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Methylation-specific loop-mediated isothermal amplification for detecting hypermethylated DNA in simplex and multiplex formats.

DNA methylation at the C-5 position of cytosines of CpG dinucleotides is recognized as one of the most important mechanisms for gene expression control in multicellular organisms (1, 2). In vertebrates, methylation and demethylation events are produced by specific methylase- and demethylase-based enzymatic activities in CpG-rich regions, known as "CpG islands" (1-3). CpG island hypermethylation produces transcriptional silencing by either impairing the binding of the transcriptional machinery or inhibiting the binding of specific transcriptional factors to gene promoter regions and first exons. Moreover, chromatin status seems to be also dependent on the DNA methylation pattern through the recruitment of histone deacety-lases within hypermethylated DNA regions (4, 5). Thus, many DNA regions are extensively demethylated to allow DNA transcription, whereas other regions are extensively methylated to silence transcription of tissue-specific, X chromosome-linked, and imprinted genes (6-8).

The aberrant DNA methylation of certain promoter regions is linked to tumor formation, owing to the silencing of the transcription of well-established tumor suppressor genes, such as CDKN2A [4] [cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)], GATA5 (GATA binding protein 5), and DAPK1 (death-associated protein kinase 1) (9-11). Molecular testing of the methylation status of one or more gene promoters from samples of surgically resected tissues, or from bodily fluids, represents a reliable way to diagnose cancer at an early or advanced stage and to determine possible outcomes of the pathology, such as metastasis of the tumor from the primary site. Such molecular testing also has potential therapeutic indications (12-14).

The DNA methylation status of the promoters of interest can be detected directly via such methods as methylation-sensitive restriction analysis (15), but the most commonly used techniques rely on chemically treating target DNA with sodium bisulfite. Under suitable conditions, this chemical reagent converts unmethylated DNA cytosines to uracils but leaves unchanged methylated cytosines, such as those within CpG dinucleotides (16, 17). Therefore, bisulfite treatment produces different sequences for methylated and unmethylated DNA that can then be detected via several methods, such as direct sequencing (18), MALDITOF mass spectrometry (19), or restriction enzyme analysis (20). Moreover, some PCR-based approaches rely on the use of primer sets designed specifically to recognize and amplify only methylated (or, alternatively, unmethylated) DNA sequences due to the bisulfite conversion of the target DNA. PCR amplification can be detected at the end of the reaction via gel electrophoresis [i.e., methylation-specific PCR (MSP) [5]] (21) or by real-time PCR techniques [MethyLight and methylation-specific fluorescent amplicon generation (MS-FLAG)] (22-24).

A novel technology for isothermal DNA amplification, loop-mediated isothermal amplification (LAMP) (25, 26), has recently been developed for the amplification and detection of a target DNA sequence. This technique relies on strand-displacement amplification and entails the use of 6 sequence-specific primers and a polymerase with strand-displacement activity. LAMP reactions produce fast, efficient, and specific sequence amplification through the formation of intermediate double stem-loop structures and subsequent inverted repeats of target DNA strands, without the requirement of thermocycling (see Fig. 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol56/ issue8). A LAMP reaction can be monitored in real time by measuring the progressive increase in sample turbidity due to the precipitation of the magnesium pyrophosphate generated as the deoxynucleoside triphosphates (dNTPs) are incorporated into the extended DNA strands (27). Turbidity is monitored in a real-time turbidimeter, which consists of a thermoblock that allows continuous monitoring of transmittance. Turbidimetry detection has cost and simplicity advantages over other detection platforms. Fluorescencebased detection for LAMP is also possible through the guanine-quenching principle (28), which exploits a 5' fluorescently marked oligonucleotide that is substituted for one of the 2 loop primers in the reaction mixture. The fluorescence emitted by each dye is progressively quenched as the dye-linked primer is progressively incorporated into the final amplification product. A remarkable feature of the LAMP technology is its exquisite specificity for the intended DNA target, which is due to the 8 sequence-recognition events required by the mechanism associated with the isothermal process, which also prevents exponential amplification of primer dimers. The high specificityalso makes the LAMP technology well suited for multiplexing.

