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Baltimore Highlands, MD, United States

Keeley B.,Johns Hopkins University | Stark A.,Johns Hopkins University | Pisanic T.R.,Johns Hopkins Institute for NanoBioTechnology | Kwak R.,The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins | And 9 more authors.
Clinica Chimica Acta | Year: 2013

The use of methylated tumor-specific circulating DNA has shown great promise as a potential cancer biomarker. Nonetheless, the relative scarcity of tumor-specific circulating DNA presents a challenge for traditional DNA extraction and processing techniques. Here we demonstrate a single tube extraction and processing technique dubbed "methylation on beads" that allows for DNA extraction and bisulfite conversion for up to 2. ml of plasma or serum. In comparison to traditional techniques including phenol chloroform and alcohol extraction, methylation on beads yields a 1.5- to 5-fold improvement in extraction efficiency. The technique results in far less carryover of PCR inhibitors yielding analytical sensitivity improvements of over 25-fold. The combination of improved recovery and sensitivity make possible the detection of rare epigenetic events and the development of high sensitivity epigenetic diagnostic assays. © 2013 Elsevier B.V.

Guzzetta A.A.,Johns Hopkins University | Pisanic Ii T.R.,Johns Hopkins Institute for NanoBioTechnology | Sharma P.,All India Institute of Medical Sciences | Yi J.M.,Korea Institute of Radiological and Medical Sciences | And 2 more authors.
Expert Review of Molecular Diagnostics | Year: 2014

Despite numerous technical hurdles, the realization of true personalized medicine is becoming a progressive reality for the future of patient care. With the development of new techniques and tools to measure the genetic signature of tumors, biomarkers are increasingly being used to detect occult tumors, determine the choice of treatment and predict outcomes. Methylation of CpG islands at the promoter region of genes is a particularly exciting biomarker as it is cancer-specific. Older methods to detect methylation were cumbersome, operator-dependent and required large amounts of DNA. However, a newer technique called methylation on beads has resulted in a more uniform, streamlined and efficient assay. Furthermore, methylation on beads permits the extraction and processing of miniscule amounts of methylated tumor DNA in the peripheral blood. Such a technique may aid in the clinical detection and treatment of cancers in the future. © 2014 Informa UK, Ltd.

Stark A.,Johns Hopkins University | Shin D.J.,Johns Hopkins University | Pisanic T.,Johns Hopkins Institute for NanoBioTechnology | Hsieh K.,Johns Hopkins University | And 2 more authors.
Biomedical Microdevices | Year: 2016

Aberrant methylation of DNA has been identified as an epigenetic biomarker for numerous cancer types. The vast majority of techniques aimed at detecting methylation require bisulfite conversion of the DNA sample prior to analysis, which until now has been a benchtop process. Although microfluidics has potential benefits of simplified operation, sample and reagent economy, and scalability, bisulfite conversion has yet to be implemented in this format. Here, we present a novel droplet microfluidic design that facilitates rapid bisulfite conversion by reducing the necessary processing steps while retaining comparable performance to existing methods. This new format has a reduced overall processing time and is readily scalable for use in high throughput DNA methylation analysis. © 2016, Springer Science+Business Media New York.

Pisanic T.R.,Johns Hopkins Institute for NanoBioTechnology | Athamanolap P.,Johns Hopkins University | Poh W.,Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins | Chen C.,Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins | And 7 more authors.
Nucleic Acids Research | Year: 2015

Many cancers comprise heterogeneous populations of cells at primary and metastatic sites throughout the body. The presence or emergence of distinct subclones with drug-resistant genetic and epigenetic phenotypes within these populations can greatly complicate therapeutic intervention. Liquid biopsies of peripheral blood from cancer patients have been suggested as an ideal means of sampling intratumor genetic and epigenetic heterogeneity for diagnostics, monitoring and therapeutic guidance. However, current molecular diagnostic and sequencing methods are not well suited to the routine assessment of epigenetic heterogeneity in difficult samples such as liquid biopsies that contain intrinsically low fractional concentrations of circulating tumor DNA (ctDNA) and rare epigenetic subclonal populations. Here we report an alternative approach, deemed DREAMing (Discrimination of Rare EpiAlleles by Melt), which uses semi-limiting dilution and precise melt curve analysis to distinguish and enumerate individual copies of epiallelic species at single-CpG-site resolution in fractions as low as 0.005%, providing facile and inexpensive ultrasensitive assessment of locus-specific epigenetic heterogeneity directly from liquid biopsies. The technique is demonstrated here for the evaluation of epigenetic heterogeneity at p14ARF and BRCA1 gene-promoter loci in liquid biopsies obtained from patients in association with non-small cell lung cancer (NSCLC) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), respectively. © The Author(s) 2015.

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