title: Methylation-specific loop-mediated isothermal amplification for detecting hypermethylated DNA in simplex and multiplex formats.
abstract: BACKGROUND
  Aberrant DNA methylation of gene promoters and the associated silencing of tumor suppressor genes are recognized as mechanisms contributing to tumor development. Therefore, detection of promoter hypermethylation is becoming important for diagnosis, prognosis, and aiding the design of cancer therapies. We describe a novel isothermal method for the detection of DNA hypermethylation.
  
  
  METHODS
  Methylation-specific loop-mediated isothermal amplification (MS-LAMP) is a novel adaptation of LAMP. MS-LAMP was used for the highly specific detection of hypermethylated CpGs in the promoters of the CDKN2A [cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)], GATA5 (GATA binding protein 5), and DAPK1 (death-associated protein kinase 1) genes. The reactions occurred under isothermal conditions with 3 primer sets specific for methylated promoters. Both turbidimetry and fluorescence were used for detection. The MS-LAMP assay was validated with bisulfite-treated plasmid and genomic DNA controls of known methylation status and was applied to detect hypermethylation in 18 clinical tumor samples. A multiplex MS-LAMP for CDKN2A, GATA5, and DAPK1 was also validated with the aid of synthetic positive and negative controls.
  
  
  RESULTS
  The MS-LAMP assay showed high specificity with plasmid and genomic DNA targets in reactions carried out in <1 h. The assay had a detection limit of approximately 30 copies of methylated target sequence and a selectivity of 0.5% methylated DNA in a mixture with unmethylated DNA. Compared with methylation-specific PCR, the MS-LAMP assay detected lower rates of methylation in lung adenocarcinoma samples. Simultaneous multiplex detection of hypermethylation in the 3 targets (CDKN2A, GATA5, and DAPK1) was readily achieved with the MS-LAMP assay in both the turbidimetric and fluorescence detection formats.
  
  
  CONCLUSIONS
  MS-LAMP provides a highly specific isothermal method for methylation detection and is well suited for multiplex approaches.
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Milano, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Zerilli
authors.forename: Cinzia
authors.initials: C
authors.lastname: Bonanno
authors.forename: Erlet
authors.initials: E
authors.lastname: Shehi
authors.forename: Giulia
authors.initials: G
authors.lastname: Amicarelli
authors.forename: Daniel
authors.initials: D
authors.lastname: Adlerstein
authors.forename: G Mike
authors.initials: GM
authors.lastname: Makrigiorgos
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001483:Base Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000235:genetics; D018899:CpG Islands; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D019175:DNA Methylation; D017931:DNA Primers; D064550:Death-Associated Protein Kinases; D005470:Fluorometry; D050992:GATA5 Transcription Factor / Q000235:genetics; D015894:Genome, Human; D006801:Humans; D007202:Indicators and Reagents; D008175:Lung Neoplasms / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009391:Nephelometry and Turbidimetry; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012680:Sensitivity and Specificity; D013447:Sulfites
chemical_list: D051017:Apoptosis Regulatory Proteins; D019941:Cyclin-Dependent Kinase Inhibitor p16; D017931:DNA Primers; D050992:GATA5 Transcription Factor; C494755:GATA5 protein, human; D007202:Indicators and Reagents; D013447:Sulfites; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C042345:hydrogen sulfite