In this study, we adapted the LAMP technology to the detection of CpG methylation, [i.e., methylationspecific LAMP (MS-LAMP)] after bisulfite treatment of DNA. We designed 3 assays to detect hypermethylation in the promoters of 3 genes recognized to be involved in lung carcinogenesis (DAPK1, GATA5, and CDKN2A). We then tested this novel method with synthetic genes and commercial genomic DNA as targets and applied the technology to a limited number of adenocarcinoma clinical samples. We also developed a triplex MS-LAMP reaction for a fast, turbidimetric approach for preliminary clinical screening. We also developed a preliminary test for a triplex MS-LAMP assay that uses 3 different fluorescent dyes, each specific for a distinct target.

Materials and Methods

PLASMIDS

For a preliminary test of the reliability and performance of the MS-LAMP assay without the variation introduced by bisulfite treatment, we performed experiments to assess specificity, the detection limit, and selectivity with plasmid targets containing the DAPK1 promoter sequence in either a methylated state or a unmethylated state after bisulfite treatment. A commercial supplier (GENEART) synthetically generated the plasmids.

GENOMIC DNA

The fully methylated and unmethylated human genomic DNAs used as positive and negative controls in our experiments were CpGenome Universal Methylated and Unmethylated DNA (Millipore).

CLINICAL SAMPLES

Lung adenocarcinoma tumor samples were obtained from the Dana-Farber Cancer Institute and the Massachusetts General Hospital Tumor Bank, Boston, Massachusetts, after we obtained Internal Review Board approval. DNA was extracted from the samples with the Dneasy Blood & Tissue Kit (Qiagen).

BISULFITE TREATMENT AND MSP

All of the unmethylated cytosines in 500 ng of completely unmethylated and fully methylated human genomic DNA controls and DNA from lung adenocarcinoma samples were converted to uracils by bisulfite treatment with the EZ DNA Methylation Kit (Zymo Research), according to the manufacturer's protocol. Final elution of the DNA was performed in 20 [micro]L of Tris-EDTA buffer (10 mmol/L Tris, 1 mmol/L EDTA, pH 8.0). We used 1 or 2 [micro]L (approximately 25-50 ng) of DNA as the target for investigating the hypermethylation status of the CDKN2A, DAPK1, and GATA5 promoters.

MS-LAMP ASSAYS

MS-LAMP primers (F3, B3, FIP, BIP, LF, LB), which specifically amplify only the methylated sequences of the CDKN2A, DAPK1, and GATA5 promoters after bisulfite treatment, were designed with the aid of PrimerExplorer V4 (Eiken). Primers were synthesized by MWG Biotech; the primer sequences are presented in Table 1 in the online Data Supplement. Simplex LAMP reactions were performed at 63 [degrees]C (DAPK1, GATA5) or 65 [degrees]C (CDKN2A) for 60 min and monitored in a Real-Time Turbidimeter LA-200 (Teramecs). Every 6 s, the turbidimeter measures the variation in light transmittance through the test tube in which the LAMP reaction occurs. The light transmittance decreases during the reaction as the insoluble salt generated by the interaction between [Mg.sup.2+] ions and pyrophosphate ions progressively produced by the incorporation of dNTPs into the growing DNA strand precipitates in the reaction mixture. DNA amplification is represented graphically by plotting turbidity in arbitrary units against reaction time; the plot shows an exponentiallike shape. Reaction mixtures contained 200 nmol/L of the F3 and B3 primers, 800 nmol/L of the LF and LB primers, 1.6 [micro]mol/L of the FIP and BIP primers, 1.4 mmol/L of each dNTP, 0.8 mol/L betaine, 20 mmol/L Tris-HCl, 10 mmol/L KCl, 8 mmol/L MgS[O.sub.4], 10 mmol/L [(N[H.sub.4]).sub.2]S[O.sub.4], 1 mL/L Tween 20, and 0.32 U/[micro]L Bst DNA polymerase (New England Biolabs).

In the experiments performed to establish the detection limit and selectivity with plasmid targets, an initial one-time DNA denaturation preceded the LAMP reaction; plasmid aliquots were heated at 95 [degrees]C for 10 min, rapidly cooled on ice for 10 min, and finally added to the reaction mixture immediately before the reaction.

