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Gene Cloning
1. Fragment of DNA to be cloned is inserted into
vector to produce rDNA
2. Vector to transport gene into host cell e.g.
plasmid or bacteriophage chromosome
3. Multiplication of rDNA molecule together with
vector
4. Division of host cell
5. After a large number of cell divisions forms a
colony or clone with identical genes
PCR
Thermal Cycler
1. Heat to 94 oC to denature the double strand DNA
2. Cool to 55 oC for primers to anneal to DNA at
specific position (gene of interest)
3. Raise to 74 oC for Taq DNA polymerase to function
4. Synthesis of DNA strands complementary to
template DNA molecules in opposite direction
5. Repeat the cycle 25-30 times
Importance of Gene Cloning and PCR
Useful in gene isolation
E.Coli contains over 4000 different genes!
PCR in a few hours, gene cloning in weeks!
Limitations of PCR:
1. Sequence of annealing sites must be known in
order for primers to anneal to correct positions
2. Limit to length of DNA sequence i.e. 5
kilobases – 40 kilobases
Application of PCR:
Useful to detect or isolate gene sequences
already known
eg. Test for mutation in blood using globin
genes or use of primers specific for the DNA of
a harmful virus
Plasmids
• Circular molecules of DNA that lead independent existence in host
• Carry genes that are responsible for a useful characteristic displayed
by host bacterium
• Survival in normally toxic concentrations of antibiotics – antibiotic
resistance as a selectable marker
Example: ampicillin, tetracycline, kanamycin resistant gene RP4
• At least one DNA sequence that can act as an origin of replication
OR
• Integrate into bacterial chromosome for division
• < 10kb desirable for a cloning vehicle
Plasmid Classification
1. Fertility or “F” plasmids carry only tra genes and have no
characteristic beyond the ability to promote conjugal plasmid
transfer
2. Resistance or “R” plasmids confers resistance to one or more
antibacterial agents
3. Col plasmids code for colicins, proteins that kill other bacteria
4. Degradative plasmids allow the host bacterium to metabolize
unusual molecules e.g. toluene and Hg
5. Virulence plasmids confer pathogenicity on the host bacterium e.g.
Ti plasmids of agrobacterium tumefaciens induce crown gall disease
on plants
Purification of DNA from living cells
total cell DNA
pure plasmid DNA
3.1 Preparation of total cell DNA
I.
A culture of bacteria is grown and then harvested
II.
The cells are removed and broken to give a cell extract
III. The DNA is purified from the cell extract
IV. The DNA is concentrated
3.1.1 Growing and harvesting a bacterial culture
• culture bacteria in a liquid both
• 2 types of growth media – defined medium and undefined medium
• Define media is used when the bacteria culture has to be grown
under precisely controlled conditions e.g. M9
• Undefined media is used when culture is grown for a source of DNA
e.g. Luria-Bertani (LB) contain yeast and tryptone
3.1.2 Preparation of a cell extract
• Purpose is to break open bacterial cells (cell lysis) by either physical or
chemical means
• lysozyme digest polymeric compunds that define a cell wall’s rigidity
• EDTA remove Mg2+ that is essential for the structure of cell envelope
• Detergents e.g. SDS help to remove lipid molecules and cause
disruption of cell membranes
• Centrifugation to remove cell debris that settle at the bottom
3.1.3 Purification of DNA from a cell extract
• Bacterial extract can contain a lot of protein and RNA inaddtion to desired
DNA
• Add phenol or 1:1 mixture of phenol and chloroform. Ppt proteins leaving
behind DNA & RNA and can be seperated after centrifugation
• Cell extracts that contain a large amount of proteins must be treated with
Protease such as proteinase K before addition of phenol
• Degrade RNA with suitable ribonuclease
3.1.4 Measurement of DNA concentration
• UV absorbance spectrophotometry
• Absorbance at 260 nm (A260) of 1.0 == 50ug of double strand DNA/ml
• UV absorbance can also check for purity of DNA preparation: A260/A280 = 1.8 for
pure samples
3.1.5 Preparation of total cell DNA from non-bacteria organism
•
plant tissues contain large amount of carbohydrates that cannot be removed
by phenol extraction
•
METHOD 1
1. Add CTAB (cetyltrimethylammonium bromide) so that CTAB complex ppt out
together with the nucleic acid. Carbohydrates and proteins and other
contaminants as a supernatant. Centrifuge and collect the ppt.
