03. Basal techniques of DNA cloning:
'from genes to clones & from clones to genes'
a.o. Brown, chapter 5 ; Primrose et al., chapter 2
Transformation of E. coli
Selection & identification
Selection of transformants :
antibiotic resistance marker
auxotrophic marker
ApR (bla), TcR (tetA), CmR,(cat), KmR,(aph, neo, npt), SmR
leuB, hisB (growth on medium with or without leucine, histidine)
Selection versus identification of recombinants
negative selection :
recombinant unviable in the presence of the selective agent
replica plating required
positive selection
recombinant survives by inactivation of the marker
no replica plating required
:
sacB (encodes levansucrase : lethal on 7% sucrose)
5-FOA : toxic if URA3+ but URA3- requires uracil in the medium
identification
:
recombinant recognized by phenotypic (visible) trait
best known and most frequently used system is based on lacZ complementation
The Lac systeem: ‘blue-white’ screening
- the Lac operon : lacZ, lacY, lacA, lacI and lacP, lacO, lacP’
- lactose hydrolysed to galactose + glucose
- -galactosidase : tetramer of lacZ (1023 aa)
- lac repressor (LacI) binds to lacP : controls expression
- induction by lactose (allolactose) and some derivatives :
=> IPTG is inducer, not substrate
=> BCIG (Xgal) is substrate, not inducer
=> hydrolysis of BCIG yields a blue, water-insoluble compound
- intracistronic complementation
the lacZ gene product may be split into two parts :  and  fragments
- lacZ lies in the amino-terminal region (< 100 aa)
- the  fragment is usually a deletant missing 31 aa in the  region
both separate parts have no -galactosidase activity by themselves,
but join into an active product
Chemical transformation with CaCl2
- competent cells
- cells in late exponential growth phase
- concentration
- effect of temperature, DMSO, RbCl, etc.
- storage
- DNA transfer
- heat shock (cold shock)
- "expression phase"
- E. coli : efficiency ccc versus oc versus linear DNA
- size of DNAs is limiting: efficiency decreases rapidly with increasing size
- LPS structure
Electroporation
- transiënt, hydrophobic pores
- important factors: length of pulse, field strength, shape of the electrodes
- efficiency 10-20 x higher compared to chemical methods (1010 per g)
- sizes up to 80 kb very efficient, but also larger DNAs are transferable
- instrumentation
Alternative methods:
- use of PEG (polyethylene glycol) (originally: fusion of animal cell, a.o.)
E. coli : requires regeneration which is difficult with E. coli
Gram-positives : ok with Bacillus, Streptomyces : regeneration is necessary
S. cerevisiae : PEG in combination with CaCl2 and sorbitol: regeneration required
- use of CH3COOLi : for Saccharomyces cerevisiae : on intact cells
- biolistics, bioballistics, etc.
Transfection
= genetic transformation using a viral (phage) or virus-based vector
plaques as identification marker
turbid > < clear plaques ()
spi selection : positive selection
'In vitro packaging' ()
with phages and cosmids
'In vivo packaging' (Ff, )
with phages, fasmids (phasmids / phagemids) and cosmids
Clone analysis
Colonies – plaques – density
Insert detection – size determination – orientation determination
agarose gel electrophoresis
detection (‘staining’)
Restriction and/or analysis
practical steps :
restriction analysis : (see also chapter 2)
- size of plasmid clone
- fragmentation : position of cleavage in vector
=> length of insert
=> orientation (internal site)
=> use of polylinker
- PCR a nalysis :
position of annealing sites in vector : length of insert
orientation : with one primer in insert, versus vector positions left and right
sensitivity versus stability
agarose gels
agarose: linear polysaccharide
alternating D- and L-(3-6-anhydro)galactose, coupled in
(1-3) and (1-4) glycosidic bonds
forms a 3-D structure with channels of 50 nm tot more than 200 nm
length of the chains variable, but estimated as averaging about
800 galactose units
gel: - agarose become liquid in aqueous mixture by heating (e.g. 90°C), and
solidifies (gellifies) at relatively low temperature (e.g. 50°C).
Can be re-melted.
