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Clinical Chemistry 53: 2119-2127, 2007. First published October 25, 2007; 10.1373/clinchem.2007.094011
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(Clinical Chemistry. 2007;53:2119-2127.)
© 2007 American Association for Clinical Chemistry, Inc.

Cancer Diagnostics

MS-FLAG, a Novel Real-Time Signal Generation Method for Methylation-Specific PCR

Cinzia Bonanno1, Erlet Shehi2, Daniel Adlerstein2,a and G. Mike Makrigiorgos3

1 University of Milano Bicocca, Milano, Italy.
2 DiaSorin SpA, Saluggia (VC), Italy.
3 Dana Farber-Brigham and Women’s Cancer Center, Harvard Medical School, Boston, MA.

aAddress correspondence to this author at: Diasorin SpA, Viale Pasteur 10, 20014 Nerviano (MI), Italy. Fax 0039-03311547; e-mail daniel.adlerstein{at}diasorin.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Aberrant promoter methylation is a major mechanism for silencing tumor suppressor genes in cancer. Detection of hypermethylation is used as a molecular marker for early cancer diagnosis, as a prognostic index, or to define therapeutic targets for reversion of aberrant methylation. We report on a novel signal generation technology for real-time PCR to detect gene promoter methylation.

Methods: FLAG (fluorescent amplicon generation) is a homogeneous signal generation technology based on the exceptionally thermostable endonuclease PspGI. FLAG provides real-time signal generation during PCR by PspGI-mediated cleavage of quenched fluorophores at the 5' end of double-stranded PCR products. Methylation-specific PCR (MSP) applied on bisulfite-treated DNA was adapted to a real-time format (methylation-specific FLAG; MS-FLAG) for quantifying methylation in the promoter of CDKN2A (p16), GATA5, and RASSF1. We validated MS-FLAG on plasmids and genomic DNA with known methylation status and applied it to detection of methylation in a limited number of clinical samples. We also conducted bisulfite sequencing on these samples.

Results: Real-time PCR results obtained via MS-FLAG agreed with results obtained via conventional, gel-based MSP. The new technology showed high specificity, sensitivity (2–3 plasmid copies), and selectivity (0.01% of methylated DNA) on control samples. It enabled correct prediction of the methylation status of all 3 gene promoters in 21 lung adenocarcinoma samples, as confirmed by bisulfite sequencing. We also developed a multiplex MS-FLAG assay for GATA5 and RASSF1 promoters.

Conclusion: MS-FLAG provides a new, quantitative, high-throughput method for detecting gene promoter methylation and is a convenient alternative to agarose gel-based MSP for screening methylation. In addition to methylation, FLAG-based real-time signal generation may have broad applications in DNA diagnostics.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytosine-5 methylation of CpG dinucleotides is associated with repression of gene expression and is recognized as an important event in carcinogenesis(1)(2). In vertebrates, certain CpG-rich regions within gene promoters, known as CpG islands, are typically unmethylated(3)(4) to facilitate gene transcription. Exceptions are CpG islands associated with tissue-specific genes, imprinted genes(5)(6), or genes located on the X chromosome(7). The epigenetic pattern (epigenome) of an individual is established early during embryogenesis and is maintained by DNA-methyltransferases during mitotic cell divisions(8). DNA methylation represses transcription by recruiting histone deacetylases, thereby inducing a compact chromatin structure(4)(9) and/or altering DNA binding sites of transcription factors. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor genes in human cancer(10)(11). Genes involved in cell-cycle regulation [p16, also called CDKN2A1 (cyclin-dependent kinase inhibitor 2A); RASSF1 (Ras association (RalGDS/AF-6) domain family 1)], apoptosis (DAPK, death-associated protein kinase), and DNA repair (MGMT, O-6-methylguanine-DNA methyltransferase) are frequently silenced in cancer(12)(13). For these genes detection of promoter methylation status from surgical tumor specimens or bodily fluids provides a sensitive, specific, and relatively noninvasive way to screen for cancer. CpG hypermethylation can be used as a molecular marker for early cancer diagnosis(14)(15)(16), as a prognostic index(17)(18), and to define therapeutic targets for reversion of aberrant methylation(11).

