Control of carry-over contamination for PCR-based DNA methylation quantification using bisulfite treated DNA

In this study, we adapted the well known uracil DNA glycosylase (UNG) carry-over prevention system for PCR, and applied it to the analysis of DNA methylation based on sodium bisulfite conversion. As sodium bisulfite treatment converts unmethylated cytosine bases into uracil residues, bisulfite treated DNA is sensitive to UNG treatment. Therefore, UNG cannot be used for carry-over prevention of PCR using bisulfite treated template DNA, as not only contaminating products of previous PCR, but also the actual template will be degraded. We modified the bisulfite treatment procedure and generated DNA containing sulfonated uracil residues. Surprisingly, and in contrast to uracil, 6-sulfonyl uracil containing DNA (SafeBis DNA) is resistant to UNG. We showed that the new procedure removes up to 10 000 copies of contaminating PCR product in a closed PCR vessel without significant loss of analytical or clinical sensitivity of the DNA methylation analysis

A combined HM-PCR/SNuPE method for high sensitive detection of rare DNA methylation

<p>Abstract</p> <p>Background</p> <p>DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation.</p> <p>Results</p> <p>This combined method allows detection of 14 pg (that is, four to five genomic copies) of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng) of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2) and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer.</p> <p>Conclusion</p> <p>The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.</p

SHOX2 DNA Methylation is a Biomarker for the diagnosis of lung cancer based on bronchial aspirates

<p>Abstract</p> <p>Background</p> <p>This study aimed to show that SHOX2 DNA methylation is a tumor marker in patients with suspected lung cancer by using bronchial fluid aspirated during bronchoscopy. Such a biomarker would be clinically valuable, especially when, following the first bronchoscopy, a final diagnosis cannot be established by histology or cytology. A test with a low false positive rate can reduce the need for further invasive and costly procedures and ensure early treatment.</p> <p>Methods</p> <p>Marker discovery was carried out by differential methylation hybridization (DMH) and real-time PCR. The real-time PCR based HeavyMethyl technology was used for quantitative analysis of DNA methylation of SHOX2 using bronchial aspirates from two clinical centres in a case-control study. Fresh-frozen and Saccomanno-fixed samples were used to show the tumor marker performance in different sample types of clinical relevance.</p> <p>Results</p> <p>Valid measurements were obtained from a total of 523 patient samples (242 controls, 281 cases). DNA methylation of SHOX2 allowed to distinguish between malignant and benign lung disease, i.e. abscesses, infections, obstructive lung diseases, sarcoidosis, scleroderma, stenoses, at high specificity (68% sensitivity [95% CI 62-73%], 95% specificity [95% CI 91-97%]).</p> <p>Conclusions</p> <p>Hypermethylation of SHOX2 in bronchial aspirates appears to be a clinically useful tumor marker for identifying subjects with lung carcinoma, especially if histological and cytological findings after bronchoscopy are ambiguous.</p

