrovic, A., 2012. DNA methylation biomarkers in cancer: progress towards clinical implementation. Expert Rev. Mol. Diagn. 12, 473–487. Montero, A.J., Díaz-Montero, C.M., Mao, L., Youssef, E.M., Estecio, M., Shen, L., Issa, J.P., 2006. Epigenetic inactivation of EGFR by CpG island hypermethylation in cancer. Cancer Biol. Ther. 5, 1494–1501. Normanno, N., Rachiglio, A.M., Roma, C., Fenizia, F., Esposito, C., La Porta, M.L., Iannaccone, A., Micheli, F., Santangelo, M., Bergantino, F., Costantini, S., De Luca, A., 2013. Molecular diagnostics and personalized medicine in oncology: challenges and opportunities. J. Cell Biochem. 114, 514–524. Nygren, A.O., Ameziane, N., Duarte, H.M., Vijzelaar, R.N., Waisfisz, Q., Hess, C.J., Schouten, J.P., Errami, A., 2005. Methylation-specific MLPA(MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res. 33, e128. Ohiso, I., Tsuruoka, M., Iida, T., Honda, T., Karube, I., 2000. A florescence polarization assay using oligonucleotide probes for the rapid detection of verotoxin-producing E. coli. J. Biotechnol. 81, 15–25. Ren, J., Singh, B.N., Huang, Q., Li, Z., Gao, Y., Mishra, P., Hwa, Y.L., Li, J., Dowdy, S.C., Jiang, S.W., 2011. DNA hypermethylation as a chemotherapy target. Cell Signal 23, 1082–1093. Shaw, R.J., Liloglou, T., Rogers, S.N., Brown, J.S., Vaughan, E.D., Lowe, D., Field, J.K., Risk, J.M., 2006. Promoter methylation of P16INK4A, RARb, E-cadherin, cyclin A1 and cytoglobin in oral cancer: quantitative evaluation using pyrosequencing. Br. J. Cancer 27, 561–568. Sherif, Latif, Irma, Bauer-Sardina, Kostubh, Ranade, Kenneth Livak, J., Kwok, Pui-Yan, 2001. Fluorescence polarization in homogeneous nucleic acid analysis II: 5’-Nuclease assay. Genome. Res. 11, 436–440. Taby, R., Issa, J.P., 2010. Cancer epigenetics. CA Cancer J. Clin. 60, 376–392. Wu, W., Zhang, J., Yang, H., Shao, Y., Yu, B., 2011. Examination of AKAP12 promoter methylation in skin cancer using methylation-sensitive high-resolution melting analysis. Clin. Exp. Dermatol. 36, 381–385. Xiong, Z., Laird, P.W., 1997. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 25, 2532–2534. Zeschnigk, M., Böhringer, S., Price, E.A., Onadim, Z., Masshöfer, L., Lohmann, D.R., 2004. A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus. Nucleic Acids Res. 32, e125. Zhang, W., Gao, Y., Jiang, Y., Ping, L., Cheng, H., Zhang, J., 2013. EGFR promoter methylation detection in cervical cancer by a hybridization-fluorescence polarization assay. Diagn. Mol. Pathol. 22, 102–106.