Several techniques have been developed for analyzing the DNA methylation status of CpG islands, including methylation-sensitive restriction enzymes(19), MALDI-TOF/mass spectrometry(20), and microarrays(21). A major contribution to the detection of DNA methylation in epigenetic studies, described by Frommer and colleagues(22)(23) in the early 1990s, was the laboratory use of sodium bisulfite. Sodium bisulfite converts unmethylated cytosines into uracils while leaving methylated cytosines relatively intact, thus creating sequence differences between genomes that originally differ only in their CpG methylation pattern. The nucleotide differences are then detected by sequencing(24), restriction enzyme analysis(25), PCR(26), and other methods. One of the most widely used methods is methylation-specific PCR (MSP),2 described by Herman et al.(26). MSP uses primers that bind to and amplify bisulfite-converted sequences only if CpG dinucleotides on these sequences remain unaffected by the chemical treatment (i.e., the cytosines are methylated). Alternatively, primers that bind specifically only to unmethylated cytosines within the primer sequence can be used, thus revealing the absence of methylation. MSP can detect 1 methylated allele in the presence of 1000 unmethylated (normal) alleles(26). Limitations in the originally described MSP are the requirement for gel electrophoresis and insufficient quantification due to the endpoint–based PCR detection format. Real-time PCR technologies using the TaqMan probe approach for signal generation(27) have been adapted for detecting methylation(28)(29). Real-time technology eliminates the need for gel separation, provides quantitative information on the degree of DNA methylation in a given sample with a sensitivity approaching 1/10 000, and has the throughput and convenience lacking in MSP(30). However, TaqMan-based approaches such as MethyLight can occasionally miss methylated samples that are detectable via MSP, possibly because both primers and probe must hybridize correctly for signal generation. In partially methylated clinical samples, this requirement is not always fulfilled. Furthermore, multiplexing of more than 1 gene is relatively difficult when TaqMan approaches are used because of the multiple oligonucleotides that must be used in the reactions.

In this report we describe fluorescent amplicon generation (FLAG), a new method for real-time signal generation during PCR that is adapted to the detection of CpG methylation (methylation-specific FLAG; MS-FLAG). We first validated this new assay on DNA with known methylation status and then demonstrated the detection of methylation for 3 tumor suppressor genes that are often found to be methylated in lung adenocarcinoma: CDKN2A, RASSF1, and GATA5 (GATA binding protein 5).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
generation of plasmid dna and sources of genomic dna
To test the reliability of FLAG signal-generation technology on controls that eliminate the variability introduced by the bisulfite treatment(22), we generated plasmid methylated and unmethylated control sequences for RASSF1 promoter. We synthesized 2 177-bp DNA fragments, each simulating the sequence for fully methylated or completely unmethylated RASSF1 promoter that would result after complete bisulfite conversion. We then cloned these fragments into PCR-Blunt 2.1 plasmids (Invitrogen). The resulting methylated (M) and unmethylated (U) recombinant control plasmids were cultured in Escherichia coli strain TOP10 cells and extracted with NucleoSpin Plasmid Kit (Macherey-Nagel), and their identity was confirmed by automated sequencing (MWG Biotech). We used these plasmids as template in experiments evaluating the specificity, sensitivity, and selectivity of MS-FLAG.

