The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing.


We have recently shown that the tet family enzymes convert 5-™ 5-hydroxymethylcytosin (5-HMC) to DNA. 5-HMC is present at high levels in embryonic stem cells and Purkinje neurons. The cytosine methylation status is generally evaluated by reaction with sodium bisulfite followed by PCR amplification. The reaction with sodium bisulfite promotes the deliniation of cytosin, while 5-methylcytosin (5-MC) reacts badly with bisulfite and resists deamination. Since the 5-Hmc reacts with bisulfite to produce 5-methylenesulfonate cytosin (CMS), we asked how the DNA containing 5-HMC behaves in bisulfite sequencing.

RESULTS We used synthetic oligonucleotides with different cytosine distributions as DNA generation models containing C, 5-MC and 5-HMC. The resulting DNAs were subjected in parallel to bisulfite treatment, followed by exposure to conditions promoting the disagreement of cytosine. The scope of the conversion of 5-Hmc to CMS was estimated at 99.7%. The sequencing of PCR products has shown that neither 5-™ nor 5-HMC undergo C-T-T transitions after bisulfite treatment, confirming that these two species of modified cytosin are indistinguishable by bisulfite technique. DNA in which CMS was a large fraction of all bases (28/201) was much less amplified that the DNA in which these bases were 5-MC or uracil (the latter produced by cytosine damination). Using a series of primer expansion experiments, we followed the inefficient amplification of CMS-containing DNA with TAQ polymerase spreading on CMS modification sites, especially when two CMS bases were adjacent or separated by 1-2 nucleotides.

We confirmed that the widely used bisulfite sequencing technique does not distinguish between 5-MC and 5-HMC. In addition, we show that CMS, the product of the 5-Hmc bisulfite conversion, tends to stall DNA polymerases during the PCR, suggesting that densely hydroxymethyl DNA regions can be underrepresented in Quantitative analyzes of methylation.

Oligonucleotides with fluorescent dyes at opposite ends are a hardened tempered sensor system for detecting the hybridization of the PCR product and nucleic acid.

The nuclease PCR test 5 ‘detects the accumulation of specific PCR product by hybridization and cleaving a double label fluorogenic probe during the amplification reaction. The probe is an oligonucleotide with a journalist fluorescent dye and an attached messy dye. An increase in the intensity of the journalist fluorescence indicates that the probe is hybridized to the target PCR product and has been cleaved by the 5 ‘-> 3’ nucleoley activity of Taq DNA polymerase. In this study, probes with the disorder dye attached to an internal nucleotide were compared to probes with the disorder dye attached to the 3′-end nucleotide.

In any case, the journalist dye was attached to the 5 ‘end. All intact probes showed a quenching of the journalist fluorescence. In general, the probes with the eternal dye attached to the 3’-extremal nucleotide have presented a larger signal in the dosage of the nuclease PCR 5 ‘than the internal labeled probes. It is proposed that the largest signal is caused by an increased probability of Cleavage by Taq DNA polymerase when the probe is hybridized to a template strand during the PCR. The probes with the eternal dye attached to the 3’-extremal nucleotide also presented an increase in the intensity of the fluorescence of the journalist when hybridized to a complementary strand. Thus, the oligonucleotides with messy journalists and dyes attached to opposite ends can be used as homogeneous hybridization probes.

 The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing.
The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing.

Nucleotide addition modulation not modeled by Taq DNA polymerase: primer of changes that facilitate genotyping.

The taq DNA polymerase can catalyze the non-modeled addition of a nucleotide (mainly adenosine) to the 3 ‘end of products amplified by the PCR. Recently, we have shown that this activity, which is primer-specific, has a potential source of error in genotyping studies based on the use of short tandem rehearsal markers.

In addition, during the revision of our data, we found that the addition of non-modeled nucleotide adjacent to a terminal C 3 ‘is favored and that the addition adjacent to a terminal of 3’ is not. However, it was clear that the characteristics of the model in addition to the terminal 3 ‘also affect the fraction of the adenylated product. To define consensus sequences that promote or inhibit the adenylation of the product, we have transplanted sequences between the 5 ‘ends of the reverse shock absorbers of adenylated markers and those of the markers that are not adenyl. It has been difficult to identify a single sequence capable of protecting products from all markers of an addition of non-modeled nucleotide. On the other hand, place the GTTTCTT sequence on the 5 ‘end of the inverse primers driven in nearly 100% adenylation of the 3′ end of the front strand. This modification or related ones (called “porktail”) should facilitate precise genotyping and effective T / A cloning. A limitation of the length of the target to the amplification of the DNA PCR has been identified and treated.

Concompide, the fidelity of the base pair, the ability to use PCR products as primers and that the maximum target fragment yield has been increased. These improvements were obtained by the combination of a high level of a non-terminal removal mutant without exonuclease of the TAQ DNA polymerase, Klentaq1, with a very low level of thermostable polymerase DNA having an activity of 3’-exonuclease. (PFU, vent or deep vent). At least 35 KB can be amplified to high yields of 1 ng of Lambda DNA model.