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Hydroxymethyl Collector™ Data on 5-hmC Enrichment

 

Hydroxymethyl Collector Range of Detection

Hydroxymethyl Collector is designed for locus-specific anlaysis of 5-hmC by real time PCR. We recommend using 5 ng - 2.5 µg fragmented genomic DNA per enrichment reaction.

Hydroxymethyl Collector–Seq is deisgned for genome-wide analysis of 5-hmC with Next-Generation sequencing. We recommend using 1 µg - 10 µg fragmented genomic DNA per enrichment reaction, with 5 µg as the suggested starting quantity. Samples containing very low levels of 5-hmC may require larger quantities of DNA. Suggested input amounts include 8-10 µg from stem cells, 20 µg from cancer tissues, 1-3 µg from brain and 5-10 µg from normal tissue.

 

To determine the range of detection for Hydroxymethyl Collector–Seq, various quantities of mouse and human tissues were assayed in the Hydroxymethyl Collector–Seq Kit. The enriched 5-hydroxymethylcytosine DNA fragments were analyzed by real time PCR using species-specific and tissue-specific PCR primer sets. The amount of enriched 5-hmC DNA was plotted as fold enrichment. From this analysis it was determined that Hydroxymethyl Collector–Seq is sensitive over the range of 1 - 10 µg fragmented genomic DNA.

Sensitivity of the Hydroxymethyl Collector–Seq kit as determined from the enrichment of 5-hmC from human and mouse genomic DNA
 
Figure 1: Sensitivity of the Hydroxymethyl Collector–Seq Kit for enrichment of 5-hmC DNA.

Human and Mouse genomic DNA were assayed in the Hydroxymethyl Collector–Seq Kit at various quantities. Enriched DNA was analyzed by qPCR using species-specific and tissue-specific PCR primers. The enriched DNA was plotted as fold enrichment.

 

To determine the range of detection for Hydroxymethyl Collector, various quantities of mouse brain DNA were assayed in the Hydroxymethyl Collector Kit. The enriched 5-hydroxymethylcytosine DNA fragments were analyzed by real time PCR across multiple loci. The amount of enriched 5-hmC DNA was plotted as a percentage of the starting material. From this analysis it was determined that Hydroxymethyl Collector is sensitive over the range of 5 ng - 2.5 µg fragmented genomic DNA. For downstream analysis requiring larger inputs, e.g. microarray, it may be necessary to pool multiple enrichment reactions or perform whole-genome amplification following 5-hmC DNA enrichment.

Sensitivity of the Hydroxymethyl Collector kit as determined from the enrichment of 5-hmC from genomic mouse brain DNA
 
Figure 2: Sensitivity of the Hydroxymethyl Collector Kit for enrichment of 5-hmC DNA.

Mouse brain DNA was assayed in the Hydroxymethyl Collector Kit at various quantities. Enriched DNA was analyzed by qPCR across multiple loci. The enriched DNA was plotted as a percentage of starting material. This graph shows that Hydroxymethyl Collector has a preferred range of detection from 5 ng to 2.5 µg. Larger DNA quantities are not efficiently captured due to the balance of available enzyme and reagents for efficient glucosylation and biotinylation reactions. The Control 2 primer set serves as a negative control in this experiment.

 

Enrichment Efficiency of Hydroxymethyl Collector using the 5-hmC Control DNA

The Hydroxymethyl Collector Kit includes a 5-hmC positive control DNA, MseI digested human genomic DNA and PCR primers that can be used to determine the efficiency of the enrichment reactions. The 5-hmC Control DNA consists of a 338 base pair DNA fragment derived from the APC gene. The 5-hmC control DNA has been modified so that 25% of the cytosine residues contain 5-hydroxymethylcytosine. When the positive control fragment is spiked into human genomic DNA and enriched, the included APC PCR primers can be used to confirm the enrichment efficiency.

Hydroxymethyl Collector qPCR results using human genomic DNA and the included 5-hmC control DNA
 
Figure 3: Enrichment of 5-hmC Control DNA included in the Hydroxymethyl Collector Kit.

MseI digested human genomic DNA (500 ng) was spiked with 25 pg of the included 5-hmC Control DNA in duplicate. These samples were then processed using the Hydroxymethyl Collector Kit to enrich for 5-hydroxymethylcytosine residues in either the presence (5-hmC) or absence (No glucose) of the UDP-Azide-Glucose donor. Eluted DNA was purified and tested using real time PCR with the included APC PCR primer mix to screen for the enrichment of the 5-hmC Control DNA. The DNA was quantified against the Input standard curve and plotted as a percentage of the starting material. This graph shows that Hydroxymethyl Collector is able to enrich for 5-hmC DNA and that the enrichment only occurs in the presence of the UDP-Azide-Glucose donor. These results are provided for demonstration only.

 

Applications of Hydroxymethyl Collector Enrichment

Hydroxymethyl Collector–Seq was performed on 5 µg each of mouse brain, mouse kidney and mouse liver genomic DNA and submitted for Nex-Gen sequencing to identify the localization of 5-hmC within each sample.

Genome-wide analysis of 5-hmC in Mouse Brain, Kidney and Liver using Hydroxymethyl Collector–Seq
 
Figure 4: Genome-wide localization of 5-hmC in mouse tissue using Hydroxymethyl Collector–Seq.

Genomic DNA was isolated form various mouse tissues. 5 µg of DNA was used in Hydroxymethyl Collector–Seq and analyzed by Next-Generation sequencing. The sequencing peaks show the localization of 5-hmC within each sample.

 

Human brain DNA was used in the Hydroxymethyl Collector Kit to enrich for 5-hydroxymethylcytosine DNA fragments. Enriched DNA was amplified by whole-genome amplification and hybridized to a Human tiling array in an effort to perform genome-wide 5-hydroxymethylcytosine DNA methylation analysis.

Human brain DNA as analyzed by microarray following enrichment by Hydroxymethyl Collector
 
Figure 5: Human tiling array using DNA enriched with Hydroxymethyl Collector.

Human brain DNA that was enriched using the Hydroxymethyl Collector Kit was amplified by whole-genome amplification and hybridized to Affymetrix Human Tiling 2.0R Array A containing chromosomes 1 and 6. This image shows a 1.2 million base pair view of chromosome 6 where there is a clear enrichment of 5-hmC across the entire length of the JARID2 gene.