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An improved fluorescence polarization assay in 5’-nuclease reaction for gene promoter methylation detection Haichuan Su b , Zhongliang Wu c , Yukun Wang a , Yinghao Jiang a , Shaoying Qiang d , Hong Cheng e , Wenchao Liu d,∗ , Ju Zhang a,∗ a Institute of Gene Diagnosis, State Key Laboratory of Cancer Biology, School of Pharmacology,The Fourth Military Medical University, Xian, Shaanxi 710032, China b Department of Oncology, Tangdu Hospital, The Fourth Military Medical University, Xian, Shaanxi 710038, China c Department of Neurology, Xijing Hospital, The Fourth Military Medical University, Xian, Shaanxi 710033, China d Department of Oncology, Xijing Hospital, The Fourth Military Medical University, Xian, Shaanxi 710032, China e Department of Pathology, Xijing Hospital, The Fourth Military Medical University, Xian, Shaanxi 710032, China
a r t i c l e
i n f o
Article history: Received 10 May 2015 Received in revised form 28 June 2015 Accepted 10 July 2015 Available online 18 July 2015 Keywords: Gene promoter Methylation Homogeneous Fluorescence polarization 5’-nuclease reaction
a b s t r a c t The detection of gene promoter methylation plays increasing roles in personalized medicine. In this study, an improved gene promoter methylation assay based on fluorescence polarization in 5’-nuclease reaction was developed. The novel assay offered a homogeneous annealing and cleavage reaction fully integrated with PCR which used a probe labeled with fluorescence without quencher to obtain the decreased fluorescence polarization values. In this platform, gene promoter methylated and unmethylated alleles were detected simultaneously in a tube. O6 -methylguanine-DNA methyltransferase gene promoter methylation in 103 glioma tissue samples and epidermal growth factor receptor gene promoter methylation in 116 primary non-small-cell lung carcinoma tissue samples were detected by the novel assay and sequencing, absolute quantitative analysis of methylated allele in parallel. The accuracy of the results measured by the improved fluorescence polarization assay was evaluated using the paired-samples t test. No significant difference was found ( P > 0.05). Therefore, the improved fluorescence polarization assay in 5’-nuclease reaction demonstrated a homogeneous, reliable and cost-effective method for gene promoter methylation analysis in clinic. That would provide a scientific basis for applying a reasonable therapeutic regimen in future treatment. © 2015 Elsevier B.V. All rights reserved.
1. Introduction DNA methylation only takes place at position 5’ of the cytosine ring in cytosine-guanine dinucleotides (CpG) and 15% of the total CpG is located in CpG islands. In 60% of human genes, the CpG islands reside in promoters, first exon and 5 untranslated region. CpG islands are usually maintained free of methylation. Gene promoter methylation is a critical signal defining heritable epigenetic states of transcription and is associated with transcriptional repression (Taby and Issa, 2010). Moreover, gene promoter methylation is reversible. Gene promoter methylation plays an important role in normal development as well as disease processes. Transcriptional inactivation by cytosine-5 methylation at promoter CpG islands of
∗ Corresponding authors. Fax: +86 29 84779726. E-mail addresses: [email protected] (W. Liu), [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.jbiotec.2015.07.003 0168-1656/© 2015 Elsevier B.V. All rights reserved.
tumor suppressor genes is thought to be an important mechanism in human carcinogenesis. Aberrant cytosine-5 methylation at gene promoter CpG islands results in cancer progression and chemotherapy resistance (How Kit et al., 2012; Normanno et al., 2013; Ren et al.,2011). The detection of gene promoter methylation plays increasing roles in personalized medicine (Mikeska et al., 2012). O6 -methylguanine-DNA methyltransferase gene (MGMT) and epidermal growth factor receptor gene (EGFR) promoter methylation are prognosis and prediction molecular biomarkers in the monitoring of carcinogenesis and the personalized cancer treatment. The DNA repair protein MGMT removes alkyl adducts from the O6 position of guanine and MGMT promoter methylation has been associated with loss of MGMT protein. The MGMT promoter methylation has relation not only with glioma progression-free survival but also with outcome treated with alkylating agents (Cankovic et al., 2013; Hegi et al., 2008; Jacinto and Esteller, 2007). EGFR promoter methylation results in transcriptional silencing of EGFR in non-small-cell lung carcinoma (NSCLC). It is of clinical interest that
