Library Construction and Screening

• a ‘(semi)-random’ collection of ‘related’ molecules

• collection of plasmid vectors with same backbone/different DNA fragments • mutants of the same gene • multiple fragments from e.g. a (partial) digest of a complete genome • a set of genes created by e.g. cDNA or PCR generated fragments

• collection of clones that represent total genome of an entire organism • genomic DNA (gDNA) library: from total genomic DNA of organism • cDNA library: from cDNA copies of total RNA or mRNAs in specific tissue or cells of organism

• Key requirement • Low concentration, Short digestion time (not a full digest, multiple random overlapping fragments) • Sized suitably for selected cloning vector • e.g. Sau3A or MboI which cut at tetra-nucleotide restriction sites (frequent)

• A1, A2, A3 & A4 are identical DNA molecules (i.e. the same sequence) e.g. genomic DNA molecules • Partial digestion with enzyme that occurs with high frequency results in multiple unique digestion patterns • Each library member (i.e. plasmid vector with fragment inserted) contains only 1 fragment and the fragments overlap. 1000s clones may be needed to include whole genome

• nucleated cells • undergo cell lysis, DNA extraction, partial digestion with restriction endonuclease • join to vector molecules and clone in bacterial and yeast cells

• coding DNA library, created from pooled mRNA, often from a specific tissue • Requirements: the isolation of mRNA, cDNA synthesis, cloning • Often used with multicellular organisms: differentiated cells, different tissues, confirmation/detection of (alternative) splicing patterns

• cells from specific organ, tissue, or development stage • cell lysis • DNA extraction • purification of total RNA, poly(A) mRNAs, small RNAs • RT with primers: oligo(dT), random hexamers, 3’ ligated tails • RNase H digests RNA strand in RNA-DNA hybrid • 3’ hairpin self-primes SS cDNA • DNApol copies primed cDNA • S1 nuclease cleaves SS DNA loop • ds cDNAs produced

• No fragmentation • Only expressed genes from source • Shorter than genes • Fewer clones needed = greater vector choice • Isolate TUs from the cells/tissue in which the gene(s) of interest preferentially expressed • E.g. pancreatic beta cells if insulin gene of interest. • E.g. myosin from muscle cells • Gene copy no. // expression intensity.

• Cloning result: hundreds to hundreds of thousands of clones • Only useful if clones can be screened for sequence of interest: colony hybridisation (blotting), PCR-based screening, immunochemical assays (insert expression), functional assay (insert function)

• membrane • plaques or colonies on a petri dish • blotting cells to membrane • cell lysis, DNA denaturation and fixing on membrane • incubation with probes and washing of unhybridized probes • hybridized DNA

• DNA library clones spotted onto nitrocellulose or nylon membrane • High density grid array • Cells lysed • DNA denatured in situ and covalently linked to membrane • Labelled probe hybridised to samples • Positive clones identified and isolated for study

• 17,664 human YAC clones • Array format • Total yeast DNA weakly-probed for background • human X chromosome DXYS646 probe used for strong signal

• 𝑁= ln⁡(1−𝑃)/ln⁡(1−𝑓) • 𝑁= the number of clones required • 𝑃= the probability of finding a particular sequence • 𝑓= the ratio of the length of the insert to the entire genome