DNA,RNA, Recombinant DNA Technology

Сентябрь 1, 2022

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DNA,RNA, Recombinant DNA Technology

Содержание

2. “Metabolic pathways” expanded
3. Model organisms: Cellular biology, biochemistry... molecular biology
5. Developmental biology......
6. Fly mutation “eyeless” ‘The fly and you are not much different.”
7. Jaenisch, R.Nat. Genet. 2001 27: 327-331
8. The ‘first’ science: “technology drives research drives technology dri...
10. Nucleic Acids – DNA and RNA
12. DNA + RNA DNA + RNA DNA + RNA RNA DNA
13. DNA structure -> sequence
15. Polymerase reaction: 5’-> 3’
16. Page 93 The central dogma
18. Gene expression. Page 93
20. What is a gene?
24. Page 95 Eukaryotes – Intron-Exon concept
28. Recombinant DNA Technology
29. Definitions Recombinant DNA, a DNA construct created by fusing different fragments of DNA Genetic Engineering, the
30. Recombinant DNA Technology Clones -> Cells or organisms with identical DNA
31. Restrictionendonucleases 5’-> 3’ 3’
35. Gel Electrophoresis
36. Gel Electrophoresis
37. Gel Electrophoresis
38. X-Ray structure of a complex of ethidium bromid with DNA. Page 1125
39. Construction of a restriction map. Page 104
40. Restriction map for the 5243-bp circular DNA of SV40. Page 104
43. Construction of a recombinant DNA molecule through the use of synthetic oligonucleotide adaptors Page 109
45. Plasmid Cloning Vectors
46. Plasmid Cloning Vectors
47. Insertional inactivation Gene in cloning site: LacZ -> pUC18 (lacZ complements the host defect in lacZ)
48. http://www.bio.davidson.edu/Courses/genomics/method/reporters.html Blue/White Selection
49. Insertional inactivation Gene in cloning site: Resistance marker -> pBR322 (cloning sites within antibiotica resistence marker)
51. Transformation and Selection
52. Horizontal gene transfer - Transformation -> uptake of naked DNA (chemical transformation, electroporation) - Conjugation ->
53. Electron micrograph of bacteriophage λ. Page 107 Electron micrograph of the filamentous bacteriophage M13. Bacteriophages
54. Bacteriophage T2 injecting its DNA into an E. coli Page 84
55. Life Cycle of Bacteriophage
58. Page 107 Replication of bacteriophage upon infection of a cell
61. Molecular genetics and bacteriophage
63. Cloning of foreign DNA in λ phages. Page 110
64. What is a gene library ?
65. Creation of Libraries
66. Creation of Libraries
67. Sizes of Some DNA Molecules. Page 92
69. Cosmid = Cos - Plasmid
73. Fragmentation of genomic DNA
75. cDNA synthesis
76. DNA Library Clones -> genetically identical
77. Genomic phage library
78. Evaluation of library
79. Evaluation of library
80. Ordered library Microarrays
81. Ordered library “Chromosome Walking” -> also used in “Human Genome Project”
82. Different ways to clone a gene
83. Bacterial host engineering Although most strains of E. coli are harmless, some can cause illness or
84. Genetic and physical maps of the E. coli chromosome Fig. 8.14
85. E.Coli K12 strain has been used for further engineering The K12 strain was first isolated in
86. Additional changes in K12 E.coli for ease of the laboratory practice 1. Bacterial restriction modification systems
87. Additional changes in K12 E.coli for ease of the laboratory practice 2. DNA recombination systems are
88. Transformation of plasmid DNA in competent E. coli cells Competent (here) = able to uptake DNA
89. Transformation of plasmid DNA to competent E. coli cells -- Electroporation and electroporation-competent cells -- Heat
90. Calcium/phosphate (heat shock) method www.bch.msu.edu/bchug/webwww.bch.msu.edu/bchug/web/
92. Скачать презентацию

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“Metabolic pathways” expanded

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Model organisms: Cellular biology, biochemistry...
molecular biology

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Developmental biology......

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Fly mutation “eyeless”
‘The fly and you are not much different.”

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Jaenisch, R.Nat. Genet. 2001 27: 327-331

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The ‘first’ science: “technology drives research drives technology dri...

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Nucleic Acids – DNA and RNA

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DNA + RNA
DNA + RNA
DNA + RNA
RNA
DNA

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DNA structure -> sequence

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Polymerase reaction:
5’-> 3’

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Page 93
The central dogma

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Gene expression.
Page 93

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What is a gene?

