Library Construction and Screening

Question 1

Library (DNA/RNA/peptides/compounds etc.)

Answer 1

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

Question 2

DNA library

Answer 2

• 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

Question 3

Gene library

Answer 3

• 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

Question 4

Partial Restriction Digest

Answer 4

• 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)

Question 5

Preparation of a Genomic DNA library

Answer 5

• 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

Question 6

Steps in genomic DNA library

Answer 6

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

Question 7

cDNA library

Answer 7

• 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

Question 8

Steps in forming cDNA library

Answer 8

• 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

Question 9

cDNA Libraries vs Genomic Libraries

Answer 9

• 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.

Question 10

Library screening

Answer 10

• 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)

Question 11

Library Screening by Colony Lift

Answer 11

• 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

Question 12

Colony Hybridisation on Arrays

Answer 12

• 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

Question 13

Colony Hybridisation

Answer 13

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

Question 14

Representativeness of a Genomic Library

Answer 14

• 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