The genomic DNA samples used as positive (M) and negative (U) controls were CpGenome Universal Methylated DNA and CpGenome Universal Unmethylated DNA (Chemicon), respectively. Surgical lung adenocarcinoma tumor samples were obtained from the Massachusetts General Hospital Tumor Bank, Boston, after we obtained internal review board approval. DNA was extracted from these samples by use of the DneasyTM Tissue Kit (Qiagen).

bisulfite treatment and sequencing
To convert unmethylated cytosines to uracils, we treated 300 ng human genomic DNA from lung adenocarcinoma samples and genomic DNA controls with sodium bisulfite by use of the CpGenome DNA Modification Kit (Chemicon) according to the manufacturer’s protocol. We performed MS-FLAG assays on 1 µL bisulfite-treated DNA (corresponding to approximately 5 ng starting material) to investigate hypermethylation of promoter regions of RASSF1, p16, and GATA5. We also examined the methylation status of clinical samples via bisulfite sequencing. After bisulfite treatment, the MS-FLAG target region was PCR-amplified using primers external to the MS-FLAG region, and the amplified products were processed via dideoxy sequencing.

methylight assay for p16
Sequence of primers and probe used in p16 MethyLight assay were as follows: forward primer p16_Fw (TGG AGT TTT CGG TTG ATT GGT T), reverse primer P16_Rv (AGG AGG TGC GGG CGT TGT T), and TaqMan probe p16_Probe (FAM-ACC CGA CCC CGA ACC GCG-BHQ1). Reaction mixtures contained 300 nmol/L of each primer, 200 nmol/L probe, and 1x TaqMan Universal PCR Master Mix (Applied Biosystems) in 30 µL total volume. After an initial denaturation of 10 min at 95 °C, the amplification protocol consisted of 40–45 cycles (95 °C 15 s, 57 °C 15 s, and 60 °C 1 min).

ms-flag assay for p16, gata5, and rassf1
The restriction endonuclease used for FLAG signal generation was PspGI (New England Biolabs). An 11-nucleotide oligonucleotide (TTT CCA GGT TT) containing the PspGI recognition sequence (underlined) was added to the 5' end of the gene-specific portion of the primers (primer tail). The primer tail was doubly labeled with Iowa-Black FQ quencher (Integrated DNA Technologies) at the 5' end and a fluorophore at the 3' end. We used fluorescein for p16 and RASSF1 assays and MAX (Integrated DNA Technologies) dye for GATA5 assays. Fluorescence was detected and quantified on a Chromo4 real-time PCR machine (MJ Research) using the FAM detection channel for fluorescein and VIC detection channel for MAX. We visualized the gene-specific amplification products via ethidium bromide-stained 2% agarose gel electrophoresis. All MS-FLAG primers were designed with the support of VisualOmp software (DNA Software) and synthesized by Integrated DNA Technologies. Sequences of primers for bisulfite-converted DNA used in each assay were as follows.

p16.
Forward primer CB78QF (TTT CCA GGT TTC GAT TCG TGT ACG ACG TTG), reverse primer CB79QF (TTT CCA GGT TTG CAA CCG CGC GCA AA), which generate a 192-bp product.

RASSF1.
Forward primer CB107QF (TTT CCA GGT TTA CGA GAG CGC GTT TAG TTT CGT TTT C), reverse primer CB108QF (TTT CCA GGT TTA GCT AAC AAA CGC GAA CCG AAC G), which generate a 188-bp product.

GATA5.
Forward primer CB131QR (TTT CCA GGT TTC GTT GGG GTT TCG GTC GTA), reverse primer CB132QR (TTT CCA GGT TTA CTA ATC CGA ACT CCG CGC TA), which generate a 129-bp product.

Reaction mixtures contained 300 nmol/L of each primer, 300 µmol/L dNTPs, and 1x JumpStart PCR buffer with 10 units PspGI enzyme (New England Biolab) and 1 unit JumpStart Taq Polymerase (Sigma) in 20 µL total volume. After an initial denaturation of 3 min at 94 °C, the amplification protocol consisted of 45–50 cycles of (94 °C 30 s, 68 °C 30 s, and 72 °C 1 min). To facilitate multiplex reactions, the MS-FLAG primers were designed to operate under a single annealing temperature.

