Screening, selection & characterisation

1. 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. colileuB 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)

2. Activity Extract Protein Amino acid sequence Reverse translation CELL Antisera Chemical DNA synthesis Tumor line Natural condition Probe Induction Hybridisation mRNA oligo-dT chromatography DNA sequencing EST libraries polyA+ mRNA in vitro translation cDNA cloning clones Expression TEST HART HST Activity cDNA cloning : survey of tools / strategies / procedures

4. Direct selection for a clone containing the R6-5 kanamycin resistance gene (kanR)

5. Direct selection for a trpA gene using a trpA- strain of E. coli An appropriate mutant strain must be available.

6. => 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 ormixtureor 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)

7. - immunochemical methods : directly on expression product (epitope) (mature protein or fusion construct) => binding to plastic (polyvinyl) plates (colonies) or to cellulose nitrate sheets (l-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

8. 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

10. 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.

11. Heterologous probing

13. The amino acid sequence of yeast cytochrome c. The hexapeptide that is highlighted yellow is one to 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 2 16 oligonucleotides represent the entire set of possibilities.

14. The use of a synthetic, labelled or tagged oligonucleotide to identify a clone of the yeast cytochrome c gene. From probable to definite.

15. 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.

16. 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.

17. Plaque screening of clones of fusion proteins (at LacZ) using antibodies.

18. "Sandwich"-approach in probing with antibodies.

19. Activity Extract Protein Amino acid sequence Reverse translation CELL Antisera Chemical DNA synthesis Tumor line Natural condition Probe Induction Hybridisation mRNA oligo-dT chromatography DNA sequencing EST libraries polyA+ mRNA in vitro translation cDNA cloning clones Expression TEST HART HST Activity cDNA cloning : survey of tools / strategies / procedures

20. 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 Hybrid arrested translation (HArT) 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.

21. 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.

22. 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

23. - 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

24. Abundance screening Probing within a cDNA library to identify an abundant gene (mRNA).

25. 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.

26. 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-stranded by alkaline treatment. The cDNA is complementary to mRNA, also those of cell type B inasmuch as these are expressed in 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 be isolated and either used to make an A-specific cDNA library, or be used probe to screen a complete cDNA library of cell type A.

27. 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.

28. Differential display analysis

29. RDA from : Primrose & Twyman

30. 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

31. Tester Driver 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 / Driver linear amplification Tester / Driver exponential amplification Driver / Driver no amplification PCR amplification with primers corresponding to the appropriate strand of the latter cassette Characterize amplified products RDA

33. General basis of SAGE. Anchoring enzyme : NlaIII Tagging enzyme : FokI (more details on following slide)

34. Each mRNA represented by one tag : from the poly(A) tail to the first occurrence of an NlaIII site. FokI cleaves at 9/13 : the 4-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 two 18-bp fragments to 36-bp and cleavage with NlaIII reduces them to 18-bp with extra 3'-CATG-extensions at both sides. The tags are pairwise ligated into the vector and pairs are separated (and thus identified) by a CATG.

35. Count the number of tags in the sequenced products.

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

37. Functional complementation in transgenic mice using BAC clones.