Shanes Faculty, JNC DBT, Blore 95.

Types of Vectors

Plasmid DNA E. coli vectors, extra-chromosomal and circular Bacteriophages Phage l clone large DNA fragments and incorporate into host genome Phage M13 allows cloned DNA to be isolated in single-stranded form Cosmids hybrids of plasmid-bacteriophage l Artificial chromosomes - Cloning of very large genomic fragments - BACs (bacterial artificial chromosomes) - YACs (yeast artificial chromosomes

Types Plasmid Lambda phage Cosmids BACs YACs

Inserts <10000 bp 10-15 kbp 45 kbp 300kbp 1000kbp

Figure 11.11

Ampicillin resistance

Order of restriction enzyme cut sites in polylinker

lacZ ApoI - EcoRI BanII - SacI Acc651 - KpnI AvaI - BsoBI SmaI - XmaI BamHI XbaI AccI - HincII - SalI BspMI - BfuAI SbfI PstI SphI HindIII lacI

Polylinker

pUC19
2686 base pairs

Origin of DNA replication

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lacZ AmpR

Vector

Foreign DNA

Digestion with restriction enzyme

Opened vector

Join with DNA ligase

Recyclized vector without insert

Transform into Escherichia coli and select on ampicillin plates containing Xgal

Vector plus foreign DNA insert

Transformants blue (-galactosidase active)

Transformants white (-galactosidase inactive)

 viruses that infect bacteria known dsDNA sequence of ~ 50 kb

linear double-stranded molecule with

single-stranded complementary ends cohesive termini (cos region)

http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/lambdaMap.gif

Desirable properties of phage:

can accept large pieces of foreign DNA

tremendous improvement over the years

can be reconstituted in vitro

Bacteriophage l

l phage genome linear 48.5 kb genome.

Each ends consists

of cos (cohesive) sites 12 bp cos ends Cos ends allows DNA circularization in the cell

Central region of

genome are nonessential portions and can be replaced by foreign DNA (up to 23kb)

 Phage particles injects linear DNA into the cell

DNA ligate to form circle

Replicate to form many new phage particles which are released by cell lysis and cell death or DNA intergrate to host genome by site-specific recombination (lysogenic phase)

Lambda Cloning Vectors

Wild-type lambda phage genome: 48.5 kb

~20 kb
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Bacteriophage Lambda as a Cloning Vector

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bacteria lawn plaques l bacteriophage

Plaques: the clear areas within the lawn where lysis and re-infection have prevented the cells from growing.

M13 phage vectors

1. Replication form (RF, dsDNA) of M13 phage can be purified and manipulated like a plasmid. 2. Phage particles (ssDNA): DNA can be isolated in a single-stranded form
DNA sequencing Site-directed mutagenesis

Cloning (RF, like plasmid) transfection (recombinant DNA) growth (plating on a cell lawn) plaques formation (slow growth)

Hybrid plasmid-M13 vectors

Small plasmid vectors (pBluescript) being developed to incorporate M13 functionality Contain both the plasmid and M13 origin of replication

Normally propagate as true plasmids

Can be induced to form single-stranded phage particles by infection of the host cell with a helper phage.

M13 phage

M13 phage contains a circular 6.7kb ssDNA

Replicate in E. coli cells as double-stranded circles (replicative form, RF), ~ 100 copies per cell

Cells are not lysed by M13, but grow slowly.

Recombinant M13 phage can produce either - dsDNA RF can be isolated & manipulated as plasmid - ssDNA isolated from phage particles in growth medium ( used for DNA sequencing and site-directed mutagenesis)

M13 phage cloning vectors

M13 RF containing cloned fragment (eg. M13amp18 and 19)

- Transfect into E.coli cells - plating in a lawn of cells produce plaques - Plaques consist of slow growth rather than lysis of infected cells - Blue-white selection using MCS and lacZ

Hybrid plasmid M13 vectors (eg. pBlueScript)

- eveloped to incorporate M13 functionality - contain plasmid & M13 origin of replication, minus the genes for full phage life cycle. - propagate as true plasmid - can be induced to form single-stranded phage particles by infection of the host cell with a helper phage, provides the gene products required for ss production and packaging

Cloning Using Bacteriophage M13

Bacteriophage M13 Insert size: 5 kb White and Blue colony

Figure 12.24a

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Cosmid vectors
1. Utilizing the properties of the phage l cos sites in a plasmid vector. 2. A combination of the plasmid vector and the COS site which allows the target DNA to be inserted into the l head. 3. The insert can be 37-52 kb.

Formation of a cosmid clone

Digestion

Ligation

Vectors for Genomic Cloning and Sequencing

BACs
Constructed from the F plasmid
Host for a BAC is generally a mutant strain of E. coli

YACs
Can accommodate 200800 kilobases of cloned DNA Replicate like normal yeast chromosomes

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Genetic Map of a Bacterial Artificial Chromosome

F plasmid (99.2 kb) origin

BAC: ~6.7 kb
Figure 12.25

can carry > 300 kb of foreign DNA

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A Yeast Artificial Chromosome Containing Foreign DNA

YAC: 10 kb Yeast artificial chromosome can carry 200~800 kb telomere for replication of linear chromosome centromere for segregation during mitosis origin of DNA replication
Figure 12.26

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Manipulating eukaryotes, bridge from prokaryotes