Methyl-binding domain protein-based DNA isolation from human blood serum combines DNA analyses and serum-autoantibody testing

Abstract

BackgroundCirculating cell free DNA in serum as well as serum-autoantibodies and the serum proteome have great potential to contribute to early cancer diagnostics via non invasive blood tests. However, most DNA preparation protocols destroy the protein fraction and therefore do not allow subsequent protein analyses. In this study a novel approach based on methyl binding domain protein (MBD) is described to overcome the technical difficulties of combining DNA and protein analysis out of one single serum sample.MethodsSerum or plasma samples from 98 control individuals and 54 breast cancer patients were evaluated upon silica membrane- or MBD affinity-based DNA isolation via qPCR targeting potential DNA methylation markers as well as by protein-microarrays for tumor-autoantibody testing.ResultsIn control individuals, an average DNA level of 22.8 ± 25.7 ng/ml was detected applying the silica membrane based protocol and 8.5 ± 7.5 ng/ml using the MBD-approach, both values strongly dependent on the serum sample preparation methods used. In contrast to malignant and benign tumor serum samples, cell free DNA concentrations were significantly elevated in sera of metastasizing breast cancer patients. Technical evaluation revealed that serum upon MBD-based DNA isolation is suitable for protein-array analyses when data are consistent to untreated serum samples.ConclusionMBD affinity purification allows DNA isolations under native conditions retaining the protein function, thus for example enabling combined analyses of DNA methylation and autoantigene-profiles from the same serum sample and thereby improving minimal invasive diagnostics.

Diagnostic Performance of Plasma DNA Methylation Profiles in Lung Cancer, Pulmonary Fibrosis and COPD

“…The elevated cfDNA levels, however, were included as a separate predictor with in the study. Such an increase of cfDNA was frequently observed for diverse cancers Wielscher et al, 2011 , Jung et al, 2004 , Dawson et al, 2013 and late stage COPD patients Gormally et al, 2004 . The presence of DNA exhibiting disease marks in serum may be due to an interplay of several mechanisms Schwarzenbach et al, 2011 .…”

“…Standard curves, which also served as amplification control, as well as additional negative controls, were introduced before multiplexed enrichment. cfDNA was isolated out of 400 μl patients' serum using Roche High pure template preparation kit (Roche) with a modified isolation protocol published previously Wielscher et al, 2011 . In a volume of 12 μl cfDNA was digested with four methylsensitive restriction enzymes (MSRE) namely HpaII (ThermoFisher), Hin6I (ThermoFisher), AciI (NewEnglandBiolabs) and HpyCHIV4 (NewEnglandBiolabs).…”

Strategies for Validation and Testing of DNA Methylation Biomarkers

“…Additionally a combination of enrichment methods (e.g. by MBD-proteins) and MSRE based methylation detection may increase the sensitivity of qPCR results [20].…”

Solid phase nucleic acid extraction technique in a microfluidic chip using a novel non-chaotropic agent: dimethyl adipimidate

“…37,38 Several recent studies have demonstrated methylated DNA purification techniques using the methyl-binding protein (MBD) in solution phase in a wide array of human fluid samples. [37][38][39][40][41] These studies used the MBD protein as a capture molecule for directly and specifically binding the double-stranded methylated DNA in either a nanofluidic device or in use of magnetic beads, but not in the solid phase. For most techniques for methylated DNA purification that have been developed, the purification step was too complicated, time-consuming, and high-cost because of the use of additional molecules such as proteins and antibodies in solutionphase-based methods.…”