Recombinant DNA Technology

1. Recombinant DNA Technology

2. replication replication transcription transcription translation translation Gene expression in vivo DNA from different source DNA Recomnbinant DNA Recombination

3. Development of clone technique 1. Restriction Enzymes and DNA Ligase Are Key Tools in Forming Recombinant DNA Molecules Restriction Endonuclease (Scissor) 1962，W·Arber 1968，H·O·Smith 1971，D·Nathans Share The Nobel Prize in 1978 DNA ligase (Glue) ：1966，B·Weiss and C·C·Richardson

4. Paul Berg • Development of clone technique 2. Naissance of DNA recombination: (the first stage) Stanley N . Cohen Herbert Boyer 1972, Paul Berg

5. W. Gilbert Humulin • Development of clone technique 3. Gene clone flourished (second stage): Transgenic plants Transgenic animals 1977，W. Gilbert 1982，Humulin 1985，transgenic plants and animals 1990，gene therapy

6. Dolly and Ian Wilmut • Development of clone technique 4. Emergence of organism clone: (the third stage) 1996, clone sheep Dolly was born

7. Nucleus transferring

8. Clone: Population of identical cells or DNA molecules or organism descended from a single progenitor. Cell clone, DNA clone and organism clone

9. The technique of DNA recombination is also called: Gene cloning， DNA cloning， Molecular cloning The Purposes: To acquire a lot of identical DNA molecules To obtain the protein products of an interest gene

10. DNA Cloning Restriction enzymes: Vector Target gene * Separating * Cutting * Transformation →Screening →Amplification →Expression * Ligation

11. Section. 1 Tools for gene cloning Materials: Gene or DNA fragment Vector Restriction enzyme Scissor: Glue: DNA ligase Host cells: Prokaryotesor eukaryotes

12. Some Tool Enzymes used in Recombinant DNA Technology Restriction endonucleases, DNA ligase, DNA poly-I (Klenow fragment), Reverse transcriptase… Restriction Enzymes and DNA Ligase Are Key Tools in Forming Recombinant DNA Molecules

13. 1. Restriction Endonucleases • Recognize, bind to and hydrolyze specific nucleic acid sequence in double-stranded DNA • Isolated from bacteria • 1800 kinds of restriction endonuclease type I, II and III

14. * They are from bacteria and named after the bacterium from which they are isolated. (italic) eg. EcoR I The first letter of the genus the first two letters of the species The first letter of the strain The order of discovery

15. * The endonucleases recognize and cleave a specific double-stranded DNA sequence that is 4-6bp long of palindromic sequence (or palindrome) • Palindrome---The same sequences in the 5' ---> 3' direction of both strands.

16. * The DNA cuts produced by restriction enzymes have two types of ends depending on the cut position. SamI Blunt end Sticky end

17. Two types of sticky ends 5' 3' • 5' sticky end 3' 3' • 3' sticky end 5' 5' 3' 5'

18. -G AATTC- 3' 5' • 5' sticky end 3' -CTTAA G- 5' 5' -CTGCA G- 3' • 3' sticky end 3' -G ACGTC- 5' 5' -GTT AAC- 3' • blunt end 3' -CAA TTG- 5' Three types of DNA cutting ends • EcoRI • PstI • HpaI

19. Sticky ends are particular useful in constructing DNA recombinant .

20. nick nick Ligation

21. 2. DNA Ligase: Catalyze the complementary sticky ends or blunt ends to be joined covalently by 3’, 5’phosphodiester bond DNA ligase 2ATP Blunt-end ligation Sticky-end ligation

23. Target DNA Vector DNA (plasmid) EcoRI EcoRI

24. Section. 1 Tools for gene cloning Materials: Gene or DNA fragment Vector Restriction enzyme Scissor: Glue: DNA ligase Host cells: Prokaryotesor eukaryotes

25. Vector ---- the vehicle DNA Vectorisa DNA molecule capable of self-replication in a host organism and into which a piece of DNA can be inserted. Cloning vector---- is used for reproducing the DNA fragment. Expressionvector---- is used for expressing certain gene in the DNA fragment (plasmid, phage DNA, virus DNA…)

26. Plasmid • Small circular dsDNA outside chromosomal DNA in bacteria • Can be automaticly replicated in bacteria • Carry some genes that endow genetic features for host cells: antibiotic resistance, to allow selection by growth on medium containing antibiotics • Contain several restriction endonuclease sites

27. Insert DNA BamHI Marks for selection pBR322 Self-replication Cloning Vector - pBR322

29. Multiple cloning site (MCS)

30. Expressing vector

31. ＇

32. Review Lac operon *Structural genes: gene Z,Y,A encoding three Es *Promoter: RNA pol binding site *Operator: repressor binding site regulatory region *CAP site: CAP binding site *Regulatory gene I: encoding lac repressor protein

33. Review The lac operon is subject to negative regulation, and induced by allolactose (isopropylthiogalactoside, IPTG) RNA-pol RNA-pol

34. Review Some other β-galactosides such as isopropylthiogalactoside (IPTG) are potent inducers of β-galactosidase expression. IPTG is useful in the laboratory as a tool for inducing gene expression.

35. Vectors must: (for both cloning vector and expression vector) 1.Be safe 2. Contain origin of replication 3.Have selectable marker: antibiotic resistance 4.Multiple cloning sites (restriction enzyme sites): cutting/linking of DNA fragments (only for expression vector) 5. Promoter:  recognized by RNA polymerase 6. Transcription termination sequences 7. RBS for binding ribosome

36. Section 2 Strategy of Recombinant DNA Technology

37. separation cutting ligation transformation selection Amplification and expression

38. 1. Separation Acquirement of the target gene: (1) Separated directly from chromosomal DNA, easily in prokaryotes. digestion with restriction enzyme separated by electrophoresis _ + markers samples

39. 1. Separation (2) Artificial synthesis (by DNA synthesizer): Simple and short sequence: 50bp-12kb

40. (3) Amplified by polymerase chain reaction(PCR) method Semiconservative replication in vivo PCR: Semiconservative replication in vitro PCR is a technique for amplifying a specific DNA segment in vitro by multiple cycles of DNA synthesis.

41. Semiconservative Replication in vivo Principle of PCR:mimic DNA replicationin vitro Denaturation: Unwind double- stranded DNA Annealing: primer binding Extension: DNA strand extending

42. PCR reaction system • Template DNA: genomic DNA, cDNA or plasmid DNA • Primers: a pair of short oligonucleotides, which can hybridize to the 3’-end of template (specific) • Polymerase:thermostable DNA polymerase, e.g. Taq • Substrates: dNTPs (dATP, dCTP, dGTP, dTTP) • present in equal concentrations • Reaction buffer: maintain the pH value of the reaction • mixture and supply Mg2+

43. hot spring (Opalescent Pool in Yellowstone National Park, Wyoming USA.  Conditions for life in this environment are similar to earth over 2 billion years ago. In these types of hot springs, the orange, yellow and brown colors are due to pigmented photosynthetic bacteria )  Thermus aquaticus (early 1970s) (It has heat-stable enzyme systems including the Taq polymerase)

44. dNTPs • buffer containing Mg2+ primer DNA polymerase DNA template primer Amplified DNA fragment

45. Thermal Cyclers PCR Process Double strand DNA melted Denaturation The primers hybridize with DNA template Repeat 25 to 30 cycles Taq polymerase synthesize DNA strand Extension Annealing

49. Number of double strands21 22 23 24