Vectors for cloning large inserts

Why clone large (> 30 kb) inserts?

– simplifies assembly of genomic maps

– less pieces to add up

– reduces number of clones screened for desired gene

– identify location of gene in part of genome

– then subclone library from clone in lamda vectors

– allows entire eukaryotic genes to be cloned intact

– eukaryotic genes may have: – introns

– distant cis-acting regulatory sequences

– gene (& regulatory region) may be > 100 kb

cosmids – 30-45 kb fragments

advantages: – size selection

– in vitro lambda packaging

disadvantages: – stability

P1 phage vectors – 70-100 kb fragments

– derivatives of bacteriophage P1

– temperate phage of E. coli

– lysogenic growth as plasmid

(1-2 copies per cell)

– similar to F plasmid

– lytic growth as concatemers of genome

– packaged in size-dependent manner

– different than lambda packaging

advantages: – size selection

– in vitro packaging

– stable as plasmid vector

– lytic growth produces large amounts DNA

disadvantages: – packaging system inefficient

– libraries usually produced by expert labs/companies

– provide proper clones to other researchers

– identified by screening libraries

– specific probes for gene desired

P1 artificial chromosomes – up to ~ 150 kb

– plasmids containing P1 origins of replication

(lysogenic & lytic)

advantages: – stable as plasmid vector

– lytic growth produces large amounts DNA

– (but no phage particles)

– easy to work with

– clone & transform cells as plasmid vector

disadvantages: – no size selection from packaging

bacterial artificial chromosomes (BACs)

– up to ~ 300 kb

(larger plasmids too hard to handle without shearing)

– derivatives of F plasmid

advantages: – very stable plasmid vector

– low copy number

– no selection for deletion derivatives

– F plasmid par gene products prevent loss of plasmid

disadvantages:

– no size selection

– concatemers of DNA fragments may be cloned

– false map

yeast artificial chromosomes (YACs) (Fig. 9.7)

– over 1 Mb can be cloned

– 250-400 kb more common

– limit is from shearing of long DNA fragments

– function as plasmid in E. coli – pMB1 ori

– bla (ampicillin resistance)

– functions as chromosome in S. cerevisiae

– ARS origin of replication

– centromere

– 2 telomeres

– TRP1 – wild type tryptophan biosynthesis gene

– vector selection in auxotrophic yeast host cells

– SUP4 – ochre suppressor tRNA

– nonsense mutant hosts get wild type phenotype

– if SUP4 insertion inactivated by cloned DNA

– host yeast strains keeps mutant phenotype

cloning strategy – cut vector with BamHI – frees telomeres

– produces linear “chromosome” structure

– clone genomic digest into SmaI site

– transform yeast cells

advantages: – can clone more DNA than other vectors

– some versions stable in mammalian cells

– study expression of entire mammalian genes

disadvantages: – recombination common

– no plasmid incompatibility (> 1 clone per cell)

– get recombination between regions of homology

– concatemers of DNA fragments may be cloned

– yeast genetics difficult

– libraries made by experts

– maintained by genome sequencing centres