A new type of 2-D separation material was synthesized and studied. The material is suitable for 2-D chromatography utilizing both covalent and noncovalent interactions. The first dimension is boronate affinity chromatography, and the second dimension is RP chromatography (or vice versa). The polymeric media were prepared using p-vinylphenylboronic acid as the functional monomer. This monomer was selected due to the presence of the boronic acid group for the cis-diol/boronate interaction in boronate chromatography. Two crosslinkers were evaluated, namely ethylene glycol dimethacrylate and divinylbenzene. The crosslinker content was varied to maximize the polymer strength and the RP performance of the packed column. Several parameters were evaluated to define the optimum for polymer strength and column performance including crosslinker, porogen, initiator, and column-packing parameters. The polymer-based HPLC columns were successful in separating phenol, catechol, dimethylphthalate, and hydroquinone under RP conditions, and thus can be used as an RP HPLC column. The columns were also successful in separating catechol and adenosine under boronate chromatography conditions, and thus can be used as a boronate affinity column. Moreover, the two types of chromatography can be performed consecutively on the same column during one complete chromatographic run, making it a 2-D chromatography. Under these 2-D conditions, the catechol was separated from a mixture of phenol, catechol, dimethylphthalate, and hydroquinone; the adenosine ribonucleoside was separated from a mixture of adenosine ribonucleoside, adenosine deoxyribonucleoside, and uridine deoxyribonucleoside. This type of single-column 2-D HPLC eliminates the requirement of a complex and expensive multidimensional HPLC instrument and provides increased peak capacity for separation.

Objective Universal noninvasive genomic screening to detect cancer and/or fetal DNA in plasma at all stages of development is highly warranted. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome. In the current study, we demonstrate that discrete 5hmC sites within 80 bp hotspot regions exist in a greater proportion of cancer versus normal cells. Result 5hmC was detected in 16 of 17 known hotspots having C to T or G to A mutations. The results show the presence of two characteristically distinct 5hmC groups: Tier 1 Group with 3 to eightfold more 5hmCs detected in tumor-cells than in normal-cell derived DNA (as observed in 6 of 11 CpG sites). Tier 2 group with equal allele frequency of 5hmC among normal and tumor-cell derived DNA at 5 CpG hotspot sites as well as 5 non-CpG hotspots. Thus, detection and quantification of the Tier 1 group of 5hmC sites or its prevalence at or near cancer mutation hot spots in cells may enable early detection, screening and potentially prediction of the likelihood of cancer occurrence or the severity of the cancer.

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