Screening, selection & characterisation

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)

Activity Amino acid sequence

CELL Antisera

Reverse translation Protein Extract

Probe

Tumor line

Induction

Naturalcondition

mRNA

in vitro translation

Activity

ChemicalDNA

synthesis

TEST

oligo-dT chromatography

polyA+ mRNA

cDNA cloning clones

Hybridisation

DNA sequencing

Expression

EST libraries

HARTHST

cDNA cloning : survey of tools / strategies / procedures

Direct selection for a clone containing the R6-5 kanamycinresistance gene (kanR)

Direct selection for a trpA geneusing a trpA- strain of E. coli

An appropriate mutant strain must be available.

=> 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

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

Screening by hybridisation

DNA probes

RNA probes

oligonucleotide probes

mixed probes and guessmers

mismatch probes

homologous probes

heterologous probes

=> ZOO blots

polyclonal antisera

monoclonal antibodies

Colony hybridization.

Probing with a radioactively labelled

or enzymatically, fluorescently or

immunologically tagged probe.

Detection by autoradiography, color,

fluorescence, etc.

Similar procedures with plaque hybridization.

Heterologous probing

The amino acid sequence of yeast cytochrome c.

The hexapeptide that is highlighted yellow is oneto illustrate how a nucleotide sequence can be predicted from an amino acid sequence.

-trp-asp-glu-asn-asn-met-

-TGG-GAY-GAR-AAY-AAY-ATG- 18-mer

2 x 2 x 2 x 216 oligonucleotides represent the entire set of possibilities.

The use of a synthetic,

labelled or tagged

oligonucleotide to identify

a clone of the yeast

cytochrome c gene.

From probable to definite.

Antibodies :

(a) Antibodies bind to foreign

molecules and help to

degrade them.

(b) Purified antibodies ("antisera")

can be obtained from (a.o.) a

small volume of blood taken

from a rabbit injected with

the foreign protein.

Immunoscreening

Detection by :

- labelled antibody itself

or

- labelled protein A (as shown in the

figure)

or

- the use of a second antibody which binds specifically to the primary antibody.

Plaque screening of clones

of fusion proteins (at LacZ)

using antibodies.

"Sandwich"-approach

in probing with

antibodies.

Activity Amino acid sequence

CELL Antisera

Reverse translation Protein Extract

Probe

Tumor line

Induction

Naturalcondition

mRNA

in vitro translation

Activity

ChemicalDNA

synthesis

TEST

oligo-dT chromatography

polyA+ mRNA

cDNA cloning clones

Hybridisation

DNA sequencing

Expression

EST libraries

HARTHST

cDNA cloning : survey of tools / strategies / procedures

Hybrid arrested translation (HArT)

mRNA extract analyse add denatured plasmid DNA analyse

activity from 1 or more cDNA clones activity

in vitro translation

protein biochemical in vitro translation

test protein test

The plasmid DNA of the cDNA clone that inhibits the biological activity in the biochemical test is a good candidate of representing a positive clone. Sets of DNA's are tested simultaneously, and subsequently split into smaller numbers, until individual DNA's can be tested and analysed.

Hybrid selection translation (HST)

mRNA is selected from a total mRNA extract

by hybridisation to an immobilized cDNA

plasmid clone DNA.

In vitro translation of the selected mRNA

provides protein product that is analyzed

by a biochemical test.

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

- mRNA from plants in light versus dark

- etc.

- subtractive techniques (screening/cloning)

- differential display (PCR approach)

oligonucleotide such as 5’-TTTTTTTTVN-3’ + nonamers

(one of 12 primers) ; amplication by chance

- RDA (representational difference analysis)

initially developed for genomic libraries, adapted later for

cDNA libraries (after fragmentation with a restriction

enzyme)

- genome comparisons

- cDNA comparisons

- SAGE : concatemerisation of short tags (9-13 bp), one tag per mRNA

(“serial analysis of gene expression”)

- ESTs : random DNA sequencing of library clones : 300-600 nt as tag

Abundance screening

Probing within a cDNA library to

identify an abundant gene (mRNA).

Difference screening&differential expression analysis

Making multiple DNA-filters in parallel and

comparing hybridisations with different probes.

Labelled or tagged cDNA copies of mRNAs

from different cells or cell types or different

conditions of growth or induction or other

treatments.

Subtractive techniques : comparing cell types

mRNA prepared from cell type A.mRNA prepared from cell type B.

cDNA prepared from mRNA of cell type A, made single-strandedby alkaline treatment.

The cDNA is complementary to mRNA, also those of cell type Binasmuch as these are expressedin the latter cell type. => renaturation of cDNA(A) with mRNA(B) makes hydrids, but sequences which are not common to both cell types remain single-stranded.

mRNA and RNA-DNA hybrids bind to hydroxyapatite. Cell type A-specific cDNA can beisolated and either used to make an A-specific cDNA library, or be usedprobe to screen a complete cDNA library of cell type A.

Comparing cells of an induced and the corresponding non-induced stage.Steps in differential hybridization screening procedures.

+/- strategy : comparison of signals obtained with replica filters hybridized with material derived from cells of the induced and the non-induced stage, respectively.

Dotted lines : strategy with subtracted probes, enriched in sequences expressed in the induced stage. Direct identification of "positive" clones.

Differential display analysis

RDA

from : Primrose & Twyman

Restriction digestion of cDNA sample (average size of fragments >200 bp)

Anneal and ligate a 12/24 linker cassette (top strand only at left side ; bottom strand at right side)

Remove the unbound 12-mer and fill-in to blunt

PCR amplification provides an ample source of tester or driver DNA

RDA : preparing tester and driver DNA

(synthetic) linkers are not phosphorylated

RDA

Remove the initial adaptor by restriction cleavage

Attach a secondary adaptor (cfr. above) only to the tester DNA

Mix tester and driver Tester/Selector in a 1/100 ratio. Denature and re-anneal.

Fill-in reaction with polymerase

Tester / Driverlinear amplification

Tester / Driverexponential amplification

Driver / Driverno amplification

PCR amplification with primers corresponding to the appropriate strand of the latter cassette

Characterize amplified products

Tester Driver

General basis of SAGE.

Anchoring enzyme : NlaIII

Tagging enzyme : FokI

(more details on following slide)

Each mRNA represented byone tag : from the poly(A)tail to the first occurrence of an NlaIII site.

FokI cleaves at 9/13 : the4-nt extension is filled-in with DNA polymerase.

(the 14/18 fragment is released from the beads and filled-in to 18-bp fragments)

Blunt-end ligation joins two18-bp fragments to 36-bp and cleavage with NlaIII reduces them to 18-bp withextra 3'-CATG-extensions at both sides.

The tags are pairwise ligatedinto the vector and pairs areseparated (and thus identified) by a CATG.

Count the numberof tags in the sequencedproducts.

An example of SAGE tag analysis in yeast (S. cerevisiae)

Functional complementationin transgenic mice using BAC clones.