p.1
04. Screening, selection & characterisation
See
Primrose, Twyman & Old
Principles of Gene Manipulation, 6th edition (2002)
a.o.
Chapter 6 (partim: pages 103 (101 bottom lines) - 119) (not 1121/2 – 1141/2)
survey of cDNA cloning strategies/tools/procedures/interconnections
plating out a library : cfu/ml or pfu/ml (suspension)
low density (individual clones) or high density (up to confluent lawn)
(9 cm plates, 13 cm plates, 21 x 21 cm square plates, microtiter trays (96 - 384 - 1536)
- direct selection : rather exceptional
- e.g. antibiotic resistance gene
- e.g. auxotrophic marker : ‘marker rescue’ approach : mutant strain or
particular medium/condition required (e.g. isolation of trpA gene)
- e.g. complementation cloning : yeast genomic DNA fragment can
complement an E. coli leuB mutant : => yeast LEU2 gene
- e.g. direct identification : any gene for a biochemical conversion of a
substance in the growth medium to a scorable product (color, …) (e.g. lacZ)
- gene-directed versus comparison-directed
gene-directed
- clone detection : based on ‘information’ : protein or nucleic acid (sequence)
- hybridisation : probe required
colony hybridisation, colony lifting
plaque hybridisation
G. Volckaert
Genetic engineering : Screening of DNA libraries
17/02/2016
p.2
probes:
(in order of decreasing amount of information)
- sequence known
- homologous sequence : fragment available
- heterologous sequence / fragment
=> ZOO blots
- protein sequence known
- synthetic/degenerated probes (oligonucleotides)
set of individual oligonucleotides
or
mixture
or
guessmer
use of dI, etc.
- screening by PCR : wells containing clone pools, then dilution until
homogeneous clones are obtained
(mixed or degenerate primers may be used)
- immunochemical methods : directly on expression product (epitope)
(mature protein or fusion construct)
=> binding to plastic (polyvinyl) plates (colonies)
or to cellulose nitrate sheets (-plaques)
=> originally 125I-labelled IgG as probe
=> now, enzymatic label onto antibody (e.g. alkaline phosphatase)
primary antibody against target, secondary (labelled) antibody
to detect bound primary antibody
=> use of either polyclonal antibodies or monoclonal antibody
- indirect approaches: based on “activity”
- HArT (‘hybrid arrested translation’)
- in vitro translation of mRNA extract
=> selective inhibition by complementary DNA
- HST (HRT) (‘hybrid-selected translation’, ‘hybrid-released translation’)
- affinity selection and re-elution of ‘positive’ activity
=> pools of clones, stepwise reduction to single ones
G. Volckaert
Genetic engineering : Screening of DNA libraries
17/02/2016
p.3
comparison-directed
- abundance screening : cloning equalizes level in each clone
but : the number of clones corresponds to the relative abundance
- genome libraries : gene families
- cDNA libraries : high vs low abundance mRNA’s
- differential expression and difference screening
- Xenopus gastrula versus egg mRNA
- comparison leaf – root – stem – etc.
- + / - induction
- light versus dark
- etc
- subtractive techniques (screening/cloning)
- differential display (PCR approach)
oligonucleotide such as NVTTTTTTT T + nonamers
(one of 12 primers) ; amplication by chance
- RDA (representational difference analysis)
- genome comparison
- cDNA comparison
- ESTs : random DNA sequencing of library clones : 300-600 nt as tag
- SAGE : concatemerisation of short tags (9-13 bp), one tag per mRNA
(“serial analysis of gene expression”)
G. Volckaert
Genetic engineering : Screening of DNA libraries
17/02/2016
p.4
not in 2007-2008
G. Volckaert
Genetic engineering : Screening of DNA libraries
17/02/2016