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NSCLC cells with EGFR promoter hypermethylation are resistant to EGFR inhibitor (Anglim et al., 2008; Jakopovic et al., 2013; Li et al., 2013; Montero et al., 2006). The detection methods for DNA methylation, such as methylation-specific polymerase chain reaction (MS-PCR), methylation-specific multiplex ligation-dependent probe amplification (MLPA), combined bisulfite restriction assay (COBRA), sequencing, pyrosequencing, MethyLight and absolute quantitative analysis of methylated allele (AQAMA), have been developed (Eads et al., 2000; Herman et al., 1996; Hernández et al., 2013; Nygren et al., 2005; Wu et al., 2011; Shaw et al., 2006; Xiong and Laird, 1997; Zeschnigk et al., 2004). A detection method for gene promoter methylation in clinical diagnosis should be reliable, cost-effective, and a one step format is preferred to prevent crossover contamination. Enhancement and innovation for DNA methylation detection technologies are currently in progress. The fluorescence polarization (FP) technique can be used to detect any significant change in molecular weight of a fluorescent molecule (Ohiso et al., 2000; Bao et al., 2010). FP is a good detection method for 5’-nuclease reaction, where a fluorescent probe is cleaved by 5’-nuclease activity of Taq DNA polymerase during PCR only when it is annealed to a perfectly complementary template. The FP assay in 5’-nuclease reaction has been used as a homogeneous and reliable method for genotype in one step (Sherif et al., 2001). The detection format of FP assay in 5’-nuclease reaction avoids crossover contamination resulting from sample separation and is adaptable to automated analysis. FP plate reader can detect more than one kind of fluorophore in one tube. In our study, the inclusion of a pair of methylated and unmethylated allele-specific probes labeled respectively with reporter fluorophores 6-carboxyfluorescein (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) at the 5 -end without quenchers allowed the simultaneous, homogeneous detection of methylated and unmethylated alleles in one step. In this study, the successful adaptation of FP assay in 5’-nuclease reaction to a reliable, cost-effective gene promotor methylation detection were reported. To evaluate the feasibility of the improved FP assay, MGMT promoter methylation in 103 glioma tissue samples and EGFR promoter methylation in 116 primary NSCLC tissue samples were detected by the novel assay and sequencing, AQAMA in parallel. The research confirmed the improved FP assay in 5’nuclease reaction would be applicable in clinical diagnosis.
2. Material and methods 2.1. Primers and probes Two pairs of universal primers ( MGMT -forward and MGMT reverse, EGFR -forward and EGFR -reverse) and the methylated and unmethylated allele-specific probes were designed based on GenBank accession nos. AL355531 and X17054. The universal primers amplified the bisulfite-modified DNA target but did not discriminate between methylated and unmethylated alleles, as they did not cover any potential CpG dinucleotide sites. It was possible to amplify both methylated and unmethylated alleles simultaneously in one tube. Most current researches for the detection of methylation are based on the conversion of unmethylated cytosine into uracil after sodium bisulphite treatment, and the uracil is converted to thymidine during subsequent PCR. In this study, FP technique and PCR-annealing-cleavage were combined to develop gene promoter methylation assay. The methylated and unmethylated allele specific probes recognized their target sequences in the same promoter as the binding sites of the probes covered the four target CpG and TpG dinucleotide sites (Table 1). DNA methylation was discriminated when the two differently-labeled methylated
Table 1 Sequences of primers and probes. Name Sequence MGMT-forward primer 5’-ttggatatgttgggatagtt-3’ MGMT-reverse primer 5’-cccaaacactcaccaaatc-3’ EGFR-forward primer 5’-ggttttttgatttygtttagta-3’ EGFR-reverse primer 5’-ccttacctttcttttcctcc-3’ FAM-unmMGMT-probe FAM-5’-taggttttt gtggtg tgtattg-3’ TAMRA-mMGMT-probe TAMRA-5’-aggtttt cgcggtg cgtat cg- 3’ FAM-unmEGFR-probe FAM-5’-atg tgattttt tggga tggt tg-3’ TAMRA-mEGFR-probe TAMRA-5’-tg cgattttt cggga cggt cg-3’ MGB-unmMGMT-probe FAM-5’- tgtggtg tgtat tg-BHQ-3’ MGB-mMGMT-probe VIC-5’- cgcggtg cgtat cg-BHQ-3’ MGB-unmEGFR-probe FAM-5’- tgattttt tggga tggt tg-BHQ-3’ MGB-mEGFR-probe VIC-5’- cgattttt cggga cggt cg-BHQ-3’ The CpG dinucleotide sites were highlighted in bold and underlining.
