DNA,RNA,%20Recombinant%20DNA%20Technology - PowerPoint PPT Presentation

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DNA,RNA,%20Recombinant%20DNA%20Technology

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Title: Advanced Gene Technology Author: ep Last modified by: tichym Created Date: 9/4/2006 10:32:55 AM Document presentation format: p edv d n na obrazovce – PowerPoint PPT presentation

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Title: DNA,RNA,%20Recombinant%20DNA%20Technology


1
DNA,RNA, Recombinant DNA Technology
2
Metabolic pathways expanded
3
Model organisms Cellular biology,
biochemistry... molecular biology
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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...
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Nucleic Acids DNA and RNA
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DNA RNA
DNA RNA
DNA RNA
RNA
DNA
13
DNA structure -gt sequence
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Polymerase reaction 5-gt 3
16
The central dogma
Page 93
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Gene expression.
Page 93
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What is a gene?
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Eukaryotes Intron-Exon concept
Page 95
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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
30
Recombinant DNA Technology
Clones -gt Cells or organisms with identical DNA
31
Restrictionendonucleases
5-gt 3
3 lt- 5
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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
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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
46
Plasmid Cloning Vectors
47
Insertional inactivation
  • Gene in cloning site
  • LacZ -gt pUC18 (lacZ complements the host defect
    in lacZ)
  • -gt pUC18 into host organism -gt active lacZ
    (ß-galactosidase) from plasmid-gt cleavage of
    X-gal
  • (blue colonies)
  • -gt gene cloned into polylinker -gt lacZ gene
    disrupted -gt no cleavage of X-gal (white
    colonies)

48
Blue/White Selection
http//www.bio.davidson.edu/Courses/genomics/metho
d/reporters.html
49
Insertional inactivation
  • Gene in cloning site
  • Resistance marker -gt pBR322 (cloning sites within
    antibiotica resistence marker)
  • -gt plasmid into host -gt resistance against 2
    antibiotica
  • -gt gene cloned within one resistance marker
    -gt gene for antibiotica
  • resistance marker disrupted -gt sensitive
    against one antibioticum

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Transformation and Selection
52
Horizontal gene transfer
  • - Transformation -gt uptake of naked DNA
    (chemical transformation, electroporation)
  • - Conjugation -gt DNA transfer by cell cell
    contact
  • Transduction -gt DNA transfer by bacteriopage
    infection
  • Other methods of Gene transfer -gt used with
    fungi, animal and plant cells
  • Microinjection
  • protoplasts

53
Electron micrograph of bacteriophage ?.
Bacteriophages
Page 107
Electron micrograph of the filamentous
bacteriophage M13.
54
Bacteriophage T2 injecting its DNA into an E. coli
Page 84
55
Life Cycle of Bacteriophage
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Replication of bacteriophage upon infection of a
cell
Page 107
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Molecular genetics and bacteriophage
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Cloning of foreign DNA in ? phages.
Page 110
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What is a gene library ?
65
Creation of Libraries
66
Creation of Libraries
67
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
76
DNA Library
Clones -gt genetically identical
77
Genomic phage library
78
Evaluation of library
79
Evaluation of library
80
Ordered library
Microarrays
81
Ordered library
Chromosome Walking -gt 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 even
death.The most serious form is E. coli 0157H7.
E. coli leads to about 73,000 cases of infection
and 61 deaths each year in the United States.
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 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.
NIH Recombinant Advisory Committee (RAC) (1973)
Asilomar Conference on Recombinant DNA (February
1975)
Every strain comes with description of its
genotype DH5alpha (recA- hsdR- LacIq uvrA-
mcrA-)
Most strain in molecular biology are recA- endA-
hsdR-
86
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).
mcrA/mcrB/mrr complex
hsdR/hsdM/hsdS (EcoK) restriction system
E.coli DNA is methylated by dcm, dam and hsdM
Degrades DNA not methylated at the sequence
5'-AAC-(N)5-GTGC-3'
mcrA/mcrB/mrr cleaves DNA methylated by other
systems
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
  • mcrA-/mcrB-
  • strains are good for cloning
  • eukaryotic DNA

87
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)
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 shock transformation and chemically
competent cells
Chemical transformation
treating E. coli CaCl2 will batter the membranes
and essentially make the bacteria very
unhappy. 
CaCl2 is gaping holes in the membrane
BRIEF HEAT SHOCK
90
Calcium/phosphate (heat shock) method
www.bch.msu.edu/bchug/web/
91
Conjugation Lederberg
Monod
  • F- to F
  • 100 minutes
  • 4000 genes
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