The Qiagen Epitect Bisulfite kit converts DNA in one step which is followed by a clean up step. Following the bisulfite conversion, I ran a PCR using the. U can try EZ Direct Methylation kit from Zymo research. For formalin fixed tissues, u need to increase the digestion time as well as volume of Proteinase K. Both. during purification. QIAGEN’s EpiTect® Fast Bisulfite kits prevent DNA fragmentation during bisulfite conversion thanks to the unique DNA. Protect Buffer, which.
|Published (Last):||1 December 2006|
|PDF File Size:||17.91 Mb|
|ePub File Size:||19.91 Mb|
|Price:||Free* [*Free Regsitration Required]|
Accordingly, a bias toward the amplification of completely converted DNA and therefore an overestimation of the conversion efficiency is avoided. The kits led to an inappropriate conversion between 0. However, several technological advances have now led to protocols which are much more bidulfite and user friendly compared to the original 16 hours protocol .
Phys Chem Chem Phys. DNA was bisulfite converted using nine different commercially available kits.
These sample types each represent their specific technological challenges, i. The appropriate lysis is essential with regard to performance in downstream molecular applications . Bioorg Med Chem Lett. Curr Protoc Nucleic Acid Chem. Increasing the duration of proteinase K treatment up to 48 hours and supplementing the reaction with additional proteinase K during the lysis will help to increase the yield of highly integer DNA  epitsct, .
Furthermore, the occurrence of bisulfite conversion errors  is an important parameter and should be considered carefully. Table 1 provides an overview of the analyzed kits and their applicability to different sample types.
This DNA might further suffer from degradation due to storage. 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, the introduction of an incubation step at high temperature after proteinase K treatment might help to remove remaining crosslinks based on the antigen retrieval principal and therefore lead to higher quality DNA .
The low abundance of tumor DNA in blood, especially in blood from patients with early stage tumors where screening is particularly valuable, necessitates the usage of high volumes of plasma or serum in order to increase the likelihood of presence of methylated tumor DNA and therefore reach sufficient sensitivity of the test.
Accordingly, slightly different results can be expected when using a different test system, i. Inappropriate Conversion of Methylated Cytosines to Thymines.
Basic principle of the CFP clone sequencing assay for quantifying the bisulfite conversion efficiency of the bisulfite kits. Published online Apr 3. Handling and user friendliness of the different kits differ significantly.
Received Jan 17; Accepted Mar 8. Conversion efficiency ranged from Nucleic Acids Symp Ser Oxf. Each kit was tested in 9 replicates. The bisulfite conversion is a chemical reaction under harsh chemical conditions low pH, high temperature and elongated incubation times causing significant DNA degradation .
All clinical specimens and samples were obtained from the University Kt of Bonn, Germany.
EpiTect Bisulfite Kit (48)
The conversion efficiency of each kit was tested by means of clone sequencing of a PCR product generated using primers targeting cytosine-free priming CFP sites Figure 7 A.
Correlation between conversion efficiency and inappropriate conversion was epittect using Pearson correlation. After HSO 3 — induced hydrolytic deamination of the intermediate bisulfite is eliminated at alkaline pH . All nine kits studied in this series of experiments showed significantly different but comparable results and high performance when applying high molecular weight HMW DNA.
At kig same time, the kits resulted in only moderate inappropriate conversion between 0.
EpiTect 96 Bisulfite Kit (2) from QIAGEN | SelectScience
A high stability of bisulfite-converted Kih is required if studies bisulfits to be conducted over a period of time. 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.
Chr2,— were amplified bp-fragment forward primer: Fixed tissues require an efficient cell lysis in order to release DNA of sufficient quality and quantity for downstream analyses.
Another critical parameter is the stability of bisulfite solutions.