and unmethylated allele specific probes annealed to their target sequences and were cleaved in the same tube (Fig. 1). FAM-unm MGMT-probe and TAMRA-mMGMT-probe were respectively specific for the unmethylated and methylated alleles of the MGMT promoter. FAM-unmEGFR-probe and TAMRA-mEGFR-probe were respectively specific for the unmethylated and methylated alleles of the EGFR promoter. In AQAMA, the methylated and unmethylated allele discrimination occurred using the specific minor-groove-binding (MGB) molecule containing probes covered the same CpG dinucleotide sites of the methylated and unmethylated alleles (Table 1) (Zeschnigk et al., 2004). The AQAMA was based on real-time PCR using fluorescence quenching detection. The MGB probe was fitted with a minor groove binder to keep the probe length to a minimum and thus maximize the effect of the one-base mismatch (Afonina et al., 1997). DNA methylation was discriminated when two differently-labeled MGB probes hybridized with their target sequences in the same tube (de Kok et al., 2002). All primers and probes were synthesized and labeled by Invitrogen (Shanghai, China). 2.2. Standard plasmid construction In order to develop the improved FP assay, standard controls with methylated and unmethylated target DNA sequences should be detected first, and standard plasmids should be constructed. Bisulfite-modified DNA from peripheral blood leukocytes of healthy volunteers was amplified using the two pairs of primers to create standards for the unmethylated allele; to create standards for the methylated allele, bisulfite-modified DNA from peripheral blood leukocytes of healthy volunteers was treated in vitro with SssI methyltransferase (New England Biolabs, M0226S, Boston, USA) before amplification. The PCR products of methylated and unmethylated alleles ( MGMT promoter, 100 bp; EGFR promoter, 156 bp) were introduced into pGEM-T-easy Vector (Promega, A1360, USA) to construct recombinant plasmids, according to the manufacturer’s instructions. The recombinant plasmids were identified by sequencing. The plasmids p-methylated- MGMT and p-methylated- p16INK4a contained the PCR products of methylated alleles. The plasmids p-unmethylated- MGMT, p-unmethylatedEGFR contained the PCR products of unmethylated alleles. The plasmids were purified using a Quick Plasmid Miniprep kit (Tiangen, DP105, Beijing, China), the concentration of DNA was determined by measuring the optical density at 260 nm. 2.3. Template preparation One hundred and three glioma tissue samples were obtained from patients undergoing surgery in the Department of Neurology, Xijing Hospital and the Tangdu Hospital of The Fourth Military
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Template DNA Bisulphite treatment Conversion template DNA Forward Primer
Amplification
---g(a)c-g(a)c—g(a)c---
---c(t)g-c(t)g--c(t)g---
Reverse Primer Methylated probe
Annealing-cleavage
n----cg-cg--cg-cg---------gc-gc--gc- gc------
Unmethylated probe
Methylated amplicon
n ---- tg- tg--tg-tg----
Small Fluorophores
------ac-ac-ac-ac-----n
Unmethylated amplicon Small Fluorophores
Decreased FP Values of TAMRA n
Decreased FP Values of FAM
=TAMRA
=FAM
Fig. 1. Schematic of the improved FP assay in 5’-nuclease reaction. Template DNA was treated with sodium bisulphate. Unmethylated cytosine residues were conversed to uracil, which were converted to thymidine during subsequent PCR. Thus, after bisulphite treatment, alleles that were originally methylated have different DNA sequences as compared with their corresponding unmethylated alleles. A pair of universal primers and a pair of TAMRA-m-probe, FAM-unm-probe (methylated, unmethylated) were added to each reaction. The universal primers amplified the bisulfite-modified DNA target to generate amplicons. The results between methylated and unmethylated targets were distinguished by TAMRA-m-probe and FAM-unmprobe. TAMRA-m-probe and FAM-unm-probe annealed respectively to methylated, unmethylated amplicons respectively and the probes were cleaved by the 5’-nuclease activity of Taq DNA polymerase. Subsequently, an intact probe bearing a fluorescent molecule, which has a molecular weight of ∼10,000 Da, were conversed to one base nucleotide molecular bearing a fluorescent molecule, which has a molecular weight of ∼1000 Da. That resulted in the decreased FP values.
