Library Construction and Screening Flashcards

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1
Q

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

A
  • a ‘(semi)-random’ collection of ‘related’ molecules
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2
Q

DNA library

A
  • 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
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3
Q

Gene library

A
  • 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
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4
Q

Partial Restriction Digest

A
  • 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)
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5
Q

Preparation of a Genomic DNA library

A
  • 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
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6
Q

Steps in genomic DNA library

A
  • nucleated cells
  • undergo cell lysis, DNA extraction, partial digestion with restriction endonuclease
  • join to vector molecules and clone in bacterial and yeast cells
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7
Q

cDNA library

A
  • 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
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8
Q

Steps in forming cDNA library

A
  • 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
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9
Q

cDNA Libraries vs Genomic Libraries

A
  • 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.
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10
Q

Library screening

A
  • 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)
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11
Q

Library Screening by Colony Lift

A
  • 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
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12
Q

Colony Hybridisation on Arrays

A
  • 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
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13
Q

Colony Hybridisation

A
  • 17,664 human YAC clones
  • Array format
  • Total yeast DNA weakly-probed for background
  • human X chromosome DXYS646 probe used for strong signal
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14
Q

Representativeness of a Genomic Library

A
  • 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
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