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Produced by Huh Jae-won. 이전 연구. Materials and Methods. Genomic DNA preparation (Phenol 법과 proteinase K 이용 ) HpaⅡor HhaⅠ 를 이용한 Enzyme cut PCR 을 통한 enzyme 반응 확인 EthoH precipitation Adaptor ligation Purification (Qiaquick) 3’end 를 채움 (Klenow fragment of DNA polymerase Ⅰ)

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Materials and Methods

  • Genomic DNA preparation

  • (Phenol법과 proteinase K이용)

  • HpaⅡor HhaⅠ를 이용한 Enzyme cut

  • PCR을 통한 enzyme 반응 확인

  • EthoH precipitation

  • Adaptor ligation

  • Purification (Qiaquick)

  • 3’end를 채움 (Klenow fragment of DNA polymerase Ⅰ)

  • Purification (Qiaquick)


[Klenow fragment of DNA polymerase Ⅰ]

1. E coli의 DNA polymerase Ⅰ에서 기원

Klenow fragment의 활용

1. Synthesis of double-stranded DNA from single-stranded templates


2. Filling in recessed 3' ends of DNA fragments

3. Digesting away protruding 3' overhangs

4. Preparation of radioactive DNA probes

일반 nucleotides 대신에 radioactive된걸 사용하면 된다.


Hypomethylation

Hypermethylation


: negative control

Duplicated

Cerebellum (A, B) and lymph node (C, D)


PCR

1-3:LTR28, 4-6:LTR30, 7-9:LTR14 : control



Cis acting element로 부터 메틸레이션이 확장되어 감

계속확장되어 감 (Open chromatine이나타날때 까지)

발혀되는 프로모터에 의해 바운드리가 정해짐

발현되는 프로모터와 그에 관련된 다양한 complex에 의해 바운드리는 이동가능함


Silencing 기작: Methylation의 변화기작


Conclusions

1. 다양한 방법으로 메틸레이션 상태를 확인해본 결과 거의 같은 결론을 보여줌으로써 ‘methylation spread’의 가설을 실험적으로 확인함

2. 그러므로 Enzyme을 이용한 본 연구 기법(methylation tag)을 통한 large scale의 방법도 또한 매우 효과적이라고 할 수 있음


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