Medical University between March 2009 and July 2013. One hundred and sixteen primary NSCLC tissue samples were obtained from patients undergoing surgery in the Department of Oncology, the Department of Thoracic Surgery, Xijing Hospital and Tangdu Hospital of The Fourth Military Medical University from March 2009 to October 2013. The study was approved by the Human Research Protective Committee of The Fourth Military Medical University, and written informed consent was obtained from each patient. Each cancer tissue specimen was cut into two parts; one part was paraffin-embedded for histological diagnosis, the other for molecular analysis. Genomic DNA was extracted from 20 to 30 mg cancer tissue using a QIAamp DNA Mini kit (QIAGEN, 51,304, Germany), following the manufacturer’s instructions. The extracted genomic DNA was bisulfite-modified using an EZ DNA Methylation-GoldTM kit (Zymo Research, D5005 USA), according to the manufacturer’s instruction. 2.4. Gene promotor methylation detection using the improved FP assay Firstly, the improved FP assay in 5’-nuclease reaction for individually assessing methylated or unmethylated alleles by one step was developed. Serial dilutions from 107 copies to 102 copies of p-methylated- MGMT or p-methylated- EGFR were used as the methylated positive standards and serial dilutions from 107 copies to 102 copies of p-unmethylated- MGMT or p-unmethylated- EGFR were used as the unmethylated positive standards. The pGEM-
T-easy Vector plasmid, which didn’t contain any PCR product of target methylated or unmethylated alleles, was used as the negative control. The 25 l reaction system included 1.5 U TaqDNA polymerase (Tiangen, ET101, China), 1 M of each primer, 100 M of each deoxynucleotide triphosphate (dNTP, Tiangen, CD111, China), 4.0 mM MgCl2 and 100 nM of the methylated or unmethylated allele-specific probe. The reaction was carried out with initial denaturing at 97 ◦ C for 5 min followed by 35 cycles of denaturing at 95 ◦ C for 40 s, annealing at 55 ◦ C for 40 s, extension at 72 ◦ C for 40 s, and cooling to 25 ◦ C. FP value (millipolarization [mP]) was directly proportional to the fluorescent molecular weight. A fluorescent molecule with high molecular weight rotated slowly in space and FP values was high. A one-base nucleotide bearing fluorophore had a molecular weight of ∼1000 Da and a 18–25 base probe bearing fluorophore had a molecular weight of ∼10,000 Da. In the 5’-nuclease reaction, when the fluorescent probes annealed to the complementary amplicons, they were cleaved into one-base nucleotides bearing fluorophore by the 5 -nuclease activity of Taq DNA polymerase and a decreased FP values was detected. Conversely, the fluorescent probe remained intact and FP values remained high. Therefore, when a template with only methylated allele was detected, TAMRA-m-probe annealed to the amplicon and was cleaved, and the FP values of TAMRA decreased. When a template with only unmethylated allele was detected, FAM-unm-probe annealed to the amplicon and was cleaved, and the FP value of FAM decreased (Fig. 1). Reactions were performed in duplicate. And then the FP values of TAMRA and FAM
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were measured using an FP plate reader. FP values were measured for FAM with excitation at 485 nm and emission at 530 nm; for TAMRA with excitation at 535 nm and emission at 590 nm. Secondly, the improved FP assay in 5’-nuclease reaction for simultaneous methylated and unmethylated allele assessment in one step was developed. FP technique can detect two different fluorophores with distinct excitation and emission frequencies. Each reaction of the FP assay was carried out as described above with minor modifications. In reaction buffer, the mixtures of methylated and unmethylated standards were used as templates and 80 nM each of the methylated and unmethylated allele-specific probe were included. As the template with methylated and unmethylated allele was detected, both TAMRA-m-probe and FAM-unm-probe annealed respectively to their target amplicon and were cleaved, and the FP values of both TAMRA and FAM decreased. Therefore, the methylated and unmethylated alleles were homogeneous detected simultaneously. Lastly, MGMT promoter methylation of 103 glioma samples and EGFR promoter methylation of 116 primary NSCLC samples were assessed by the improved FP assay in 5’-nuclease reaction. The unmethylated allele, which should always be detected, served as an internal control to test inhibitors and assess the quality of the template. The gene promoter methylation in each sample was determined by data analysis of FP values.
