Section G Gene manipulation

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Molecular Biology Course
Section G Gene manipulation
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Gene manipulation
G1-1 DNA cloning G1-2 Hosts and vectors G1-3
Subcloning G1-4 DNA libraries G1-5 Screening
libraries G1-6 Analysis of a clone
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Gene manipulation
G1 DNA cloning an overview
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G1 DNA cloning An Overview
G1-1 DNA cloning (definition)
DNA cloning is to place a relatively short
fragment of a genome, which might contain the
gene or other sequence of interest, in an
autonomously replicating piece of DNA, known as a
vector, forming recombinant DNA, which can be
replicates independently of the original genome,
and normally in other host species altogether.
Propagation of the host organism containing the
recombinant DNA forms a set of genetically
identical organism, or a clone. This process is
called DNA cloning.

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G1 DNA cloning An Overview
G1-2 Hosts and vectors

• Host organism/cell where the plasmids get
  multiplied and propagated faithfully, which is
  crucial for DNA cloning.
• Hosts for DNA cloning vector
• Prokaryotic host E. coli ( most cases)
• Eukaryotic host Yeast Saccharomyces cerevisiae
  (large fragments of human genome)

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G1 DNA cloning An Overview

• General features of a Vector
• autonomously replicating DNA independent of
  hosts genome.
• Easily to be isolated from the host cell
• Most are circular, some are linear
• Contains at least one selective marker, which
  allows host cells containing the vector to be
  selected amongst those which do not.
• Contains a multiple cloning site (MCS)

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G1 DNA cloning An Overview
Types of vectors

• Cloning vectors
• Expression vectors
• Integration vectors

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Cloning vectors allowing the exogenous DNA to be
inserted, stored, and manipulated at DNA level.
E. coli cloning vector plasmids, bacteriophages
(l and M13), plasmid-bacteriophage l hybrids
(cosmids). Yeast cloning vector yeast artificial
chromosomes (YACs)
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G1 DNA cloning An Overview

• Expression vectors allowing the exogenous DNA to
  be inserted and expressed. Promoter and
  terminator for RNA transcription are required.
• bacterial expression vectors
• yeast expression vectors
• mammalian expression vectors

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G1 DNA cloning An Overview

• Integration vectors allowing the exogenous DNA
  to be inserted and integrated into a chromosomal
  DNA after a transformation. The integration is
  conducted by homologous recombination between the
  homologous sequence shared by the plasmid and the
  genome of the recipient cells.
• bacterial integration vectors (Agrobacterium
  tumefaciens Ti plasmid is used to integrate DNA
  into plant genome)
• yeast integration vectors
• Mammalian integration vector virus based

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G1 DNA cloning An Overview

• G1-3 Subcloning
• Transfer of a fragment of cloned DNA from one
  vector to another.
• Enables us to investigate a short region of a
  large cloned fragment in more detail.
• To transfer a gene from one plasmid to a vector
  designed to express it in a particular species.

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DNA Subcloning a flow chart
Preparation of plasmids containing a cloned DNA
fragment (insert)
Plasmid preparation (vector)
Restriction digestion (trimming the DNA ends)
Restriction endonuclease
Ligation (join the insert and the vector)
Transformation selection of transformants (intro
duce the plasmids into host cells)
Assay of the recombinants
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G1 DNA cloning An Overview

• Agrose Gel Electrophoresis
• check your DNA at each step
• Separation and Purification of DNA fragments of
  interests
• Analysis of recombinant plasmids

ladder
Restriction analysis of a plasmid
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G1 DNA cloning An Overview
G1-4 DNA libraries

• DNA libraries are sets of DNA clones, each of
  which has been derived from the insertion of a
  different fragment into a vector followed by
  propagation in the host.
• A clone is a genetically distinct individual or
  set of identical individuals
• Genomic libraries cDNA libraries

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G1 DNA cloning An Overview
Genomic libraries prepared form random fragments
of genomic DNA, which may be inefficient to find
a gene because of the huge abundance of the
non-coding DNA
cDNA libraries DNA copies (cDNA) synthesized
from the mRNA by reverse transcription are
inserted into a vector to form a cDNA library.
Much more efficient in identifying a gene, but do
not contain DNA coding functional RNA or
noncoding sequence.
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G1 DNA cloning An Overview
G1-5 Screening libraries
Searching the genes of interest in a DNA library