- relatively stable gel mass between 0,2 and 5% (in practice 0,5 – 3%)
- allows mixing with alkali, urea, methyl Hg-chloride, SDS, formamide, etc
- migration of DNA through the gel matrix at a speed reversely proportional to
log10 of the relative molecular mass (number of bp) : up to about 20 kb
- basis: - the charge is responsible for the direction of migration
- frictional resistance is responsible for the fractionation
- velocity = cst/log(size)
cst is dependent on the agarose concentration
- effect of conformation
1) ccc versus oc versus linear
2) in the presence of ethidium bromide: charge decreases; DNA
become less flexible and influences the size
=> migration
speed is reduced by about 15%
- influence of voltage, a.o.
- different types: a.o. - “low melting” agarose by hydroxy-ethylation
- metaphor agarose: higher resolution (of smaller fragments)
- electro-endosmose: the charge of the gel (SO42-) causes counter-ions and a
buffer stream in opposite direction of the DNA.
- reference dyes: bromophenol blue, xylene cyanol FF
- recuperation of the DNA after fractionation: with “low melting” agarose (+ phenol
treatment), by disruption of the gel matrix (freeze-thaw, mashing, etc),
electrophoretically, or by continuous electrophoresis. Altogether rather
inefficient as far as purity is concerned.
- detection: ethidium bromide, silver staining, autoradiography
- movement: - “accordeon” effect of small molecules
- “reptation” of longer molecules : from about 20 kb on
=> PFGE : re-orientation of the voltage field
=> fractionations up to 10 Mb are possible
different configurations
example: Saccharomyces cerevisiae chromosomal DNA’s
embedded of cells in agarose plugs before DNA isolation
(lysis of cells, protease treatment, restriction cleavage)
ethidium bromide
- structure
cfr. acridine, acridine orange (stains dsDNA green, ssRNA red-orange)
- absorbs UV-radiation at 300 and 360 nm
+ UV-radiation absorbed by DNA at 260 nm is transferred to the
ethidium moiety (energy transfer)
=> emission at 590 nm (orange-red in the area of visible light)
- staining of DNA in a gel by submerging in a tray with a solution of
ethidium bromide at a concentration of 0,5-1 g/ml (water or buffer)
=> fixation in the DNA ànd the nearby presence of the base-rings lead to
increased fluorescence compared to the dye in solution.
- ethidium bromide is a mutagen : in an Ames test, 90 g ethidium bromide
corresponds to the smoke condensate of 1 sigarette, or 2 g nitrosoguanidine
(every mutagen is considered as a potential cancerogen, although tests with
ethidium bromide so far have failed to show any cancerogenicity)
- ethidium bromide causes photo-oxidation of DNA in the presence of light and
O2 (the dye may be extracted with butanol and iso-amylalcohol)
polyacrylamide gels
- structure ; polymerisation in the presence of bis-acrylamide (1/10-1/40)
- fractionation: 1 nt – about 2 kb
- same fractionation characteristics and detection procedurer as with agarose gels
- addition of urea and other compounds is feasible
- one-dimensional, also two-dimensional formats are possible (the latter
in particular for separation of proteïns : SDS-PAGE)
- sequence analysis : polyacrylamide gels containing 7 to 9 M urea
=> fractionation of “nested sets”
Sequence analysis
(see later : chapter 10)
basic strategies: chemical degradation, chain termination / synthesis
sequencing gels
Hybridisation
- immobilisation of nucleic acids
- preparation and labeling/tagging of probes
- analysis of reassociation kinetics is usually done in solution (see chapter 1)
- renaturation is also possible in a two-phase system with one strand immobilised
(target) and the other strand in solution. The strand in solution is labelled
radioactively (or tagged by chemical structures specifically detectable) and is
named the 'probe'
- qualitative and quantitative comparison of nucleic acid sequences is feasible.
(the kinetics of renaturation may be quite different and are not dealt with here)
(Tm values do apply)
- DNA-DNA, RNA-RNA, DNA-RNA hybrids duplexes may form.
- complex fragment mixtures (e.g. after restriction digestion) can be analysed after
fractionation and immobilisation:
=> blotting techniques : Southern, northern, western, eastern, south-western, etc.
cellulose nitrate ("nitrocellulose"), nylon membrane
capillary transfer, electro-blotting, vacuum blotting, dot blot
- labelling of probe: P32, P33, S35
tagging: biotin, digoxigenin
- detection:
autoradiography (if radioactively labelled)
=> visualization: X-ray film
biotin-streptavidin alkaline phosphatase
peroxidase
=> substrate => product
color 1 => color 2
=> visualization: color (by sight), light emission (X-ray film,
polaroid film, other instrumentation)
digoxigenin => antibody => idem