2.7. Gene promotor methylation detection using sequencing and AQAMA To evaluate the accuracy of the improved FP assay, MGMT and EGFR promotor methylation of the samples were detected by sequencing and AQAMA in parallel. In sequencing assay, 50 l reaction system included 2.5 M of each universal primer. The bisulfite-modified template of each glioma tissue sample was subjected to the primers of MGMT forward and MGMT -reverse, the bisulfite-modified template of each NSCLC tissue sample was subjected to the primers of EGFR -forward and EGFR -reverse. PCR products stained with ethidium bromide were detected visually on gel under UV, and they were purified with the Wizard DNA Clean-up System (Promega, USA) followed by sequencing on an ABI 377 instrument by the AmpliCycle sequencing kit (PerkinElmer, San Jose, California). The gene promotor methylation of each sample was assayed in duplicate. In AQAMA, 25 l reaction system included 2.5 M of each universal primer and 150 nM each of methylated and unmethylated allele-specific MGB-probes. The bisulfite-modified template of each glioma tissue sample was subjected to MGMT promoter methylation detection, and the bisulfite-modified DNA of each NSCLC tissue sample was subjected to EGFR promoter methylation detection. Samples were assayed in duplicate. 3. Results
2.5. Data analysis of the improved FP assay 3.1. Cut off Value of the improved FP assay The negative controls had high FP values for TAMRA and FAM, as no probe was cleaved. The methylated positive standards had decreased FP values for TAMRA, as the TAMRA-m-probes were cleaved. The unmethylated positive standards had decreased FP values for FAM, as the FAM-unm-probes were cleaved. By performing a t distribution test, the FP values of the negative and the positive data sets of TAMRA and FAM were found to be significantly different at a 95% level of confidence. The net change of FP values between the positive and the negative standards for TAMRA and FAM was analyzed. Cut off values for the net change of the positive reactions were determined as described (Ohiso et al., 2000; Bao et al., 2010; Sherif et al., 2001).
2.6. Specificity, sensitivity and reproducibility of the improved FP assay The specificity of the improved FP assay for methylated and unmethylated alleles was affected mainly by the specificity of the 5’-nuclease reaction. Cross annealing between the probes and amplicons would interfere with the specificity of the 5’-nuclease reaction. In the specificity determination reaction for unmethylated alleles, TAMRA-m-probe and FAM-unm-probe were used in a cocktail, 106 –108 copies of methylated plasmid were used as template. In the specificity determination reaction for methylated allele, TAMRA-m-probe and FAM-unm-probe were used in a cocktail, 106 –108 copies of unmethylated plasmid were used as template. FP values of TAMRA and FAM were monitored for 24 h. To determine the sensitivity of the improved FP assay, the mixtures of methylated and unmethylated allele plasmid were used as template in the ratios 1:99, 2:98, 3:97, 4:96, 5:95, 6:94, 7:93, 8:92, 9:91 and 10:90. The methylation allele of each mixture was determined based on the FP values of TAMRA and FAM. To determine the reproducibility of the FP assay, the mixtures with the following ratios of methylated to unmethylated allele plasmid were analyzed three times on different days: 10:90, 50:50, 70:30 and 90:10. The effect of varying amounts of template on the results in samples was also investigated.
In the improved FP assay, the FP values of the positive standards and negative controls were significantly different. For TAMRA, the 95% confidence interval was: (49, 180; 120 ± 43) from the methylated-MGMT standards population; (56, 185; 126 ± 40) from the methylated- EGFR standards population; and (237, 254; 243 ± 11) from the negative control population. For FAM, the 95% confidence interval was (42, 168; 105 ± 39) from the unmethylated-MGMT standards population; (46, 163; 116 ± 41) from the unmethylated- EGFR standards population; and (221, 246; 235 ± 10) from the negative control population. These intervals do not overlap, demonstrating a clear separation between positive and negative samples. For MGMT gene promoter methylation detection, cut off values for the net decreasing of the FP value in TAMRA was >56 mP of the negative controls and in FAM was >47 mP of the negative controls. For EGFR gene promoter methylation detection, cut off values for the net decreasing of the FP value in TAMRA was >51 mP of the negative controls and in FAM was >41 mP of the negative controls. The FP values curves for 102 –107 copies of methylated and unmethylated allele was shown in Fig. 2. 3.2. Specificity, sensitivity and reproducibility In the specificity determination for unmethylated or methylated allele, no net change of FP values more than the cut off values was found, indicating that TAMRA-m-probes and FAM-unm-probes exhibited strong specificity for their targets, with no cross reaction. Therefore, few possibilities for misidentification were present. In the sensitivity analysis, the improved FP assay could detect methylated allele for content as low as 5%. In the reproducibility analysis, all the different ratios of methylated to unmethylated allele plasmid were defined as positive, and varying amounts of template in samples had no effect on the results. 3.3. Gene promoter methylation in samples Each sample was detected in parallel using the improved FP assay and sequencing, AQAMA. In the improved FP assay, the gene
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FP assay, sequencing and AQAMA. One sample was identified as EGFR promoter methylation positive by the FP assay and AQAMA but EGFR promoter methylation negative by sequencing. The accuracy of the results measured by the improved FP assay, sequencing or AQAMA was evaluated using the paired-samples t test. There was no significant difference ( P > 0.05), suggesting that the FP assay is as reliable as the conventional detection method.
