Library Construction and Screening Flashcards
1
Q
Library (DNA/RNA/peptides/compounds etc.)
A
- a ‘(semi)-random’ collection of ‘related’ molecules
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
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
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)
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
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
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
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
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.
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)
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
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
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
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