Multiplex (triplex) LAMP reactions assessed by turbidimetry were performed at 63 [degrees]C with the reagents at the same concentrations as in the simplex reactions and with all 18 primers included in the same reaction mixture. For the multiplex LAMP reactions assessed by fluorescence, the LF primers of the DAPK1 and CDKN2A assays were substituted by oligonucleotides that had the same DNA sequence but were labeled at the 5' end with BODIPY FL (dipyrromethene boron difluoride FL) and 6-carboxytetramethylrhodamine (TAMRA) dyes, respectively. The sequence of the fluorescent primer used in the GATA5 assaywas shifted 2 nucleotides upstream and lengthened by 1 nucleotide at the 3' end compared with the original LF primer (new sequence, 5' -CAAACCCCGCGAACAAAA-3') to ensure a closer proximity to guanine nucleotides; this primer was labeled at the 5' end with BODIPY 650/665. The reaction mixture contained the CDKN2A and GATA5 primer sets at standard concentrations and DAPK1 primers F3 and B3 at 0.08 [micro]mol/L, FIP and BIP primers at 0.64 [micro]mol/L, and the fluorescent primer and the LB primer at 0.32 [micro]mol/L. The reaction mixture also contained 1.4 mmol/L of each dNTP, 0.8 mol/L betaine, 20 mmol/L Tris-HCl, 10 mmol/L KCl, 8 mmol/L MgS[O.sub.4], 10 mmol/L [(N[H.sub.4]).sub.2]S[O.sub.4], 1 mL/L Tween 20, and 0.64 U/[micro]L Bst polymerase.

All MS-LAMP reactions were performed in triplicate and repeated at least twice in independent experiments.

MSP ASSAYS

The methylation status of clinical samples was also assayed by MSP with the external LAMP primers (F3 and B3) used as PCR primers. Reaction mixtures for all 3 MSP assays contained GoTaq polymerase (Promega) as indicated by the manufacturer, 1X of the reaction buffer provided by the manufacturer, 0.2 mmol/L of each dNTP, additional MgCl2 (2.5 mmol/L), and 250 nmol/L of each primer. The thermocycling protocols for the PCR reactions were as follows: CDKN2A, initial denaturation at 94 [degrees]C for 2 min and 50 cycles of 94 [degrees]C for 30 s, 65 [degrees]C for 30 s, and 72 [degrees]C for 30 s; DAPK1, initial denaturation at 94 [degrees]C for 2 min and 45 cycles of 94 [degrees]C for 30 s, 68 [degrees]C for 30 s, and 72 [degrees]C for 30 s; GATA5, initial denaturation at 94 [degrees]C for 2 min and 50 cycles of 94 [degrees]C for 30 s, 66 [degrees]C for 30 s, and 72 [degrees]C for 30 s.

Results

MS-LAMP PRIMER DESIGN

We used the LAMP approach to study promoter hypermethylation by designing 3 primer sets specific for the selected promoters with the aid of the dedicated software program PrimerExplorer V4. We designed the 3 primer sets to anneal perfectly to the hypermethylated version of the promoter regions after treatment with sodium bisulfite. To allow the best discrimination between methylated and unmethylated promoters, we designed primers to anneal to target regions that included the maximum number of CpGs. To limit the potential for false-negative results due to the partial methylation of the promoters in some clinical samples, however, we decided not to include CpG cytosines within 3 bases of the extensible end of each primer (i.e., the 3' end of F3, B3, F2, B2, LF, and LB and the 5' end of F1 and B1 (Fig. 1).

[FIGURE 1 OMITTED]

VALIDATION OF THE MS-LAMP PRINCIPLE WITH PLASMID CONTROLS

To test the specificity of MS-LAMP to detect hypermethylation, we first synthesized 2 plasmids that incorporated the expected sequence of the DAPK1 promoter after bisulfite treatment in either the fully methylated or fully unmethylated versions. In the plasmid representing the fully methylated sequence after bisulfite treatment, all of the cytosines of each CpG were kept as cytosines, and the other cytosines were replaced with thymidines. In the plasmid representing the unmethylated sequence after bisulfite treatment, everycytosine was replaced by a thymidine. These DNA controls allow a preliminary evaluation of the performance of this method without the variation introduced by the bisulfite treatment.

MS-LAMP amplification curves obtained by realtime turbidimetry detection with 15 000 copies per reaction of synthetic plasmid controls (Fig. 2A) showed that amplification occurred exclusively in the plasmid incorporating the methylated DAPK1 promoter. The plasmid containing the unmethylated sequence and the no-target control produced no amplification within the 1-h reaction time. These results indicate that the MS-LAMP method is specific and is able to discriminate sequences according to the original differences in the methylation status of CpG islands.