Development of real time PCR methods for DNA methylation analysis

DNA-Methylierung spielt eine wichtige Rolle bei einer Reihe biologischer Prozesse wie Imprinting, X-chromosomaler Inaktivierung und Genexpression. DNA-Hypermethylierung ist häufig assoziiert mit einer verminderten Transkription, während Hypomethylierung oft mit einer Aktivierung der Transkription einhergehen kann. Veränderte DNA-Methylierungsmuster sind sowohl bei der Tumorgenese als auch bei einigen genetischen Krankheiten beschrieben. Die Analyse der DNA-Methylierung eignet sich daher zur Klassifizierung von Krebs und anderen Krankheiten. Die schnelle und einfache Detektion von Tumor-DNA in Körperflüssigkeiten anhand ihrer veränderten DNA-Methylierung ist ein innovatives und vielversprechendes Verfahren zur Früherkennung von Krebs. Für diagnostische Zwecke ist die Realtime-PCR besonders geeignet, da sie sich durch Genauigkeit, Schnelligkeit und Automatisierbarkeit auszeichnet. Im Mittelpunkt dieser Arbeit stand daher die Entwicklung von drei Messverfahren zur Methylierungsanalyse im Realtime-PCR-Format sowie die Etablierung einer Methode zur PCR-Kontaminationsvermeidung. HeavyMethyl-Assay (HM-Assay): Die Anreicherung methylierter DNA in einer PCR kann mit Hilfe von Blockeroligonukleotiden erreicht werden (HeavyMethyl-Assay). Ziel dieser Arbeit war es, dieses Assayformat in ein Realtime-PCR-Format zu übertragen und ein generelles Konzept zur Etablierung von HM-Assays zu erarbeiten. Die Leistungsfähigkeit des Detektionsverfahrens wurde am Beispiel der Entwicklung und Validierung des GSTp1-HM-Assays demonstriert. Das Detektionslimit dieser PCR lag bei 15ipg methylierter DNA. Das entspricht etwa 4i-i5 methylierten Genkopien. Dieser Nachweis gelang auch in einem hohen Hintergrund nicht methylierter DNA, dabei lag das relative Detektionslimit bei mindestens 1:4000. Mit dem GSTp1-HM-Assay wurden anschließend Gewebeproben aus Prostatatumoren analysiert. Die klinische Sensitivität betrug hier 79i% bei einer Spezifität von 95i%. Die Kombination des GSTp1-HM-Assays mit einer GSTp1-Referenz-PCR in einer Duplex-Reaktion erlaubte die gleichzeitige Analyse von methylierter und Gesamt-DNA und erhöhte die Effizienz der Methode. Die Realtime-HM-Technologie ist inzwischen als Standardmethode zur Detektion von Tumor-DNA in Körperflüssigkeiten etabliert und wird bereits in mehr als 20 weiteren Realtime-PCRs zur Detektion von methylierter DNA erfolgreich angewendet. QuantitativeMethylation-Assay (QM-Assay): Ein weiteres Ziel der Arbeit war die Weiterentwicklung des QM-Assays, einem Realtime-PCR-Verfahren, das die genaue Bestimmung des Anteils methylierter DNA in Gewebeproben ermöglicht. Durch die neuartige Kombination von SNP-spezifischen und methylierungsspezifischen Sonden konnte erstmals eine allelspezifische, quantitative Analyse von DNA-Methylierung mit einem Realtime-Verfahren realisiert werden. Mit allelspezifischen QM-Assays wurden primordiale Keimzellen aus Mausembryonen untersucht. Für die H19-Region wurde zwischen den Tagen 9,5 und 10,5 nach der Befruchtung eine signifikante Abnahme der parentalen Methylierung gemessen. In der IGF2/DMR2-Region wurde am Tag 9,5 keine Methylierung detektiert, während am Tag 10,5 sowohl maternales als auch paternales Allel geringe Methylierung aufwiesen. HeavyQuantitativeMethylation-Assay (HQM-Assay): Aus der Kombination von HM-Assay und QM-Assay wurde ein neuartiges Verfahren der Methylierungsanalyse, der HQM-Assay, entwickelt. Dieses Realtime-PCR-Verfahren vereint die Vorteile beider Technologien, die hohe relative Sensitivität des HM-Assays mit den guten quantitativen Eigenschaften des QM-Assays. Es handelt sich dabei um eine Duplexreaktion aus zwei HM-Assays, deren Signale nach dem Prinzip eines QM-Assays ausgewertet werden. Dabei werden methylierte und nicht methylierte DNA des gleichen Locus parallel amplifiziert und analysiert. Mit dem TMEFF2-HQM-Assay konnten DNA-Mischungen mit einem Anteil von 0,1 % und 0,5 % methylierter DNA exakt quantifiziert und damit im Vergleich zu einem QM-Assay eine 10 mal niedrigere Grenze für die quantitative Detektion methylierter DNA erreicht werden. Verfahren zur Vermeidung von PCR-Kontaminationen: Für die Methylierungsanalyse, basierend auf Bisulfit-DNA, standen bisher keine Verfahren zur Vermeidung von PCR Kontaminationen zur Verfügung. In der Arbeit wurde ein Protokoll für die Bisulfit-Konversion entwickelt, das auf der Herstellung sulfonierter DNA (SafeBis-DNA) basiert. Dieses Protokoll ermöglicht die Anwendung von Uracil-DNA-Glycosylase zum Abbau kontaminierender PCR-Produkte. Die Analyse von Tumorproben ergab die gleichen Ergebnisse unter Verwendung von SafeBis-DNA mit UNG im Vergleich zur Analyse von Standard-Bisulfit-DNA ohne UNG. In dieser Arbeit wurde also erstmalig eine Methylierungsanalyse mit einer effizienten Kontaminationskontrolle durchgeführt. Damit wurde eine weitere Voraussetzung geschaffen, Methylierungsanalysen als diagnostische Tests in der klinischen Routine einzusetzen.DNA-Methylation plays a role in a number of biological processes, such as imprinting, X chromosomal inactivation and gene expression. DNA hypermethylation is closely