Enrichment of Methylated DNA Provides New Avenue for Diagnostics

December 2014

The human microbiome is not static; it’s a fluid entity. It can change rapidly and often gives clues to the health of an individual. While the ability to identify a particular microorganism within a complex biological sample has been a diagnostic tool for many decades, it has been limited to organisms that are culturable (~1%). Recently next generation sequencing (NGS) has eliminated this limitation through its ability to query total DNA of metagenomic samples. However, due to the large pool of non-relevant DNA, NGS lacks the sensitivity needed to identify small amounts of target DNA. Additionally, genome assembly becomes more difficult as the samples become larger and more complex.

To tackle this problem, Barnes et al. recently published a method in which they utilized a restriction endonuclease to enrich for target microbial DNA in complex samples. Enrichment prior to NGS could improve detection of small amounts of DNA in metagenomic samples such as tissue or saliva. To do this, the enzyme must be manipulated to restrict cleavage activity while maintaining high affinity binding.

DpnI is an endonuclease that targets and cuts at G^m6ATC, a methylation pattern found often in a variety of bacteria but is rare in eukaryotes. It had previously been reported that the absence of magnesium restricted cleavage activity, though whether the lack of magnesium affected binding affinity was unknown. To test this, the authors utilized an electrophoretic mobility shift assay. They incubated a FAM-labeled oligonucleotide containing a single G^m6ATC sequence with DpnI and DpnI-biotin and found that lack of magnesium did not affect the binding affinity.

Next, Barnes et al. utilized pull-down assays with biotin-labeled DpnI and found that they were able to enrich for G6mATC containing DNA when present at levels 108 times lower than eukaryotic DNA. They extended this study to show that recovery of clinically relevant organisms was increased. Additionally, this method improved sequencing coverage in both synthetic DNA mixtures as well as saliva samples. In the saliva sample, human DNA reads were decreased from more than 75% to less than 5% in the DpnI bound fraction. Prokaryotic reads increased from less than 5% to more than 50%. In both the saliva and water samples, the authors noted that while they saw overall enrichment of target sequences, there were some limitations. Experiments indicated that a small amount of binding occurs in the absence of the G^m6ATC while CpG methylation tends to decrease binding of DpnI to the target sequence.

Finally, Barnes et al. demonstrated their approach could also be used to segregate human DNA with another enzyme, DpnII, which recognizes non-methylated GATC. The restriction enzyme enrichment method may extend broadly to multiple restriction endonucleases, thus allowing for the enrichment of DNA from different pools of organisms. It is feasible that this method may ultimately allow for the detection of many previously undiagnosable conditions. TriLink offers a number of methylated NTPs and oligo modifications. Please see our website for our full list of epigenetics-related products. We also offer FAM and other fluorescent dye-labeled oligonucleotides for easy detection.

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