Multiplex MS-FLAG.
Assay conditions were slightly different for the duplex GATA5/RASSF1 MS-FLAG, to account for the simultaneous amplification of 2 gene fragments with different efficiencies. The reaction mixture in this case contained 300 nmol/L GATA5 primers (CB131QR and CB132QR) labeled with MAX dye, 200 nmol/L RASSF1 primers (CB107QF, CB108QF) labeled with fluorescein, 500 µmol/L dNTPs, and 1x JumpStart PCR buffer with 10 units PspGI enzyme (New England Biolab) and 1 unit JumpStart Taq Polymerase (Sigma) in a final volume of 20 µL.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ms-flag principle
FLAG is a novel signal generation technology for real-time PCR (Fig. 1 ) that includes the endonuclease PspGI in the reaction. Amplification is performed using primers that have a target-specific 3' region and a 5'-oligonucleotide tail containing a fluorophore-quencher pair separated by nucleotides carrying the recognition sequence of an exceptionally thermostable restriction endonuclease, PspGI(28). PspGI has a half-life of 2 h at 95 °C(31) and remains active throughout the PCR reaction. At each amplification cycle, the complete double-stranded recognition site for this enzyme is generated at both ends (primer tails) of the amplified product. Cleavage of the primer tails by the endonuclease results in an increase in fluorescence due to loss of fluorescence resonance energy transfer quenching (Fig. 1 ). Because only the introduced 5' tail of the PCR amplicons is digested, amplicons retain their primer-binding regions, which serve as templates for primer binding and amplification. MS-FLAG primers are specific for methylated sequences (Fig. 1 ) such that fluorescence signals are generated only if the interrogated sample contains methylated CpG DNA.


Figure 1
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  		Figure 1. Principle of signal generation via MS-FLAG.

MS-FLAG primers are designed to anneal to regions harboring methylated CpG sites and contain an oligonucleotide 5' tail carrying a quencher and a fluorophore separated by the recognition sequence of the PspGI endonuclease. The highly thermostable PspGI enzyme is present during the PCR reaction. (A), MS-FLAG forward primer anneals to methylated target DNA (Step 1) and is extended by the DNA polymerase (Step 2). The reverse primer synthesizes the opposite strand (Step 3) and generates a double-stranded recognition sequence for PspGI. Cleavage by PspGI (Step 4) leads to separation of the quencher and fluorophore, generating fluorescence. (B), if the interrogated target DNA is not methylated, the binding of the primers is inefficient and no PCR product or fluorescence is generated.

validation of ms-flag principle on plasmid controls: specificity, sensitivity, and selectivity
To test MS-FLAG in regard to the reliability and specificity of real-time signal generation, 2 recombinant plasmids were synthesized to act as positive and negative controls. The plasmids contained the anticipated sequence that the RASSF1 promoter region will have after complete bisulfite conversion of fully methylated (positive) or completely unmethylated (negative) samples. In the 1st case, each CpG spot of the original target sequence is supposed to be methylated, whereas in the 2nd case, no methylated CpGs are present. These plasmid controls eliminate the variability introduced by bisulfite treatment(22) and allow an independent evaluation of the novel signal generation method without the complications introduced by chemical treatment.

Real-time signal generation via MS-FLAG performed on control plasmids is depicted in Fig. 2 . Only plasmids representing fully methylated sequences generated real-time signals (Fig. 2A ), indicating the specificity of the designed primers for methylated CpG. Electrophoretic separation on ethidium bromide-stained agarose gels (Fig. 2A , inset) depicts no amplification products for no-target control (NTC) or unmethylated plasmid controls, whereas an approximately 188-bp band is present in the methylated plasmid controls. The sensitivity of MS-FLAG signal generation was then tested on 10-fold serial dilutions of the methylated control plasmid, ranging from 100 fg to 10 ag of target DNA. Signal generation was detected down to 10 ag (corresponding to 2–3 plasmid copies) of methylated DNA. A semilogarithmic plot of the thresholds obtained demonstrated an almost perfect linearity (r2 = 0.998) (Fig. 2B , inset). The selectivity of MS-FLAG signal generation was tested by enriching the RASSF1 methylated control plasmid into its unmethylated counterpart, to form a series of dilutions with methylated-to-unmethylated ratios ranging from 1:10 to 1:106 (0.0001%). MS-FLAG detected the methylated sequence in 104 -fold higher amounts of unmethylated sequences (Fig. 2C ); the detection was quantitative (r2 = 0.999; Fig. 2C inset). Overall, real-time signal generation using MS-FLAG is highly sensitive and quantitative, and the primers designed for the RASSF1 promoter are selective for sequences expected to form after bisulfite treatment of methylated CpG-containing DNA.


