p.1
01. 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 cloning strategies
DNA fragment preparation:
- genomic versus cDNA strategy
- why cDNA?
- expressed information
- only fraction of the genome
- non-uniform :some genes (mRNA’s may be present in high amounts)
- intron-less sequences
- usually poly(A)-tailed : target for copying
Overview of library strategy:
=> fragmentation, insertion, primary library, amplification & storage
cDNA libraries
- 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
G. Volckaert
Genetic engineering : making DNA libraries
17/02/2016
p.2
- directional cloning (“forced” cloning)
(Okayama-Berg, extended primers, RAcE)
- RAcE
- RT-PCR
- relative representation in library : high/low/intermediate abundance mRNA's
- problem of “full-size” copying and “full-size” cloning
- CAPture technique
- oligocapping technique
- ZAP-vectors
- survey of cDNA cloning strategies/tools/procedures interconnections
(see Part 2 : Screening and selection)
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
- 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.
G. Volckaert
Genetic engineering : making DNA libraries
17/02/2016
p.3
- chromosome walking
- chromosome jumping
not in 2007-2008
High capacity vectors
lambda phage (replacement vectors)
cosmids - fosmids
30-45 kb
P1 “phage” vectors
70-100 kb
20-25 kb
pac (packaging site)
loxP sites, Cre recombinase (cre+ host)
P1 lytic replicon to high copy number
PAC (artificial chromosomes) combines P1 phage vector with BAC (F-replicon)
100-300 kb
BAC (artificial chromosomes) 100-300 kb and more
sex factor F replicon
direct transformation of E. coli (electroporation)
high stability
improvements with new restriction sites
and selection markers
sacB positive selection (sucrose-sensitivity)
YAC (artificial chromosomes) 200-2000 kb and more
minimal size 50 kb to m
aintain stably
CEN + TEL + ARS + selection marker
problem of chimerism
clones are often unstable (deletion of internal regions)
linear DNA’s : ‘returning’ to E.coli only by extra steps
[TAC
G. Volckaert
transformation-competent artificial chromosomes, and other --AC’s]
Genetic engineering : making DNA libraries
17/02/2016