p.1
Cloning strategies & DNA libraries
See
Primrose, Twyman & Old
Principles of Gene Manipulation, 6th edition (2002)
Chapters 6 (partim: pages 85-102)
Primrose & Twyman
Principles of Gene Manipulation and Genomics, 7th edition (2006)
Chapters 6 (partim: pages 96-111 and 77-79 and 213-214)
Overview of library strategy:
=> fragmentation, insertion, primary library, amplification & storage
Overview of cloning strategies
DNA fragment preparation:
- genomic versus cDNA strategy
- why cDNA?
Genomic libraries:
- from DNA fragment to GENE
- from genome to library : partial restriction cleavage or shearing
- collection of clones : DNA library : overlapping set of fragments
- tetramer cleavages :
- Maniatis human library (partial cleavages with AluI, HaeIII) + EcoRI-linkers
- GATC (Sau3AI) => produce large (partial) fragments of 20 or > kb
G. Volckaert
DNA libraries
12/02/2016
p.2
- how many clones ? Carbon & Clarke : N = ln(1-P)/ln(1-f)
(99% probability => P = 0.99)
(f = fraction of genome per insert)
= minimum : deviations by non-random character (G+C content, etc),
survival differences by amplification; diploid character; etc.
N = number of clones required
f = fraction of the genome in one clone
= 1/n when n = the minimal number of inserts to cover the entire genome
(= the absolute minimum, i.e. if there are no overlaps between the inserts)
f is ordinarily very small (in a "real" genome cloning experiment)
if f is small, then ln(1-f) ~ -f = -1/n and thus N = -n . ln(1-P)
at 90%
99%
(P=0.9)
ln(1-P) = ln(0.1) = - 2.3 => N = 2.3 x n
(P=0.09)
ln(1-P) = ln(0.01) = - 4.6
=> N = 4.6 x n
99.9% (P=0.009) ln(1-P) = ln(0.001) = - 6.9 => N = 6.9 x n
etc.
- chromosome walking
- overlapping clone inserts
- use of terminal fragments as probes to select neighbouring clones
- cosmid pWE vectors : insertions close to GATC end
T7 RNA polymerase
- chromosome jumping
- use of TAG-plasmids : supF marker
- linking the distal ends of fragments
e.g. NotI sites
G. Volckaert
DNA libraries
12/02/2016
p.3
cDNA cloning & libraries
- abundance classes of mRNA
- relative representation in library : high/low/intermediate abundance mRNA's
- survey of technical interrelationships
=> testing activities
=> mRNA to cDNA and clones
=> from protein to reverse translation and hybridisation
=> detection of a clone, sequencing, expression
- preparation of cDNA
- reverse transcription with oligo(dT)n
- initial approach with terminal loop
=> insertion by homopolymeric tailing
=> insertion using linkers (after methylation)
- avoiding nuclease S1 treatment
- Gubler & Hoffman procedure : no loop, RNase H + DNA polymerase I
=> using tailing or linkers or adaptors
- Okayama & Berg procedure : vector as primer
- RT-PCR
- RAcE
- directional cloning (“forced” cloning)
(Okayama-Berg, extended primers, RAcE)
- the problem of “full-size” copying and “full-size” cloning
- CAPture technique
- oligocapping technique
- ZAP-vectors : cloning in -vectors ; excision with filamentous phage
- survey of cDNA cloning strategies/tools/procedures interconnections
(see Part : Screening and selection)
G. Volckaert
DNA libraries
12/02/2016