genomic DNA에서 직접 어떤 cytosine 잔기가 methylation되었는지 결정하기 위해 EpiXplore Methyl Detection Kit를 사용한다. 이 Kit를 이용하면 시료 보존과 bisulfite로 변형된 DNA를 고효율로 얻을 수 있어 고품질의 epigenetic data를 가능하게 한다.


Figure 1. Distinguish methylated DNA via bisulfite modification and sequencing analysis. Methylated Cs (left-hand set of arrows) are protected during bisulfite conversion (Step 1) and transcribed as Gs during PCR (Step 2). Nonmethylated Cs (right-hand set of arrows) are converted to Uracils during bisulfite conversion (Step 1) and transcribed as As during PCR (Step 2). Methylated domains are identified by comparing sequencing data for converted and unconverted DNA.


기존 Bisulfite Conversion Kit과 비교

Methyl detection Kit

EpiXplore 프로토콜

기존의 프로토콜

초기 DNA는 어느 보존되는가?

예. 초기 DNA를 거의 완벽하게 보존합니다.

아니오. 초기 DNA의 84-96% 손실될 수 있습니다.

DNA의 전처리가 필요한가?

아니오. 직접 genomic DNA 적용 가능합니다.

예. 제한효소 전처리가 필요합니다.

소량의 시료 적용은 가능한가?

예. 50 pg 정도의 genomic DNA (8 mammalian cells)도 적용 가능합니다.

아니오. DNA의 손실이 많기 때문에 더 많은 양의 시료가 필요합니다.


Bisulfite Conversion이란?


Applications
Understanding the roles of DNA methylation in embryonic development, gene regulation, chromatin integrity, genomic imprinting, and human diseases including cancer.
Components
· Methylated DNA Enrichment & Detection Kit
   Methyl Detection Kit
   Methylated DNA Enrichment Kit

· Methyl Detection Kit
   Box 1: Conversion Reagents #1 and 2, Binding Buffer,
             Wash Buffer, Elution Buffer, NucleoSpin Tissue XS, Collection Tubes
   Box 2: Control Samples #1 and 2 (Unconverted and Bisulfite-converted DNA respectively),
             Control Primers #3A & #3B (first- and second-round primers for nested PCR)
   Protocol: PT5124-2

· Methylated DNA Enrichment Kit (Cat. No. 631962)
   Box 1: Binding/Wash Buffer, TALON Magnetic Beads, Elution Buffers (Low, Middle & High), Dr. GenTLE Precipitation Carrier
   Box 2: MBD2 Protein, Labeled DNA Duplex Controls (Methylated & Non-Methylated DNA)
   Protocol: PT5034-2