Figure 2
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  		Figure 2. Validation of MS-FLAG real-time signal generation on engineered plasmids.

(A), specificity: real-time growth curves of duplicate independent experiments on fully methylated (M) or completely unmethylated (U) control plasmid DNA samples. The specific amplification PCR product (inset) and the corresponding fluorescence are selectively generated only in controls mimicking methylated samples. Duplicate independent experiments are depicted (overlapping growth curves). (B), sensitivity: the assay was performed on samples containing decreasing amounts of RASSF1 plasmid DNA. Fluorescence was generated in samples down to 10 ag (approximately 2–3 copies of M plasmid DNA), with an excellent degree of linearity, r2 = 0.998 (inset). (C), selectivity: decreasing amounts of methylated (M) RASSF1 DNA were added to RASSF1 unmethylated (U) DNA. Methylation was detected with excellent linearity (r2 = 0.999), down to 0.01% methylated-to-unmethylated plasmid DNA (inset).

detection of CPG methylation in bisulfite-treated genomic dna controls
After validation of the real-time signal-generation properties of MS-FLAG, the assay was applied to bisulfite-treated human genomic DNA that was either completely unmethylated or fully methylated. We used 1 µL bisulfite-treated DNA, corresponding to approximately 5 ng starting material, as a target for MS-FLAG assays to identify the methylation status of the promoter region of the genes GATA5, RASSF1, and p16. Results from triplicate independent experiments are shown in Fig. 3 , A–C. The MS-FLAG thresholds [threshold cycle (SD)], as derived by the 3 independent repeats, were 33.0 (0.5) (GATA5), 31.3 (0.2) (RASSF1), and 32.7 (0.5) (p16). Methylated samples generated fluorescent signals corresponding to the formation of specific amplification products for each assay, as also verified by agarose gel electrophoresis-based sizing of the final PCR products (insets of Fig. 3 , A–C). In terms of fluorescence intensity and threshold cycle, MS-FLAG data were very similar to those of the well-established MethyLight technique, as shown for p16 in Fig. 3D .


Figure 3
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  		Figure 3. MS-FLAG on genomic DNA controls and comparison to MethyLight.

MS-FLAG assay was performed on 1 µL commercially available fully methylated (M) or completely unmethylated (U) genomic DNA controls (Chemicon) after bisulfite treatment. For all 3 investigated genes, GATA5 (A), RASSF1 (B), and p16 (C), triplicate independent MS-FLAG experiments are depicted. The data show fluorescent signals corresponding to the formation of specific amplification products from methylated DNA, as also confirmed by ethidium bromide–stained gel electrophoresis (insets, conventional MSP). Unmethylated samples demonstrate no fluorescence and no gel electrophoresis (conventional MSP) bands. MethyLight assay for p16 performed in triplicate independent experiments on the same genomic samples (D) showed threshold cycle values and fluorescence intensities similar to those of MS-FLAG.