To evaluate the detection limit of the method, we performed MS-LAMP reactions with serial dilutions of methylated plasmid between 100 fg/reaction (approximately 3 x [10.sup.4] copies per reaction) and 10 ag/reaction (approximately 3 copies/reaction). Amplificationreactions were reproducible down to 100 ag/reaction with a good linear correlation ([r.sup.2] = 0.98) between the logarithm ofthe target amount and the threshold time (Fig. 2B). We also evaluated the selectivity, which we defined as the ability of the method to detect the specific target in the presence of a nonspecific background. We performed MS-LAMP assays with dilutions of methylated plasmid (100% to 0.01% methylated plasmid) in an unmethylated plasmid background. The total amount of DNA in each target mixture was 100 fg/reaction. The MS-LAMP assay was able to detect methylated plasmid quantitatively and reproducibly down to 0.1% (100 ag) diluted in a background of unmethylated plasmid ([r.sup.2] = 0.97; Fig. 2C).

[FIGURE 2 OMITTED]

DETECTION OF CpG METHYLATION IN BISULFITE-TREATED GENOMIC DNA CONTROLS

To further evaluate the performance of our assays with more complex targets, we tested the specificity and selectivity of MS-LAMP assay for the promoters of DAPK1, CDKN2A, and GATA5 with commercial human genomic DNA as a target, either in fully methylated or in fully unmethylated form. We treated both samples with bisulfite before testing and added 2 [micro]L of the treated DNA (approximately 50 ng) to the LAMP reactions. The amplification reactions (based on [greater than or equal to] 3 independent experiments; Fig. 3) showed that no amplification occurred in the unmethylated target and the no-target control reactions, whereas the methylated targets produced an amplification product within 30 min. Therefore, the MS-LAMP assay specifically and rapidly detected and amplified the correct target DNA after bisulfite treatment.

In a preliminary assessment of the capability of the assays to selectively detect a small amount of hypermethylated genomic DNA in an unmethylated background after bisulfite treatment, we performed experiments with human genomic DNA by progressively diluting fully methylated DNA into fully unmethylated DNA (from 100% to 0.25% methylated DNA). After bisulfite treatment, we assayed 50 ng of each of the resulting DNA preparations with the CDKN2A, DAPK1, and GATA5 MS-LAMP assays. Fig. 4 presents amplification curves for each methylated DNA/unmethylated DNA ratio down to the lowest reproducibly (3 independent experiments) detectable ratio. The lowest reproducibly detectable ratio obtained with a total of 50 ng DNA was 0.5% methylated DNA for the DAPK1 and CDKN2A assays and 1% methylated DNA for the GATA5 assay. We obtained no nonspecific amplification with either the no-target control or 100% unmethylated DNA.

[FIGURE 3 OMITTED]

COMPARISON OF MS-LAMP AND MSP TESTING WITH CLINICAL TUMOR SAMPLES

To compare the performances of the MS-LAMP assay and an established method with actual clinical samples, we assayed the DNA methylation status of the DAPK1, CDKN2A, and GATA5 promoters in 18 lung adenocarcinoma clinical samples with the MS-LAMP and MSP methods. The MSP assay was performed with LAMP F3 and B3 as the PCR primers so that we could evaluate the same DNA region assayed by the LAMP method. Fig. 2 in the online Data Supplement illustrates the amplification curves of MS-LAMP reactions and MSP electrophoresis gels for some representative samples. The predicted amplicon lengths for the MSP assays were 192 bp (DAPK1), 204bp (CDKN2A), and 202 bp (GATA5). Methylation-positive results in the MS-LAMP assay were obtained for 8 samples (44%) for DAPK1, 9 samples (50%) for CDKN2A, and 13 samples (72%) for GATA5 (see Table 2 in the online Data Supplement). All but one of the samples that were methylation positive in the MS-LAMP assaywere also scored positive in the MSP assay. Nine of the 35 cases tested that were scored as methylation positive in the MSP assay were methylation negative in the MS-LAMP assay. The very high specificity of the MS-LAMP method is a potential explanation for this reduced rate of scoring methylation-positive samples compared with the MSP method (see Discussion). A brief attempt to perform bisulfite sequencing on some of the clinical samples demonstrated simultaneous C and T peaks, a result consistent with partial methylation (data not shown).

