Types of cloning vectors
1. plasmids (about 20kb)
2. Bacteriophage (bacterial viruses), 30-50kb inserts
3. Cosmids (35-50kb insert)
4. BACs
•
Use fertility F plasmid
•
75-300kb inserts possible
•
developed during the human genome project
5. YACs
•
Mimics yeast chromosome
•
Contains all regions for replication (yeast ori and
centromere)
•
100-1000kb inserts poss.
•
developed during the human genome project
Viral vectors
1. Bacteriophage Lambda
• Virus of E.coli
• Lytic or Lysogenic life style
• Virulent phage
– do not integrate their DNA into the host
bacterium genome and they usually kill the
host (lytic)
•
Temperate phage may integrate into the
host genome causing lysogeny
)
Bacteriophage lambda vectors are commonly used for
construction of genomic libraries
Packaging of bacteriophage lambda
Packaging of DNA into the head does not require a full
length of wild type DNA. Length of between 78-105%
can be packaged (37-53kb)
• To get packaging need 2 cos ends one at each end.
• Usually 50 kb apart and this ensures only 50kb gets
packaged
• If further than 53kb , no packaging
• An enzyme at entrance to head recognises and cuts at
cos site
• During replication, the phage DNA is produced in a concatameric
form, which is cleaved by appropriate endonucleases to allow
packaging of a single genome within the phage capsid.
• It was found that internal regions of the phage genome, which were
not essential to phage replication, could be removed and replaced
with DNA of interest.
• This hybrid DNA could be efficiently packaged, and form an infective
phage.
Advantages of this type of system vs plasmids like pBR322
o The phage genome is able to package efficiently with DNA inserts
as large as 20 Kb.
o the packaged phage are highly infectious and infect E. coli at a
much higher efficiency than plasmid transformation methods.
Bacteriophage Lambda vectors
• Derivatives have been manipulated to have unique
restriction enzyme sites
• Some are insertion some are replacement vectors
• Some have replacement areas flanked by cutting sites
• 40% of the wild type lambda is dispensible and can be
replaced
e.g. of lambda cloning vector (see diagram)
• lambda sequences have been devised with 2 Bam H
sites flanking the insertion excision region
• When cut with Bam get 3 segments
• Left arm has info for heads and tails
• Right arm has info for DNA replication and lysis
• Middle fragment can be replaced with cloned DNA about
20kb long.
replacement vector
replacement vectors
Insertion Vector
-Can not fit as much new foreign DNA in
In vitro packaging systems
• Can be made
• Purified empty heads, lambda DNA with foreign
insert (50kb with cos ends), and tail assemblies
in test tube.
• Result in infective bacteriophage
• Uses
• Cloning pieces too big for plasmids
• Generation of ds DNA
M13 , a single stranded filamentous phage
•
Phage DNA is packaged in the core of a helical particle
•
The length of particle is dependant on length of DNA
•
In all M13 preps the following occur
•
polyphage- more than one genome length
•
minphage- 0.2-0.5 genome length
•
maxiphage- genetically defective but more than one genome
length
•
Molecular biologists use this to create cloning vectors
•
Can insert long stretches of DNA into non essential regions
•
No sharp cut off in length that can be packaged
•
Some decrease in efficiency of packaging with increasing length
•
10% longer not affected, 50% longer replicate more slowly
How M13 infects and reproduces
•
infects through pili
•
Protein coat is stripped and ss DNA is converted to double
stranded replicative form
•
DNA relicated by “rolling circle method”
•
New particles assembled
•
200 particles per infected cell per generation
•
M13 released without lysis
•
No lysis on bacterial lawn, generally do in liquid culture.
Uses
•
•
•
Cloning
Ss DNA for probes, sequencing
Phage display technology, M13 will produce foreign protein on
surface as part of its protein coat, can use to generate specific
antibodies
Adeno virus
• Human virus
• Used to insert DNA into
human cells
• Gene therapy
• Normally causes
respiratory diseases
and pink eye. Want
virus to infect our cells
but not cause illness
Cosmids
• Cross between phage and plasmid
• Circular ds DNA
• Contain cos sites and can be packaged
into phage heads
• Carry more DNA than plasmid and can
be maintained and manipulated as
plasmids
Cloning using cosmids
• Clone into vector as would with plasmid
• Introduce DNA to cell as you would with
phage
• Propagate as you would a plasmid
• Cosmids have about 5kb DNA and
therefore can get ~ 33-48kb foreign DNA
into a phage head.
Measurement of viral growth
• Must grow virus on host cells to see anything. Can't
grow virus without cells.
• To quantify viruses, need some way to get flat surface
of growing cells, allow virus-infected cells to spread
radially where present = plaque.
• In bacterial cells this is easy. Spread "lawn" of bacteria
on plate, add diluted phage suspension or culture
infected with phages. After 6-8 hours can see plaques in
E. coli.
Bacteria have three mechanisms to accomplish genetic
recombination (mixing up of DNA):
1. transformation -acquire new genes by taking up DNA
molecules from their surroundings
2. conjugation -can transfer a portion of their chromosome to a
recipient with which they are in direct contact usually via a
plasmid
3. transduction- via Bacteriophages - in the process of
assembling new virus particles, some host DNA may be
incorporated in them
Bacterial artificial chromosomes (BAC):
The F factor plasmid has the ability to continue to function even when
integrated into a complete bacterial chromosome.
Highly modified F plasmids have been generated that are capable of cloning
very large inserts of up to 300,000 base pairs.
One feature is the incorporation of cut sites for restriction endonucleases with
eight base cut sites.
Such endonucleases cut DNA less frequently and thus generate larger
fragments for cloning.
Bacterial artificial chromosomes are sometimes introduced into their host cells
by electroporation, which consists of a brief treatment with high voltage electric
current that momentarily disrupts the cell membranes and facilitates entry of
large DNA molecules.
Once in the cell, the BAC replicate like F plasmids.
Yeast Artificial Chromosomes (YAC):
a yeast artificial chromosome (YAC) contains
a yeast origin of replication a centromere,
a telomere at each end
a large inserted DNA sequence of up to about 500 kb
Prior to insertion of the foreign DNA, the essential components of the YAC are
maintained in bacterial cells as circular plasmids.