2. Nucleic acids remaining can be concentrated using ethanol precipitation and
RNA remove by ribonuclease treatment
•
METHOD 2
1. Add guanidinium thiocyanate to dissolve all biochemical other than
nucleic acids
2. Pass the sample through a chromatography column with silica
particles inside. DNA in presence of guanidinium thiocyanate bind
more strongly to silica
3. DNA is recovered by adding water which destabilizes interaction
between DNA and silica
3.2.1 Plasmid Separation
• Separation by size work on the principle that cells are lysed under very
carefully controlled conditions
• very little breakage of DNA chromosome. Hence DNA is much larger
than plasmid and can be removed together with cell debris in
centrifugation
• Chromosomal DNA also attached to cell envelope and settle at bottom
Method 1
For E. Coli and related species under controlled lysis
1. Add EDTA and lysozyme in the presence of 25% sucrose – prevent cell
from bursting immediately
2. Cell lysis is induced by adding non-ionic detergent (Triton X-100)
because ionic detergent cause chromosomal breakage
3. Centrifugation leaves a cleared lysate consisting of only plasmid DNA
Method 2
Separation by conformation using alkaline denaturation.
Plasmid is circular DNA but also often supercoiled
1. A narrow pH range at which non-supercoiled DNA is denatured while
supercoiled plasmid will not. pH range between 12.0 – 12.5 (using NaOH)
2. After non supercoiled DNA’s H bonding is broken to form linear strand
DNA, add acid to reach pH 7.0
3. Denatured bacteria DNA strands entangle into a mass and can be
centrifuged, leaving pure plasmid DNA in supernatant
3.2.2 Plasmid Amplification
• To increase the copy number of plasmid
• some multicopy plasmid can replicate in the absence of protein
synthesis, whereas main bacteria chromosome cannot replicate
• After a satisfactory cell density is reached, add inhibitor of protein
synthesis e.g. chloramphenicol and incubate for another 12 hrs
• Plasmid copy of 1000++ , hence an efficient way of increasing yield of
multicopy plasmid
Chapter 4: manipulation of purified DNA
DNA manipulative enzymes
• Nuclease – cut or degrade nucleic acid
 exonuclease remove nucleotides one at a time from end of DNA
 endonuclease break internal phosphodiester bonds within DNA
• Ligase – join nucleic acid
• Polymerase – make copies of molecules
• Modifying enzymes – remove or add chemical grp
 alkaline phosphatase remove phosphate grp at 5’ end of DNA
 Polynucleotide kinase reverse effect to alkaline phosphatase
 Terminal deoxynucleotidyl transferase add deoxyribonucleotides to 3’ end
of DNA
• Topoisomerase – introduce or remove supercoils from covalently closed
circular DNA
Chapter 5: Introduction of DNA into living cells
5.1.1 Transformation - Uptake of foreign DNA molecule by a cell
• Most cell take only limited amounts of DNA normally, must increase
efficiency of intake by physical or chemical enhancement
• E.coli cells soaked in ice cold salt solution more efficient at DNA
uptake. A solution of 50mM CaCl2 is used
• Next heat shock the solution to 42 oC for 2 min to facilitate uptake of
plasmid by cell
5.1.2 Selection for transformed cells
• using selectable marker e.g. amipicillin resistance gene or tetracycline
resistance gene
• LacZ’ gene codes for beta galactosidase, breaks lactose to glucose +
galactose
• e.g. PUC8 plasmid with both AmpR and LacZ’ genes
• some strains of E.coli have modified lacZ gene that lack the segment
LacZ’
• These mutants can only synthesize beta galactosidase if it has PUC8
plasmid that carries the missing LacZ’ gene segment
• Add X-gal (5-bromo-4-chloro-3-indoyl-B-D-galactopyra-lactose) which is
broken down by B galactosidase to form a blue product
• Add IPTG (inducer of the beta galactosidase)
• Xgal + IPTG + agar plate to select cells between white and blue colonies
Cloning vectors for E.Coli
pBR322 – ori, ampR, tetR
Nomenclature of plasmid cloning vectors
pBR322
• p – plasmid
pBR327 – ori, ampR, tetR
pUC8 – ori, ampR, lacZ’
• BR – identifies the laboratory in which vector was discovered (BR for
Bolivar and Rodriguez, the 2 researchers that developed it)
• 322 – distinguishes this plasmid from others developed in the same
laboratory (pBR325, pBR327 etc..)