EpiTect Fast DNA Bisulfite Kit (200)，59826，Qiagen，凯杰

EpiTect Fast Bisulfite Conversion Kits

For ultrafast, highly efficient bisulfite conversion from gDNA, FFPE, blood, cell or tissue samples in spin-column and 96-well format

Features

Optimized procedure enables high cytosine conversion rates of over 99%

Efficient bisulfite conversion in as little as 30 minutes

Dedicated solutions for gDNA, whole blood, cells, tissue and FFPE slices

Fast, streamlined procedure for efficient conversion and purification of DNA, also in 96-well plates

Unique DNA Protect technology prevents DNA degradation

EpiTect Fast Bisulfite Conversion Kits are a coordinated system of kits that enable highly efficient conversion of unmethylated cytosines to uracils in as little as 30 minutes while offering outstanding flexibility in choice of starting material. Streamlined procedures for sample lysis and direct bisulfite conversion of DNA from whole blood, cultured cells, tissue samples, genomic DNA and formalin-fixed, paraffin-embedded (FFPE) slices make efficient bisulfite conversion fast and effortless. Innovative DNA Protect technology ensures effective DNA denaturation without degradation, resulting in high DNA yield and reproducible results. The spin-column procedures can be automated on the QIAcube Connect. The procedure in 96-well format delivers ultrafast, high-throughput bisulfite conversion of DNA and can be processed using a centrifuge or vacuum manifold.

Highly efficient bisulfite conversion

Bisulfite treatment of DNA and the resulting conversion of unmethylated cytosines to uracils is the most critical step in methylation analysis, since the reaction efficiency will greatly impact the reliability of downstream analyses. EpiTect Fast Bisulfite Conversion Kits contain innovative Bisulfite Solution, which enables ultrafast, highly efficient bisulfite conversion, even on difficult-to-analyze samples such as X-chromosome genomic DNA derived from a female sample, where one of the 2 X-chromosomes is highly methylated (see figure  "Highly efficient cytosine conversion of X chromosome DNA"). Quantification of individual cytosine conversion by Pyrosequencing shows that significantly better conversion is achieved with the EpiTect Fast Bisulfite Conversion Kit than a kit from another supplier, particularly when the amount of starting material is limited (see figure  "Superior bisulfite conversion").

Reduced DNA fragmentation

The harsh conditions necessary for complete bisulfite conversion usually lead to a high degree of DNA fragmentation and sample loss, leading to low DNA yield, highly fragmented DNA and irreproducible conversion rates. QIAGEN's unique DNA Protect Buffer prevents DNA degradation, resulting in fully-converted DNA of much larger fragment sizes than DNA converted with other suppliers' kits (see figure  "Analysis of DNA fragment sizes after bisulfite conversion").optimized RT mix for efficient and sensitive reverse transcription over a wide range of RNA template amounts. For convenience, the master mix can be stored at 2–8°C.

Highly efficient cytosine conversion of X-chromosome DNA.

K562 genomic DNA was bisulfite converted using the EpiTect Fast DNA Bisulfite Kit, and a 100 bp amplicon from the X-chromosome was amplified using cytosine-free primers to allow amplification of both unconverted and bisulfite converted DNA. PCR products were cloned into a vector and Sanger sequencing was done to identify converted cytosines. 1252 individual cytosines were analyzed, revealing a highly efficient cytosine conversion rate of 99.8%. This high conversion rate is especially significant as the genomic DNA was derived from a female; therefore, one of the 2 X-chromosomes is highly methylated.

Superior bisulfite conversion.

Following bisulfite conversion of genomic DNA using the EpiTect Fast DNA Bisulfite Kit or a kit from Supplier Z, unconverted genomic DNA and bisulfite converted DNA were coamplified and then sequenced by Pyrosequencing to quantify the rate of conversion of individual cytosines. [A] The EpiTect Fast DNA Bisulfite Kit gave comparable results to a kit from Supplier Z when 2 µg of DNA was analyzed. [B] However, when a limited amount of DNA was analyzed (10 ng), the 4 cytosine residues shown in this Pyrogram demonstrate that the EpiTect Fast DNA Bisulfite Kit delivered significantly better results than the kit from Supplier Z. (Percentage of each cytosine residue that did not undergo bisulfite conversion using the kit from Supplier Z vs. the EpiTect Fast DNA Bisulfite Kit: 12% vs. 2%, 5% vs. 1%, 37% vs. 9% and 11% vs. 2%, respectively.)

Analysis of DNA fragment sizes after bisulfite conversion.

Following bisulfite conversion with the EpiTect Fast DNA Bisulfite Kit, 1 µg of genomic DNA showed the lowest level of fragmentation, with a size distribution ranging from several hundred base pairs to over 6 kb, compared to other methods with peaks at 500 bp and 1 kb, respectively. Samples were analyzed on an Agilent RNA 6000 Nano Chip.

EpiTect Fast DNA Bisufite Kit procedure.

Streamlined and straightforward, the EpiTect Fast DNA Bisulfite Kit enables complete bisulfite conversion and cleanup of genomic DNA in approximately 1 hour.

The EpiTect Fast Bisulfite Conversion Kit delivers fast and efficient bisulfite conversion of DNA from various starting material that is highly suited for all techniques currently used for the analysis of DNA methylation, including:

 PCR: end-point, real-time and methylation-specific

Bisulfite sequencing (direct and cloning)

COBRA (Combined Bisulfite Restriction Analysis)

Pyrosequencing