[FIGURE 4 OMITTED]

MULTIPLEX MS-LAMP

One of the major advantages of the MS-LAMP method is its high specificity for the intended target, combined with the absence of primer dimers. We exploited this key feature of the technology to develop a multiplex assay capable of detecting the hypermethylation status of any of the 3 promoters simultaneously. All 18 primers used in the 3 LAMP assays were included in a single reaction. This triplex assay was initially used to assay various mixtures of the synthetic plasmid targets containing the 3 promoters (Fig. 5A). DNA amplification occurred either with a single methylated plasmid (GATA5) or with 2 methylated plasmids (GATA5 and CDKN2A) in the presence of the unmethylated counterparts Of the remaining promoters, and amplification occurred when all 3 methylated synthetic genes were mixed. We obtained no amplification in reactions that contained only the unmethylated versions of the 3 plasmids at the same time and in no-target control reactions. To further confirm these multiplex results, we used the same triplex reaction after bisulfite treatment to test the methylation status of fully methylated and fully unmethylated genomic DNA controls, along with the no-target control. We also tested 4 clinical samples (TL52, TL12, TL127, and TL75), which simplex MSLAMP reactions had shown to be hypermethylated at 3, 2, 1, and 0 promoters, respectively. Fig. 5B, which summarizes 3 independent experiments, shows again that no DNA amplification occurred in the no-target control, the fully unmethylated genomic control, and the triple-unmethylated clinical sample, whereas it confirms the hypermethylated status of the fully methylated genomic control and the 3 distinctly methylated clinical samples.

[FIGURE 5 OMITTED]

FLUORESCENCE MULTIPLEX MS-LAMP

To exploit further the intrinsically high specificity of the MS-LAMP method for multiplex detection of different hypermethylated targets, we also developed a multiplex application that uses sequence-specific detection of fluorescence signals. The principle of guanine quenching was exploited for the detection of the amplification process. Fluorescence quenching is detectable in real time if a fluorophore linked to the 5' end of a primer oligonucleotide is brought into proximity with one or more guanine-containing nucleotides. Accordingly, fluorescently labeled primers reduce their fluorescence once they are incorporated within the MS-LAMP product. Thus, the fluorescence signal is at a maximum at the beginning of the amplification reaction and is quenched progressively during the amplification process down to a stable plateau, where it remains until the end of the reaction. Distinctively labeled fluorescent primers were used as one of the 2 loop primers in the 3 assays (see Materials and Methods). The triplex reaction was then run in isothermal conditions in a real-time thermocycler and monitored simultaneously in the green, yellow, and red channels (Fig. 6). As reaction targets, we used different plasmid mixtures: each of the methylated plasmids in the presence ofthe unmethylated forms of the other 2 plasmids (Fig. 6, A, B, and C), all 3 unmethylated plasmids (Fig. 6D), or all 3 methylated plasmids (Fig. 6E). In the 3 samples containing only 1 methylated plasmid target (Fig. 6, A, B, and C), we detected fluorescence quenching only in the channel of the label specific for that methylated plasmid. No amplification curve could be detected in any fluorescence channel when only unmethylated versions of the plasmids were used as the reaction targets (Fig. 6D), indicating that no nonspecific reaction product was generated in the absence ofa methylated target. In the triplex reaction containing all 3 methylated targets (Fig. 6E), all 3 channels showed an amplification curve, indicating that the 3 different reactions were occurring simultaneously in the same reaction tube. Similar results were obtained when fully methylated and fully unmethylated genomic DNA samples were used as reaction targets (data not shown).

Discussion

LAMP is a DNA-amplification technique that possesses a number of advantageous features for routine molecular diagnostics applications: (a) It is isothermal and therefore requires no thermal cycler; (b) it is rapid, because no temperature ramping is needed and the reaction thresholds are typically reached in <30-40 min; and (c) its detection limit and quantitative performance parallel that of the PCR (29, 30). A distinct advantage of LAMP over PCR-based amplification is its high specificity for the intended DNA target, which is conferred by its unique amplification mechanism and the sequence-specific binding of multiple primers as a requirement for amplification. The latter feature eliminates primer dimer formation and nonspecific amplifications, which frequently are not possible to avoid with the PCR.