multiplex ms-flag for gata5 and rassf1 on genomic dna controls
After validation of MS-FLAG in detecting the methylation status of DNA in simplex reactions, we also designed and optimized duplex assays that allow for simultaneous detection of the methylation status of 2 different genes in a single reaction. To this end, MS-FLAG primers for RASSF1 and GATA5 were synthesized with primer tails containing distinct fluorophores, FAM and MAX, respectively (the fluorescence of MAX can be monitored on the VIC channel of most real-time PCR machines). When the interrogated genomic DNA control sample was methylated for both genes, fluorescence was produced by PspGI cleavage of the primer tails in both sequences, generating real-time signals simultaneously in the FAM and VIC channels (Fig. 4A ). Electrophoretic analysis of the PCR end products on agarose gels showed 2 bands (Fig. 4B ), each representing a single methylated gene-specific amplification product (129 bp for GATA5 and 188 bp for RASSF1).


Figure 4
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  		Figure 4. Multiplex MS-FLAG assay.

Multiplex detection of the methylation status of 2 different genes (RASSF1 and GATA5) in a single reaction tube. Two different sets of primers were used, labeled with 2 different fluorophores: fluorescein for RASSF1 (green curves) and MAX for GATA5 (red curves, detected on VIC channel). (A), methylated samples generated fluorescence in both FAM and VIC channels. (B), electrophoretic analysis of the PCR end products on ethidium bromide–stained agarose gel.

methylation analysis of clinical samples
To field test the new technology on clinical samples, MS-FLAG was used to investigate the methylation status of GATA5, p16, and RASSF1 on a limited number of samples from lung adenocarcinoma surgical specimens. GATA5 was methylated in 10 of 21 samples (47%), whereas p16 and RASSF1 were methylated in 85% and 57% of samples, respectively. MS-FLAG growth curves indicating the methylation status of the interrogated genes were obtained in repeated independent experiments. Representative real-time growth curves of methylated and unmethylated samples are depicted in Fig. 5 , together with DNA controls. For an additional confirmation of the results, we performed bisulfite sequencing on samples analyzed via MS-FLAG to examine whether, indeed, the MS-FLAG primers amplify DNA containing methylated cytosines in CpG sites. Fig. 6 depicts representative results from GATA5 for clinical samples TL58 and TL25 presented in Fig. 5 . The presence of cytosines for MS-FLAG–positive sample TL58 [as revealed by observing guanines (see arrows) on the opposite strand of the electropherogram] indicates methylated CpG sites on the MS-FLAG primer-binding sites (boxed area; Fig. 6A ). In contrast, MS-FLAG–negative sample TL25 indicated thymidines at the same positions, suggesting full conversion of unmethylated C to T by the bisulfite treatment (Fig. 6C ). Bisulfite sequencing of the methylated control is also depicted (Fig. 6B ). It is noteworthy that MSP is much more sensitive than bisulfite sequencing in identifying a low fraction of methylated alleles within unmethylated alleles. Therefore clinical samples that contain a low fraction of methylated alleles may conceivably appear unmethylated when bisulfite sequencing is used but can still demonstrate substantial methylation with MSP or MS-FLAG. In the samples examined in this investigation, a discrepancy between the 2 methods was not observed, i.e., the samples were either fully methylated or fully unmethylated at the MS-FLAG primer-binding regions. Accordingly, the bisulfite sequencing data were consistent with the conclusions obtained from MS-FLAG applied to clinical samples.


Figure 5
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  		Figure 5. Investigation of the methylation status of GATA5, p16, and RASSF1 in lung adenocarcinoma clinical samples.

For each gene, MS-FLAG real-time growth curves are depicted for representative methylated and unmethylated surgical specimens (curves with error bars representing independent triplicate experiments), together with methylated or unmethylated genomic DNA controls.


Figure 6
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  		Figure 6. Bisulfite sequencing of clinical lung adenocarcinoma samples.

