1. Genetic Recombination and Genetic Engineering
Chapter 14
Genetic Recombination and Genetic Engineering

2. Section 1 DNA Recombination

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

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

5. DNA endonuclease DNA invading (recA) endonuclease ligase5´ 3´ 5´ 3´
endonuclease
(recBCD)
DNA invading
(recA) 5´ 3´
Branch migration
(recA) 3´ 5´
endonuclease
(recBCD) 5´ 3´
DNA
ligase 5´ 3´
Holiday intermediate

6. Holliday intermediate5´ 3´ 5´ 3´

7. DNA ligase DNA ligase splice recombinant patch recombinant3´ 5´
endonuclease
(ruvC) 5´
endonuclease
(ruvC) 3´ 3´ 5´ 5´ 3´ 5´ 5´ 3´ 3´
DNA ligase
DNA ligase 5´ 3´ 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 l DNA and E. coli DNA. 

20. λDNA integration

21. Phase variation of Salmonella typhimurium flagella
hix
hix P2
rH1
H2 flagellin
Hin
repressor P2
rH1
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. IS Transposition insertion sequences (IS) including:
inverted repeats (IR)：9~41bp
transposase gene
repeated sequences：4~12bp
Transposase gene

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

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

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

51. §2 Recombinant DNA Technology

52. Process of cloning Isolation of target gene
Selection and construction of vectors
Ligation of target DNA and vector
Transformation of target gene into receptor cell
Screening for recombinant plasmids
Expressing a cloned gene　

53. Process of DNA cloning

54. §2.1 Isolation of target gene
1. Chemical synthesis
only for simple polypeptide chain whose primary structure is clear.
2. Obtaining from genomic DNA library
3. Obtaining from cDNA library
4. polymerase chain reaction (PCR)

55. The genomic DNA library is a collection of the comprehensive DNA fragments representing the entire genome of a species.

56. recombinate DNA in E.coli
mRNA
The cDNA library represents the population of mRNAs, it only contains the exons of protein’s structural genes.
Reverse transcripase
cDNA
replication
dscDNA
vector
recombinate DNA
E. coli
recombinate DNA in E.coli

57. Preparation of cDNA library

58. Polymerase Chain Reaction
The polymerase chain reaction (PCR) is a rapid and versatile in vitro method for amplifying DNA.

59. PCR reaction system DNA template A pair of primers
DNA polymerase (Taq)
dNTPs
Mg2+-containing buffer

60. Procedures of PCR
Denaturing: the template DNA is denatured to become ssDNA from dsDNA by heating.
Annealing: this step allows the hybridization of the primers with target DNA.
Extension: this process is the DNA synthesis step.

61. ing

62. The first three cycles of PCR

63. §2.2 Selection and construction of vectors
A few commonly used vectors：
plasmid
phage
cosmid
yeast artificial chromosome (YAC)

64. §2.3 Ligation of target DNA and vectors
1. Ligation of sticky end

65. 3. The addition of a homopolymer tail
2. Ligation of blunt ends
3. The addition of a homopolymer tail

66. 4. Artificial linker
Adding a sequence of DNA fragment, which contains the cleavage site for restriction endonuclease.

67. Artificial linker

68. §2.4 Introduction of recombinant DNA into recipient cell
transformation
transfection
infection

69. Recipient cells Safe host bacteria
Endonuclease and recombinase deficiency
Competent cells.

70. §2.5 Screening for recombinant
direct selection
Screen of antibiotic resistance markers
Marker rescue (Insertion inactivation)
In situ hybridization and
autoradiography

71. Antibiotic resistance genes

72. The procedure to form recombinant DNA
direct selection

73. Screen of antibiotic resistance markers

74. Marker rescue

75. In situ hybridization and autoradiography

76. §2.6 Expression of the cloned gene
An expression vector is similar to cloning vectors, but with a major difference: the expression vector must contain a promoter so that proteins can be expressed.

77. Expression vector

78. Gene expression include:
eukaryotic expression
prokaryotic expression