Enhanced Performance of DNA Methylation Markers by Simultaneous Measurement of Sense and Antisense DNA Strands after Cytosine Conversion.

BACKGROUND: Most existing DNA methylation-based methods for detection of circulating tumor DNA (ctDNA) are based on conversion of unmethylated cytosines to uracil. After conversion, the 2 DNA strands are no longer complementary; therefore, targeting only 1 DNA strand merely utilizes half of the available input DNA. We investigated whether the sensitivity of methylation-based ctDNA detection strategies could be increased by targeting both DNA strands after bisulfite conversion. METHODS: Dual-strand digital PCR assays were designed for the 3 colorectal cancer (CRC)-specific methylation markers KCNQ5, C9orf50, and CLIP4 and compared with previously reported single-strand assays. Performance was tested in tumor and leukocyte DNA, and the ability to detect ctDNA was investigated in plasma from 43 patients with CRC stages I to IV and 42 colonoscopy-confirmed healthy controls. RESULTS: Dual-strand assays quantified close to 100% of methylated control DNA input, whereas single-strand assays quantified approximately 50%. Furthermore, dual-strand assays showed a 2-fold increase in the number of methylated DNA copies detected when applied to DNA purified from tumor tissue and plasma from CRC patients. When the results of the 3 DNA methylation markers were combined into a ctDNA detection test and applied to plasma, the dual-strand assay format detected 86% of the cancers compared with 74% for the single-strand assay format. The specificity was 100% for both the dual- and single-strand test formats. CONCLUSION: Dual-strand assays enabled more sensitive detection of methylated ctDNA than single-strand assays.

A new approach to preparation of antisense oligonucleotide samples with microextraction by packed sorbent.

Our research focused on applying microextraction by packed sorbent to extracting antisense oligonucleotides from serum samples. The tested sorbents included poly(styrene-co-divinylbenzene), octyl, octadecyl, and unmodified silica gel. As nonpolar sorbents were used for highly-polar molecules, this required ion-pair mode. Comprehensive optimization of extraction conditions was performed for 20-mer phosphorothioate oligonucleotide. Several parametres - the number of "draw-eject" cycles during the conditioning and load step, the amine type and concentration, and the volume of elution mixture - and the influence they had on recovery were studied for nonpolar sorbents, which made it possible to obtain high (ca. 90%) recovery values. The most influential parameter turned out to be the volume of elution mixture. Similar optimization was performed for silica sorbents; however, despite optimization of various parameters, the recovery values stayed relatively low. The optimized procedures for nonpolar sorbents were applied in extraction of six different oligonucleotides of various length and with different structure modifications. The highest recoveries were obtained for octyl and octadecyl sorbents, ranging between 80-99%. The developed microextraction method was used to extract phosphorothioate and 2'-O-(2-methoxyethyl) oligonucleotides and their two synthetic metabolites from enriched human plasma, with recoveries around 70-80%.