Representative electropherograms of the samples TL58 and TL25 examined via MS-FLAG in Fig. 5Up for GATA5 are shown. (A), the presence of cytosines for bisulfite-treated sample TL58 [as revealed by observing guanines (arrows) on the opposite strand of the electropherogram] indicates methylated CpG sites on the MS-FLAG primer-binding sites (boxed area). (B), the same GATA5 region was sequenced for the fully methylated genomic DNA control. A similar methylation pattern at the primer-binding sites is depicted. (C), the presence of thymidines for bisulfite-treated sample TL25 [as revealed by observing adenines (arrows) on the opposite strand of the electropherogram] indicates unmethylated CpG sites on the MS-FLAG primer-binding sites (boxed area). The data confirm the methylation status of samples TL58 and TL25 as predicted by MS-FLAG.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Quantitative and precise assays for methylation bear an increasingly important role in elucidating the influence of epigenetic modifications in cancer and differentiation(30). MS-FLAG is a new real-time PCR method that uses the endonuclease PspGI for fluorescence signal generation and is adapted to the detection of DNA methylation. Because of the unusually high thermostability of PspGI(31), which has an activity half-life of approximately 2 h at 95 °C, the relief of fluorescence quenching from the 5' ends of double-stranded PCR products proceeds throughout the entire PCR reaction, resulting in robust fluorescence signals. The reproducibility of MS-FLAG corresponds to SDs of 0.2–0.5 for the real-time PCR threshold, which is very satisfactory considering the variability that can be introduced by the bisulfite treatment itself(30). A potential limitation of MS-FLAG is that the amplified region must not contain PspGI sites other than those incorporated at the 5' ends of the primers. This requirement restricts the application to amplicons that are devoid of 5'-CCTGG-3' sequence strings. For application to the field of methylation, however, this restriction should not be an issue, because bisulfite treatment removes the cytosines from PspGI recognition sequences and prevents illegitimate digestion of target sequences.

Real-time PCR methods can be distinguished from those using a hybridization probe(27)(32)(33)(34) or those using labeled PCR primers(35)(36)(37)(38)(39)(40). The former provide high specificity and sensitivity in target quantification; however, the requirement for a 3rd oligonucleotide in addition to the primers in the PCR reaction limits their multiplexing capability, owing to primer–probe interactions. Approaches using universally labeled PCR primers can be more cost-effective as long as the formation of primer-dimers or nonspecific amplification products does not adversely affect specificity. Optimal primer design is therefore required in the latter case. This requirement is particularly relevant for bisulfite-treated DNA, because the conversion of C to T degrades the DNA, lowers the DNA annealing temperature, and reduces the specificity of primers. In part because of these technical difficulties, multiplexed real-time MSP directly from genomic DNA has not, to our knowledge, been previously reported. Our data demonstrate the successful identification of methylation-specific primers for MS-FLAG and PCR cycling conditions that avoid primer-dimer formation while allowing effective PspGI-mediated signal generation. The demonstration of MS-FLAG multiplexing capabilities for the genes GATA5 and RASSF1 indicates the broad potential of this approach for increasing the throughput of methylation detection. Overall, MS-FLAG combines the broad sensitivity of MSP with the convenience and throughput of real-time PCR, while enabling a more straightforward multiplexing than TaqMan-based approaches.

The FLAG approach is expected to find broad additional applications in real-time PCR, such as in virology, genotyping, and infectious diseases. Furthermore, as preliminary results in our laboratory indicate, FLAG can be used for real-time RT-PCR for the detection of viral RNA.


   Acknowledgments
 
Grant/funding support: This work was supported by DiaSorin SpA and in part by National Institutes of Health Grants CA111994-01 and CA115439-01 (to G.M.M.).

Financial disclosures: None declared.


   Footnotes
 
1 Human genes: CDKN2A, cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4); RASSF1, Ras association (RalGDS/AF-6) domain family 1; GATA5, GATA binding protein 5; DAPK, death-associated protein kinase; MGMT, O-6-methylguanine-DNA methyl-transferase.

2 Nonstandard abbreviations: MSP, methylation-specific PCR; FLAG, fluorescent amplicon generation; MS-FLAG, methylation-specific FLAG; NTC, no-target control.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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