• Hybridization to identify the interested DNA or
  its RNA product
• Radiolabeled probes which is complementary to a
  region of the interested gene
• Probes
• An oligonucleotide derived from the sequence of a
  protein product of the gene
• A DNA fragment/oligo from a related gene of
  another species
• Blotting the DNA or RNA on a membrane
• Hybridize the labeled probe with DNA membrane
  (Southern) or RNA (Northern) membrane

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G1 DNA cloning An Overview

• Identify the protein product of an interested
  gene
• Protein activity
• Western blotting using a specific antibody

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G1 DNA cloning An Overview
G1-6 Analysis of a clone

• Restriction mapping digestion of the with
  restriction enzymes.
• Sequencing the cloned DNA

You may have to fully understand the function and
application of all the enzymes listed in Table 1
before the final exam
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Gene manipulation
G2 Preparation of plasmid DNA
G2-1 Plasmid as vectors G2-2 Plasmid
minipreparation G2-3 Alkaline lysis G2-4
Phenol extraction G2-5 Ethanol
precipitation G2-6 Cesium chloride gradient
(purification)
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G2 Preparation of plasmid DNA
G2-1 Plasmid as vectors

• Plasmids small, extrachromosomal circular
  molecules, from 2 to 200 kb in size, which exist
  in multiple copies within the host cells.
• contain an origin of replication and replicate
  independently
• Usually carry a few genes, one of which may
  confer resistance to antibacterial substance.
• Example ampr gene encoding the enzyme b-lactamse
  which degrades penicillin antibiotics such as
  ampicillin.

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G2 Preparation of plasmid DNA
G2-2 Plasmid minipreparation from E. coli

• Plasmids
• 2-20 kb in length that much smaller than E. coli
  chromosomal DNA (4600 kb), and independently
  supercoiled
• Resistant to shearing force and chemical
  denaturation, thus can be isolated from the
  chromosomal DNA easily such as alkaline lysis.
• Minipreparation (miniprep)
• Isolation of plasmid DNA from a few mililiters
  (ml) of bacterial culture.

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G2 Preparation of plasmid DNA
Miniprep

• Growth of the cells containing plasmids
• Collect the cells by centrifugation
• Alkaline lysis
• resuspension? alkaline lysis ? neutralization
• Phenol extraction to get rid of the protein
  contaminants
• Ethanol precipitation to concentrate the nucleic
  acids remained. (Please noted that RNase A is
  very bad for the lab working with RNA)

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G2 Preparation of plasmid DNA

• G2-3 Alkaline lysis
• Resuspend the cells in a buffer solution
• Lysozyme to digest the cell wall (optional)
• Cell lysis in lysis buffer containing SDS
  (disrupts cell membrane and denatures proteins)
  and NaOH (denatures DNA)
• Neutralization buffer containing KOAc (pH 5)
  renaturation of plasmid DNA (supercoiled) and
  precipitation of denatured proteins and
  chromosomal DNA which can not be renatured
  because of its size and physical property of
  easily being sheared.

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Grow the cell
Harvest the cell by centrifugation
Resuspend the cell pellet
Alkaline lysis of the cell
neutralization
Phenol extraction
Ethanol precipitation
CsCl gradient purification
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G2- 6 Cesium chloride gradient centrifugation
G2 Preparation of plasmid DNA

• CsCl gradient purification is the last step of
  large scale plasmid DNA purification
• Laborious
• Best for the production of very pure supercoiled
  plasmid DNA
• The presence of ethidium bromide (EB) is
  important.
• Binding of EB to DNA will unwind the DNA and
  reduce the DNA density
• Supercoilded DNA bind less EB than linear DNA or
  nicked DNA, thus has a higher density

Supercoiled DNA may be purified from protein,RNA
chromosomal DNA and nicked plasmid DNA in one
step!!
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Gene manipulation
G3 Restriction Enzymes and electrophoresis
G3-1 Restriction endonuclease G3-2 Recognition
sequences G3-3 Cohesive ends G3-4 Restriction
digests G3-5 Agarose gel electrophoresis G3-6
Isolation of fragments
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G3 Restriction enzymes and electrophoresis
G3-1 Restriction endonuclease

• Bacterial enzymes which cut DNA into defined and
  reproducible fragments
• Identified in the 1960s
• Key discovery which allowed the DNA cloning to
  become a reality

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G3 Restriction enzymes and electrophoresis

• One component of the bacterial restriction-modific
  ation system, a natural defense mechanism of
  bacteria to against the introduction of foreign
  DNA into the cell
• Restriction endonuclease recognize a short,
  symmetrical DNA sequence, and cut DNA backbone in
  each strand at a specific site within that
  sequence (kill foreign DNA)
• Mythylase methylates C or A of the cellular DNA