The ultrahigh specificity of the MS-LAMP methodology has both positive and negative consequences with regard to methylation detection with bisulfite-treated DNA. On the positive side, the AT-rich bisulfite-treated DNA is more apt to generate primer dimers and nonspecific amplification with MSP, whereas these artifacts are not a problem with the MSLAMP method. On the other hand, the data indicate that the MS-LAMP approach can have a reduced sensitivity compared with the MSP method in the assessment of clinical samples. The reduced sensitivity of the MS-LAMP method for detecting methylation-positive samples compared with MSP may be due to the high number of "probing zones" in the LAMP assay, which produces a strict specificity. By probing 8 regions, the primer set may fail to detect partial methylation, which is sometimes present in clinical samples (31) and potentially has biological importance. Thus, unless the promoter is heavily methylated, the MS-LAMP methodology may not generate a signal. In contrast, techniques that use only 2 primers, such as MSP or MSFLAG, often score a higher number of samples as positives. Similarly, MethyLight, a methylation-detection technology that probes 3 target regions, may also demonstrate a lower scoring rate of methylationpositive clinical samples compared with the MSP methodology (24). In designing our MS-LAMP primer sets, we maximized the specificity for methylated DNA after bisulfite conversion by appropriate positioning of the primers on the target DNA to include as many CpG dinucleotides per primer as possible (mean of 2-3 CpGs per primer region, with some being close to the 3' ends). Reducing the number of discrimination points probed by LAMP primers (i.e., designing only some of the 8 primers to overlap CpG dinucleotides) may ease the requirements and may decrease the risk of false negatives while keeping the assay adequately specific.

[FIGURE 6 OMITTED]

An additional consequence of the high MS-LAMP specificity is the ability to combine 3 individual simplex assays in a single triplex reaction without a substantial risk of primer dimer formation, which often is an obstacle for PCR-based DNA amplification protocols (32). We demonstrated the triplex MS-LAMP reaction in 2 detection formats, turbidimetry and fluorescence. The turbidimetry format benefits from the ease and low cost of this detection method and could be used as a preliminary screen of clinical samples to identify the presence of at least 1 methylated target. Fluorescence detection with multiple fluorescent dyes could then be used on the positive samples to identify each methylated promoter specifically and individually.

In conclusion, the LAMP assay, which has already been established for virology and microbiology approaches (33 ), was successfully adapted for methylation detection. To our knowledge, the MS-LAMP method is the first isothermal method for methylation detection. The high specificityofthe MS-LAMP methodology for the intended target, combined with its speed, ease of detection, and ability to multiplex reactions, demonstrates that MS-LAMP is a very promising technique for methylation detection for applications in oncology and epigenetics.

Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures of Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: G.M. Makrigiorgos, DiaSorin, Inc., Italy.

Stock Ownership: None declared.

Honoraria: None declared.

Research Funding: None declared.

Expert Testimony: None declared.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

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Francesco Zerilli, [1] Cinzia Bonanno, [2] Erlet Shehi, [2] Giulia Amicarelli, [2] Daniel Adlerstein, [2] * and G. Mike Makrigiorgos [3]

[1] Dipartimento di Biotecnologie e Bioscienze, University degli Studi di MilanoBicocca, Milano, Italy; [2] DiaSorin S.p.A., Saluggia (VC), Italy; [3] Department of Radiation Oncology, Division of Medical Physics and Biophysics, and Division of Genome Stability and DNA Repair, Dana-Farber/Brigham and Women's Cancer Center, Harvard Medical School, Boston, MA.

[4] Human genes: CDKN2A, cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4); GATA5, GATA binding protein 5; DAPK1, death-associated protein kinase 1.

[5] Nonstandard abbreviations: MSP, methylation-specific PCR; MS-FLAG, methylation-specific fluorescent amplicon generation; LAMP, loop-mediated isothermal amplification; dNTP, deoxynucleoside triphosphate; MS-LAMP, methylation-specific LAMP; BODIPY, dipyrromethene boron difluoride; TAMRA, 6-carboxytetramethylrhodamine.

* Address correspondence to this author at: Biotrin Ltd., 93 The Rise, Mount Merrion, Co. Dublin, Ireland. E-mail daniel.adlerstein@biotrin.ie.

Received January 19, 2010; accepted May 12, 2010.

Previously published online at DOI: 10.1373/clinchem.2010.143545
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Title Annotation:Molecular Diagnostics and Genetics
Author:Zerilli, Francesco; Bonanno, Cinzia; Shehi, Erlet; Amicarelli, Giulia; Adlerstein, Daniel; Makrigior
Publication:Clinical Chemistry
Date:Aug 1, 2010
Words:5137
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