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Page 95
Eukaryotes – Intron-Exon concept

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Recombinant DNA Technology

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Definitions
Recombinant DNA, a DNA construct created by fusing different fragments of

DNA
Genetic Engineering, the deliberate alteration of DNA through the creation of recombinant DNA
Genetically Modified Organism, a living entity modified through genetic engineering
Transgenic, a genetically modified organism containing DNA from another source

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Recombinant DNA Technology
Clones -> Cells or organisms with identical DNA

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Restrictionendonucleases
5’-> 3’
3’ <- 5’

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Gel Electrophoresis

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Gel Electrophoresis

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Gel Electrophoresis

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X-Ray structure of a complex of ethidium bromid with DNA.
Page 1125

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Construction of a restriction map.
Page 104

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Restriction map for the 5243-bp circular DNA of SV40.
Page 104

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Construction of a recombinant DNA molecule through the use of synthetic

oligonucleotide adaptors

Page 109

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Plasmid Cloning Vectors

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Plasmid Cloning Vectors

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Insertional inactivation
Gene in cloning site:
LacZ -> pUC18 (lacZ complements the host

defect in lacZ)
-> pUC18 into host organism -> active lacZ (β-galactosidase) from plasmid-> cleavage of X-gal
(blue colonies)
-> gene cloned into polylinker -> lacZ gene disrupted -> no cleavage of X-gal (white colonies)

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http://www.bio.davidson.edu/Courses/genomics/method/reporters.html
Blue/White Selection

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Insertional inactivation
Gene in cloning site:
Resistance marker -> pBR322 (cloning sites within

antibiotica resistence marker)
-> plasmid into host -> resistance against 2 antibiotica
-> gene cloned within one resistance marker -> gene for antibiotica
resistance marker disrupted -> sensitive against one antibioticum

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Transformation and Selection

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Horizontal gene transfer
- Transformation -> uptake of naked DNA (chemical transformation,

electroporation)
- Conjugation -> DNA transfer by cell – cell contact
Transduction -> DNA transfer by bacteriopage infection
Other methods of Gene transfer -> used with fungi, animal and plant cells:
Microinjection
protoplasts

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Electron micrograph of bacteriophage λ.
Page 107
Electron micrograph of the filamentous bacteriophage

M13.

Bacteriophages

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Bacteriophage T2 injecting its DNA into an E. coli
Page 84

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Life Cycle of Bacteriophage

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Page 107
Replication of bacteriophage upon infection of a cell

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Molecular genetics and bacteriophage

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Cloning of foreign DNA in λ phages.
Page 110

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What is a gene library ?

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Creation of Libraries

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Creation of Libraries

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Sizes of Some DNA Molecules.
Page 92

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Cosmid = Cos - Plasmid

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Fragmentation of genomic DNA

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cDNA synthesis

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DNA Library
Clones -> genetically identical

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Genomic phage library

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Evaluation of library

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Evaluation of library

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Ordered library
Microarrays

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Ordered library
“Chromosome Walking”
-> also used in “Human Genome Project”

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Different ways to clone a gene

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Bacterial host engineering
Although most strains of E. coli are harmless, some can

cause illness or even death. The most serious form is E. coli 0157:H7.

E. coli leads to about 73,000 cases of infection
and 61 deaths each year in the United States.

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Genetic and physical maps
of the E. coli chromosome
Fig. 8.14

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E.Coli K12 strain has been used for further engineering
The K12 strain

was first isolated in 1921
from the stool of a malaria patient and
it has been maintained in laboratory stocks
as a pure strain for the last 75 years.

Most strain in molecular biology are recA- endA- hsdR-

Every strain comes with description of its genotype:
DH5alpha (recA-; hsdR-; LacIq; uvrA-; mcrA-……)

Asilomar Conference on Recombinant DNA (February 1975)

NIH Recombinant Advisory Committee (RAC) (1973)

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Additional changes in K12 E.coli for ease of the laboratory practice
1.

Bacterial restriction modification systems have been removed.
(To prevent its interferention with the replication of foreign DNA in bacteria).

hsdR/hsdM/hsdS (EcoK)
restriction system

mcrA/mcrB/mrr complex

Degrades DNA not methylated
at the sequence 5'-AAC-(N)5-GTGC-3'

hsdM recognises unmethylated DNA
hsdM is also involved in methylation of DNA
hsdR encodes an endonulease
hsdS encodes DNA sequence specific protein
hsdR- or hsdS- mutants facilitate propagation of any foreign DNA

E.coli DNA is methylated
by dcm, dam and hsdM

mcrA-/mcrB-
strains are good for cloning
eukaryotic DNA

mcrA/mcrB/mrr cleaves DNA
methylated by other systems

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Additional changes in K12 E.coli for ease of the laboratory practice
2.

DNA recombination systems are modified to prevent rearrangements (RecA-)
(to prevent deletions and rearrangements)
recA is a core recombination protein
recA- strains allow cloning of repetitive sequences
recA-/recB-/recC- are enhanced strains with very low recombination efficiency
uvrC/umuC are involved in DNA repair
uvrC-/umuC- are good for cloning of inverted repeats
3. Endonuclease activity has been mutated (EndA-)
(to increase plasmid yields and improve the quality of DNA – no nicks)

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