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Ch. 4 Molecular cloning Methods

Ch. 4 Molecular cloning Methods. Restriction Endonucleases. Methylation- resistance to RE. DNA modification. DNA ligase. by Cohen & Boyer, 1973. Cloning of recombinant DNA assembled in vitro. Plasmids or Virus - Replication origin - Selection marker - Restriction enzyme site (MCS).

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Ch. 4 Molecular cloning Methods

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  1. Ch. 4 Molecular cloning Methods

  2. Restriction Endonucleases

  3. Methylation- resistance to RE DNA modification

  4. DNA ligase

  5. by Cohen & Boyer, 1973 Cloning of recombinant DNA assembled in vitro

  6. Plasmids or Virus • - Replication origin • - Selection marker • - Restriction enzyme site (MCS) pBR322 plasmid Vector

  7. Cloning of foreign DNA

  8. Screening bacteria by Replica plating

  9. pUC plasmid

  10. Alkaline phosphatase

  11. Phage vector

  12. Plaque hybridization

  13. M13 phage & ssDNA

  14. Phagemid : Phage + plasmid pBluescript vector

  15. Polymerase Chain Reaction

  16. cDNA library

  17. Nick translation (DNA polymerase I)

  18. RT-PCR & cDNA cloning

  19. RACE;rapid amplification of cDNA end)

  20. Recombinant (fusion) protein

  21. Tagging

  22. Synthesis of fusion protein in lgt11 & screening using Ab

  23. Gene expression using vaculovirus

  24. Crown gall tumor

  25. Transgenic Organism

  26. Transgenic plant

  27. Transgenic mouse

  28. DNA 재조합 기술의 응용 • 예방의학: 진단 • 법의학 • 치료제의 대량생산 • 인간 게놈 프로젝트 (HGP) • 줄기(幹; stem)세포 연구 • 유전자 변형 생물체(GMO)

  29. 의학 진단 1 • 겸형 적혈구 빈혈증 • 핵산 잡종화의 응용

  30. 의학 진단 2 • HIV의 경우 • PCR의 응용

  31. 의학 진단 3 W. H. Freeman and Company • 양수검사 (aminocentesis)

  32. 법의학 (forensic science) • VNTR변종을 응용

  33. 법의학 (forensic science)

  34. 치료제의 대량생산 인슐린 인터페론 Wm. C. Brown Communications, Inc.

  35. 치료제의 대량생산 헤모글로빈 인간성장호르몬 Wm. C. Brown Communications, Inc.

  36. 치료제의 대량생산 • Milkof transgenic goat • Mammary gland에서만 발현되는 조절성 염기서열과 프로모터 사용 Wm. C. Brown Communications, Inc.

  37. DNA 기술과 사회적 책임 • 윤리적 문제 • 법률적 문제 • 기타 사회적 파급효과 DNA재조합 기술의 안전성 및 파급효과 • Asilomar Meeting (1975); 유전자 재조합실험규제를 위한 가이드라인 • http://bric.postech.ac.kr/webzine/content/review/bioethics/2005/v7n7/bei0401.pdf 간(幹; stem)세포 및 배아연구 • 생명의 정의 • 찬성 및 반대의견 • 새로운 입양 방식

  38. Genetically Modified Organisms(GMO) • http://www.relfe.com/GMOs.html GMO Crop = Frankenfood ?

  39. Human Genome Project “The Book of Life” • International Human Genome Sequencing Consortium (2000) • Celera • 정보의 공유 vs. 이윤추구 • Draft Human Genome • 1.5% 암호화 서열 • >50% “junk DNA” (반복 염기서열)

  40. 아이슬란드 게놈 지도 제작 프로젝트 • 개인염기서열, 가계도, 질병의 연관성 • 찬성 및 반대의견 • 개인신상정보 유출의 위험성 • http://sunsite.berkeley.edu/biotech/iceland/

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