RESUMEN
DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.
Asunto(s)
Metilación de ADN , ADN/análisis , ADN/metabolismo , Oligonucleótidos/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Adenocarcinoma/sangre , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Secuencia de Bases , Calcitonina/genética , Neoplasias del Colon/sangre , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/genética , ADN/genética , Cartilla de ADN/antagonistas & inhibidores , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , Gutatión-S-Transferasa pi , Glutatión Transferasa/genética , Humanos , Isoenzimas/genética , Datos de Secuencia Molecular , Oligonucleótidos/genética , Sensibilidad y Especificidad , Sulfitos/metabolismo , Factores de TiempoRESUMEN
In recent years, many molecular biomarkers have been discovered that are capable of distinguishing tumors from normal tissue. Among the different types of markers, DNA methylation markers stand out for their potential to provide a unique combination of specificity, sensitivity, high information content, and applicability to a wide variety of clinical specimens. Methylation markers are particularly suited for situations where sensitive detection is necessary, such as when tumor DNA is either scarce or diluted by excess normal DNA. One of the most widely used methods for measuring methylation levels, methylation-specific PCR (MSP), has been proved to be a very effective tool in situations requiring sensitive detection. The addition of fluorogenic probes makes these assays more informative, quantitative, and suitable for a clinical format. The field of sensitive detection is not limited to MSP; hence, an alternative methylation-sensitive amplification is discussed. PCR-based methylation assays have been applied to the detection of tumor DNA in a variety of body fluids, including serum, plasma, urine, sputum, and lavage fluids. In many cases, the sensitivity and specificity of these detection assays has been impressive, but important technological issues remain in areas such as sample preparation, assay design, and marker selection. Once these technical concerns have been addressed, the sensitive detection of methylation will provide a powerful diagnostic and prognostic tool, especially for the early detection of preneoplastic and neoplastic lesions.
Asunto(s)
Metilación de ADN , Humanos , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
Cancer arises due to the accumulation of DNA modifications that give cells a selective growth advantage. One common DNA modification is promoter hypermethylation associated with loss of expression of a tumor suppressor gene. The methylation status of a specific sequence or the pattern of methylation across the genome can be readily measured, and these sequences and analytical methods are being rapidly developed for molecular diagnostic applications. Detection of certain methylation events can be used for early detection of tumors, and analysis of patterns of methylation across the genome might provide information on disease subtype, aggressiveness, and treatment response. DNA methylation-based molecular diagnostic assays are particularly attractive because of the stability of the target analyte (DNA) and the potential sensitivity of the assays. As the field matures, methylation-based assays will make a major contribution to the field of molecular diagnostics, providing tools to fill unmet needs in current diagnostic and treatment plans for many types of cancer.