4. Discussion
Fig. 2. The FP values curves for 102 –107 copies of methylated and unmethylated allele. (A) The FP values curves for 102 –107 copies of MGMT promotor methylated and unmethylated allele. The quantity of p-methylated- MGMT, p-unmethylated- MGMT was determined by measuring the optical density at 260 nm. 102 –107 copies of p-methylatedMGMT were used as the methylated positive standards and 102 –107 copies of punmethylated- MGMT were used as the unmethylated positive standards. FP assay in TAMRA was for methylated positive targets. FP assay in FAM was for unmethylated positive targets. This figure indicated that the FP values fell down with the increasing amount of target positive standards. For TAMRA, FP values decreased from 180 to 49 when copies of p-methylated- MGMT increased from 102 to 107 . For FAM, FP values decreased from 168 to 40 when copies of p-unmethylated- MGMT increased from 102 to 107 . The R 2 values of the trend line of the FP values curves for MGMT promotor methylated and unmethylated allele was no less than 0.9709. (b) The FP values curves for 102 –107 copies of EGFR promotor methylated and unmethylated allele. The quantity of p-methylated- EGFR, p-unmethylated- EGFR was determined by measuring the optical density at 260 nm. 102 –107 copies of p-methylatedEGFR were used as the methylated positive standards and 102 –107 copies of punmethylated- EGFR were used as the unmethylated positive standards. FP assay in TAMRA was for methylated positive targets. FP assay in FAM was for unmethylated positive targets. This figure indicated that FP values fell down with the increasing amount of target positive standards. For TAMRA, FP values decreased from 185 to 56 when copies of p-methylated- EGFR increased from 102 to 107 . For FAM, FP values decreased from 163 to 46 when copies of p-unmethylated- EGFR increased from 102 to 107. The R 2 values of the trend line of the FP values curves for EGFR methylated and unmethylated allele was no less than 0.9714.
promotor methylation in samples was determined based on the net change of the FP values in TAMRA and FAM. If the net decreasing of the FP values of a sample was less than the cut off value in TAMRA and more than the cut off value in FAM of the negative controls, the sample was identified as methylation negative. If the net decreasing of the FP values of a sample was both more than the cut off values in TAMRA and FAM of the negative controls, the sample was identified as methylation positive. Of the 103 glioma samples, 44 samples were identified as MGMT methylation positive by the improved FP assay, sequencing and AQAMA. Two samples were identified as MGMT promoter methylation positive by the FP assay, AQAMA but MGMT promoter methylation negative by sequencing. Of the 116 primary NSCLC samples, 30 samples were identified as EGFR promoter methylation positive by the improved
Research on monitoring EGFR promoter methylation with a hybridization-FP assay has been done before. In the hybridizationFP assay for EGFR promoter methylation detection, a pair of EGFR promoter primers was used in an asymmetric PCR and the single-chain PCR product hybridized with the probe labeled with fluorophore at the 3 -end, resulting in an increased FP values. The hybridization-FP assay for EGFR promoter methylation detection used multiple steps in two tubes (Zhang et al., 2013). A homogeneous approach is of particular interest to clinical molecular diagnostics, as it eliminates carryover and cross contamination. In the novel assay, a 5’-nuclease reaction was combined with FP technique to detect methylated and unmethylated alleles simultaneously in one tube. When the probe matched to its target amplicon and was cleaved into one-base nucleotides by the 5 nuclease activity of TaqDNA polymerase, the molecular weight of the fluorophore-labeled molecule decreased. Then the FP values decreased significantly. Therefore, the homogeneous, gel free detection of gene promoter methylation without separation of the PCR products was established. This is more compelling when comparing to other popular assays for DNA methylation detection such as MSP, MLPA, COBRA, the hybridization-FP assay, pyrosequencing and sequencing (Hernández et al., 2013; Herman et al., 1996; Nygren et al., 2005; Xiong and Laird, 1997; Shaw et al., 2006; Zeschnigk et al., 2004). In terms of MSP, MLPA, COBRA, the hybridization-FP assay, pyrosequencing and sequencing, methylated and unmethylated alleles couldn’t be detected simultaneously in one tube, the purified PCR products and gel electrophoresis were required. The improved FP assay obviated the necessity for an open reaction tube