associated with transcriptional silencing, while DNA hypomethylation is associated with transcriptional activation. The tumor genesis is associated with changes in DNA methylation patterns of the genome, and also characteristic of some genetic diseases. DNA methylation is therefore a suitable tool for the diagnosis and classification of cancer and other diseases. Fast and simple detection and quantification of methylated tumor DNA in body fluids and tissue is an innovative and promising method for the early recognition of cancer. The focus of this work was the development of novel detection procedures for DNA methylation based on the real-time PCR format. HeavyMethyl Assay (HM-Assay): The enrichment of methylated DNA can be realized by blocked oligonucleotides in a PCR, the so-called HeavyMethyl Assay (HM-Assay). A goal of this work was to develop a robust, high sensitive method for the detection of DNA methylation based on the HM-technology. The HM-Assay was transferred into a real-time PCR format and a concept for the establishment of HM-Assays was compiled. The efficiency of the detection procedure was demonstrated by the development and validation of the GSTp1-HM-Assays. The 90 %-detection limit of this PCR was 15 pg, the relative detection limit was determined with at least 1:4000. That corresponds to 4 - 5 molecules of methylated DNA. Afterwards, samples from prostate tumors were analyzed with the GSTp1-HM-Assay. The clinical sensitivity amounted to 79 %, with a specificity of 95 %. The combination of the GSTp1-HM-Assays with a GSTp1-Referenz-PCR in a duplex reaction allows the parallel analysis of methylated and total DNA. Only very small DNA quantities can be isolated from blood plasma or serum, and thus only a limited number of methylation markers can be analyzed. The multiplexing of GSTp1 and PTGER4-HM-Assay demonstrates one solution to this problem. The duplex real-time PCR was successfully validated on the basis of serum samples. Since then, real time HeavyMethyl technology has been established as the standard method for the detection of tumor DNA in body fluids. It is used in more than 20 additional real time PCR\u27;s for the detection of methylated DNA. QuantitativeMethylation Assay (QM-Assay): A second goal of the work was the advancement of the QuantitativeMethlylation Assay, a real-time PCR procedure, which allows for the exact determination of the proportional portion of methylated DNA from tissue samples. This is the first time an allele-specific quantitative analysis could be established by the combination of SNP specific and methylation specific probes in a real-time PCR. Calculating the real-time PCR results by fluorescence intensities, simplified the calibration of QM-Assays. The quantitative measurement was also expanded by a preamplification allowing for the analysis of small sample quantities. Allele-specific QM-Assays were then used to investigate the allelic methylation of primordial germ cells from mouse embryos. For the H19-region, a significant decrease in the parentale methylation was measured between days 9.5 and 10.5 after fertilization. No methylation was found on IGF2/DMR2 on day 9.5, while low levels were detected on day 10.5 for the maternale and paternale alleles. HeavyQuantitativeMethylation Assay (HQM Assay): In the third part of the work the technological combination of HM-Assay and QM-Assay, which is called HQM Assay, was developed. This new real-time PCR procedure combines the advantages of both technologies: the high, relative sensitivity of the HM-Assays, with the good quantitative characteristics of the QM-Assays. The principle is based on a duplex reaction of two HM-Assays, whose signals are evaluated similarly to QM-Assays. In doing so, methylated and non-methylated DNA from the same region can be amplified and analyzed in parallel. DNA mixtures consisting of 0.1% and 0.5% methylated DNA could be precisely quantified with the TMEFF2-HQM Assay, allowing for the detection of methylated DNA with a limit 10 times lower than that of a QM-Assay. Procedure for PCR contamination prevention: Avoiding reaction contamination from former PCR products is essential for diagnostic tests. For the methylation analysis, no such procedures are currently available. For this study, a modified protocol for bisulfite conversion was developed, which is based on the generation of sulfonated DNA (SafeBis DNA). It allows for the application of uracil DNA glycosylase to inactivate contaminating PCR products. The method was used successfully in a methylation analysis of tumor samples. The use of SafeBis DNA had no negative influence on the sensitivity and quality of the analysis. This is the first time an efficient carry over prevention system was applied to a bisulfite DNA based methylation analysis

TFAP2E-DKK4 and chemoresistance in colorectal cancer

Chemotherapy for advanced colorectal cancer leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential downstream target of TFAP2E and has been implicated in chemotherapy resistance. We aimed to further evaluate the role of TFAP2E and DKK4 as predictors of the response of colorectal cancer to chemotherapy