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G3 Restriction enzymes and electrophoresis
G3-23 Restriction sequencesCohesive ends
5 protruding ends
3 protruding ends
Cohensive ends
SmaI
5-CCCGGG-3 3-GGGCCC-5
5-CCC-OH 3-GGG- p

blunting ends
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G3 Restriction enzymes and electrophoresis
Recognition sequences

• Recognize 4-8 bp. Most recognition sequences are
  6 bp which occurs at a rate of 464096 bp.
• Highly specific

Restriction enzymes

• Commercially available
• Require Mg2 for enzymatic activity

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G3 Restriction enzymes and electrophoresis
G3-4 Restriction digestion
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G3 Restriction enzymes and electrophoresis
G3-5 Agrose gel electrophoresis
Agrose a polysaccharide derived from seaweed,
which forms a solid gel when dissolved in aqueous
solution (0.5-3)
Negatively charged DNA
- ve electrode
ve electrode
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G3 Restriction enzymes and electrophoresis
Agrose gel electrophoresis
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G3 Restriction enzymes and electrophoresis
Isolation of fragments and Agarose gel
electrophoresis

• Restriction digestion
• Agarose gel electrophoresis

insert

• 3. Gel excision and purification
• Ligation with vector
• transformation

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G4 Ligation, transformation and analysis of
recombinants
Gene manipulation
G4-1 Alkaline phophatse G4-23 DNA ligation
recombinant DNA molecules G4-45 Transformation
selection G4-6 Transformation efficiency G4-7 Scre
ening transformants G4-8 Growth and storage of
transformants G4-9 Gel analysis G4-10 Fragment
orientation
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G4 Ligation, transformation and analysis of
recombinants

• G4-1 Alkaline phophatse
• removes the phosphate groups from th 5-ends of
  the vector DNA linearized by a single restriction
  enzyme to prevent the self-ligation of the vector
  DNA upon the followed ligation
• Single restriction enzyme directed cloning

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G4 Ligation, transformation and analysis of
recombinants
G4-23 DNA ligation recombinant DNA molecules
DNA ligation Covalently join the DNA molecules
with the base-pairing cohesive ends, or blunt
ends, if the 5-ends have phosphate groups.
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G4 Ligation, transformation and analysis of
recombinants
Recombinant DNA molecules
X if the vector is phosphoralated
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G4 Ligation, transformation and analysis of
recombinants
The use of alkaline phosphate to prevent
religation of vector molecules
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G4 Ligation, transformation and analysis of
recombinants
G4-45 Transformation and selection
Competent cells E. coli cells treated with Ca2
solution are susceptible to take up exogenous
DNA. Enzymes involved in host cell defending,
such as restriction-modification system are
suppressed. Transformation a process of uptake
of exogenous DNA by competent cells.
Heat-shock After the DNA is uptaken, the cells
shall be put at 42oC for 1 min in order to induce
the suppressed enzymes for cell defending
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G4 Ligation, transformation and analysis of
recombinants
Selection with antibiotic resistance (ampr)
Transformantion efficiency number of colonies
formed per microgram (mg) of input DNA. Ranges
from 103 to more than 108. 105 is adequate for a
simple cloning.
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G4 Ligation, transformation and analysis of
recombinants
G4-6 Transformation efficiency
Transformantion efficiency number of colonies
formed per microgram (mg) of input DNA. Ranges
from 103 to more than 108. 105 is adequate for a
simple cloning.
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G4 Ligation, transformation and analysis of
recombinants
G4-7 Growth and storage of transformants

• Can be grow in liquid broth or solid plates
• Maintain the selection pressure by the presence
  of the corresponding antibiotics. Plasmid
  stability and lose
• Store the transformant bacteria by freezing a
  portion in the presence of glycerol to protect
  from ice crystal formation

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G4 Ligation, transformation and analysis of
recombinants
G4-89 Gel analysis and fragment orientation

• Distinguish the recombinant plasmids from the
  recreated vectors by
• Size of the plasmids (not work well if the
  plasmids are prepared with alkaline lysis)
• Restriction digestion
• 2. Determine the orientation of a inserted DNA
  fragment cloned by a single enzyme by restriction
  digestion that cuts asymmetrically within the
  insert sequence, and once at a specific site of
  the vector

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G4 Ligation, transformation and analysis of
recombinants
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G4 Ligation, transformation and analysis of
recombinants
3. Analysis of a clone by restriction mapping
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Thanks