Overview - Bisulfite Conversion Kits Sodium bisulfite conversion of DNA is the fastest, easiest, and most popular way to study DNA methylation analysis and selecting the right bisulfite conversion kit can be the difference between accurate data and frustrating wasted hours spent in the lab. Choosing a bisulfite conversion kit is an important step in DNA methylation studies to ensure that you obtain the highest quality bisulfite-converted DNA possible going into downstream applications. Zymo Research has developed several lines of bisulfite conversion kits to enable DNA methylation analysis in challenging clinical samples and make bisulfite conversion fast and high throughput. These bisulfite conversion kits are compatible with low sample inputs and have been recently endorsed by many single cell sequencing protocols.
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The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. Conversion efficiency ranged from The inappropriate conversion of methylated cytosines to thymines varied between 0. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Dimo Dietrich is co-inventor and owns patents on methylation biomarkers and related technologies. These patents are commercially exploited by Epigenomics AG.
There are no further patents, products in development or marketed products to declare. Introduction DNA methylation of cytosines within the CpG dinucleotide context is an epigenetic mechanism, which plays an important role in biological processes, such as cell differentiation and development . Furthermore, aberrant DNA methylation is a hallmark of malignant tumors and plays a key role during carcinogenesis . Studies on DNA methylation changes in the course of cancer development and progression will broaden the understanding of this devastating disease and will lead to several clinically relevant biomarkers and therapy approaches in the future.
A few DNA methylation biomarkers are already on the road to clinical use for predictive, diagnostic and screening purposes. Methylation of the promoter of the MGMT gene in gliomas allows for the prediction of the response to alkylating agents . The MGMT promoter methylation status has become a parameter for stratification of patients with glioma within several clinical trials .
Macrodissected tumor tissues from sections of FFPE tumors are the sample of choice to achieve good results . Two additional tests based on the methylation analysis in FFPE tissues already show a high level of validation qualifying them for clinical use. DNA methylation of PITX2 in FFPE prostatectomy specimens is a strong prognostic biomarker for identifying patients who are at high risk to suffer from prostate-specific antigen PSA recurrence after radical ectomy  ,  ,  , .
Free-circulating methylated SEPT9 gene copies in plasma as a screening biomarker for colorectal cancer were recently validated in a large observational prospective screening trial including more than 7, asymptomatic subjects .
Furthermore, SHOX2 DNA methylation is a validated biomarker for detecting lung cancer in the cellular fraction of bronchial aspirates  ,  and pleural effusions  ,  as well as in EBUS-TBNA endobronchial ultrasound with transbronchial needle aspiration specimens .
These examples of methylation biomarkers with the highest level of validation clearly indicate the necessity of technologies, which allow for the accurate determination of DNA methylation in various sample types. These sample types each represent their specific technological challenges, i.
The availability of kits and tools to measure DNA methylation in these sample types is mandatory to open this research area to a wide group of researchers. Methylated cytosine exhibits a similar base pairing behaviour as cytosine and therefore methlyated and unmethylated cytosines are difficult to distinguish from each other by conventional hybridization-based molecular biological methods, i.
In , Frommer et al. Consequently, the epigenetic information of the DNA is transformed into sequence information, which can be studied via PCR and other hybridization based methods. Today, direct detection of DNA methylation, without bisulfite conversion, through single-molecule, single molecule real-time SMRT sequencing  is possible. Nevertheless, the specific conversion of cytosines to uracils by means of bisulfite is still state of the art in DNA methylation analyses.
However, several technological advances have now led to protocols which are much more convenient and user friendly compared to the original 16 hours protocol  ,  ,  ,  ,  , .
In the meantime, numerous kits are commercially available allowing methylation analyses even for inexperienced researchers. All kits are designed to enable the efficient bisulfite conversion of extracted high molecular DNA.
However, only a few kits allow for the modification of DNA from challenging input sample material, i. Fixed tissues usually contain only degraded DNA which further suffers from crosslinking. Fixed tissues require an efficient cell lysis in order to release DNA of sufficient quality and quantity for downstream analyses. Body fluids often only contain low concentrations of DNA which needs to be concentrated prior to bisulfite conversion in order to reduce the volume therefore enabling the input into the bisulfite reaction.
Table 1 provides an overview of the analyzed kits and their applicability to different sample types.
EpiTect® Bisulfite Kit
EpiTect Plus DNA Bisulfite Kit
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