1. Chapter 14 Genetic Recombination and Genetic Engineering The biochemistry and molecular biology department of CMU

2. Section 1DNARecombination

3. DNA recombination • Homologous Recombination • Conjugation • Transformation • Transduction • Site-specific Recombination • Transposition

4. §1.1homologousRecombination • Homologous recombination occurs between identical or nearly identical sequences. It is also called general recombination.

5. 5´ 3´ 3´ 5´ 3´ 5´ endonuclease (recBCD) 3´ 5´ 5´ 3´ 3´ 5´ 3´ 5´ 5´ 3´ 3´ 5´ DNA invading (recA) 5´ 3´ 5´ 3´ Branch migration (recA) 3´ 5´ 3´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 5´ 3´ endonuclease (recBCD) 5´ 3´ DNA ligase 5´ 3´ 3´ 5´ 3´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 5´ 3´ Holiday intermediate

6. 3´ 5´ 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ 5´ 3´ 3´ 3´ 5´ 5´ Holliday intermediate

7. endonuclease (ruvC) endonuclease (ruvC) 3´ 5´ 3´ 5´ 5´ 3´ 5´ 3´ 3´ 3´ 5´ 5´ 5´ 5´ 3´ DNA ligase 3´ DNA ligase 3´ 5´ 3´ 5´ 5´ 5´ 3´ 3´ 3´ 3´ 5´ 5´ 5´ 5´ 3´ 3´ 3´ 5´ 5´ 3´ 3´ 5´ 5´ 3´ splice recombinant patch recombinant

8. §1.2 Conjugation • Bacterial Conjugation has been defined as the transmission of genetic information from a donor bacterium to a recipient cell through cell-to-cell contact.

9. Conjugation process

10. Conjugation process

11. Conjugation process

12. §1.3 Transformation • Introduction of an exogenous DNA into a cell, causing the cell to acquire a new phenotype.

13. Transformation

14. Transformation experiment of Streptococcus pneumoniae

15. §1.4 Transduction • Transduction is the transfer of DNA fragments from one bacterium to another bacterium by a bacteriophage.

16. Transduction

19. §1.5 Site-specific Recombination • Site-specific recombination occurs at a specific DNA sequence.  • The first example was found in the integration between lDNA and E. coli DNA. 

20. λDNA integration

21. Phase variation of Salmonella typhimurium flagella hix hix P2 rH1 H2 flagellin Hin repressor rH1 P2 H segment H1 flagellin

24. Recombination signal sequence (RSS) CACAGTG (12/23) ACAAAAACC GTGTCAC TGTTTTTGG RSS Recombination activating gene enzyme (RAG1 and RAG2)

26. §1.6 Transposition • Transposition is the movement of specific pieces of DNA in the genome. • Transposition resembles site-specific recombination being catalyzed by special enzymes.

27. Transposase gene IS Transposition insertion sequences (IS) including: inverted repeats (IR)：9~41bp transposase gene repeated sequences：4~12bp

28. types of IS transposition • duplicative transposition • Conservative transposition

29. duplicative transposition

30. Conservative transposition

31. transposon • Insertion sequence + another gene (usually antibiotic gene) Transposase gene tet-R gene

32. Transposons Transposition

34. Section 2Recombinant DNA Technology

35. §2.1 Correlative concepts Clone A clone is defined as a number of identical copy (molecules, cells or individuals) all derived from a common ancestor. Also named asexual multiplication.

36. DNA Cloning DNA cloning involves separating a specific gene or segment of DNA from its larger chromosome and attaching it to a small molecule of carrier DNA, then replicating this modified DNA thousands or even millions of times.

38. Recombinant DNA technology • By artificial means, when a gene of one species is transferred to another living organism, it is called recombinant DNA technology. In common parlance, this is known as genetic engineering.

39. Applications in enzymology • restriction endonucleases • DNA polymeraseⅠ • reverse transcriptase • DNA ligase • Alkaline phosphatase • terminal transferase • Taq DNA polymerase

40. GGATCC CCTAGG Restriction endonuclease It can recognize special sequences and cleave DNA at these specific base sequences. Type II can recognize palindrome sequences.

41. Palindrome • Palindrome is also called inverted repeat sequence, which means the nucleotide sequence in 5′to 3′direction is the same in both strands.

42. Sticky end and Blunt end sticky ends EcoRⅠ 5’…GAATTC…3’ 5’…G AATTC…3’ 3’…CTTAAG…5’ 3’…CTTAA G…5’ PstⅠ 5’…CTGCAG…3’ 5’…CTGCA G…3’ 3’…GACGTC…5’ 3’…G ACGTC…5’ blunt ends Hae Ⅲ 5’…GGCC…3’ 5’…GG CC…3’ 3’…CCGG…5’ 3’…CC GG…5’

44. Vector • The term “vector” here refers to some DNA molecules that can carry a DNA fragment into a host cell for replication. • Including: plasmids, Bacteriophages DNA,virus DNA ……

45. Vectors used in molecular cloning Vector Insert (and host) Characteristics size range Plasmid Small circular DNA <5 - 10 kb (bacteria, yeast) Bacteriophage λ Linear viral DNA up to ~20 kb (bacteria) Cosmid Hybrid of plasmid up to ~50 kb (bacteria) and phage Yeast artificial DNA containing yeast ~200 to chromosome (YAC) centromere, telomeres, ~1000 kb (yeast) and origins of replication

46. Plasmids are small, circular molecules of DNA that exist outside the main bacterial chromosome and carry their own genes for specialized functions. plasmid

47. Plasmid

48. 4363bp ori

49. Phage •  phage DNA: gt phages: Insertion type vector EMBL phages: replacement type vector • M13 phage: M13mp and pUC

50. EMBL phages