after amplification. The improved FP assay was more exact and faster than conventional assays. Furthermore, the unmethylated allele, which could always be detected, served as an internal control to assess the quality of the template DNA in the improved FP assay. This helped simplify handling, as it obviated the need for other controls. A one-step approach is of particular interest for clinical diagnosis, but the labor-intensive and time-consuming steps of the conventional techniques limits their application. A high sensitivity, accuracy and cost-effectiveness are always required in clinical diagnosis. In this study, three samples were identified as methylation positive by the improved FP assay and AQAMA but methylation negative by sequencing. This was likely because of the fact that the sequencing assay was defined by the little amount of methylated PCR product from the specimens with the minor population of gene promoter methylation. This indicated the improved FP assay was more sensitive compared to the gel-based sequencing assay for the detection of gene promoter methylation. The sensitivity of the improved FP assay was 5%, the assay was as reliable as the conventional assays based on fluorescence intensity detection, such as AQAMA and MethyLight. The conventional assays based on fluorescence intensity detection techniques relied on reporter fluorophore intensity released from a fluorescence quencher of a probe (Eads et al., 2000; Zeschnigk et al., 2004; Dimitrakopoulos et al., 2012). However, the improved FP assay relied on the decreased FP values resulted from probe cleaved, the novel assay opened up the possibility of utilizing a less costly
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probe labeled with fluorophore at the 5 -end without a fluorescence quencher. Furthermore, the FP assay was not influenced by variations in fluorescence intensities, since it detected the plane polarization of the fluorescence. So the assay was more reliable than conventional assays, such as MethyLight. In addition, the FP assay did not require any real-time PCR equipment; it was performed with a less costly FP plate reader. In this study, the FP values of the negative and positive data sets were significantly distinct. The R2 values of the trend line of the FP values curves for 102 –107 copies of methylated and unmethylated allele was no less than 0.97, demonstrating that FP value of TAMRA and FAM was linearly proportional to the amount of MGMT, EGFR promoter methylated and unmethylated alleles, respectively. This showed a high precision in discriminating the methylated and unmethylated alleles. This high precision is beneficial for high sensitivity, good reproducibility and accuracy. This study reported a homogeneous and cost-effective FP assay in 5 nuclease reaction for detection of MGMT,EGFR promoter methylation. A good compromise was achieved between efficiency, cost, and accuracy and the assay provided a practical tool for clinical molecular diagnostics. MGMT,EGFR promoter methylation serves as biomarker to predict prognosis, progression-free survival, and outcome of treatment. The incidence rate of MGMT promoter methylation was as high as 44.7% in glioma samples and the incidence rate of EGFR promoter methylation was as high as 26.7% in primary NSCLC samples in China. The results indicated that MGMT promoter methylation was common in glioma samples and EGFR promoter methylation was common in primary NSCLC samples. The detection of MGMT promoter methylation before alkylating agent therapy and the detection of EGFR promoter methylation before EGFR inhibitor therapy would provide a scientific basis for applying a reasonable therapeutic regimen in future treatment. Conflicts of interest We have no conflicts of interest to declare. Authors’ contributions Conception and design: Haichuan Su, Ju Zhang, Wenchao Liu. Development of methodology: Ju Zhang, Yinghao Jiang. Acquisition of data (provided and confirm samples, etc.): Haichuan Su, Zhongliang Wu, Shaoying Qiang, Hong Cheng, Wenchao Liu. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Ju Zhang, Wenchao Liu, Haichuan Su. Study supervision, Writing and review of the manuscript: Ju Zhang, Wenchao Liu. Acknowledgements This work was supported by the National High-tech R&D Program (2008AA02Z444), and the National Nature Science Foundation (81071435, 81371891). We thank Professor Sun Jianzhong for manuscript language helping. References Afonina, I., Zivarts, M., Kutyavin, I., Lukhtanov, E., Gamper, H., Meyer, RB, 1997. Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res. 25, 2657–2660.
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