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
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Types of cloning vectors
1. plasmids (about 20kb)
2. Bacteriophage (bacterial viruses), 30-50kb inserts
3. Cosmids (35-50kb insert)
1. Bacteriophage Lambda
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)
Advantages of this type of system vs plasmids like pBR322
Bacteriophage Lambda vectors
e.g. of lambda cloning vector (see diagram)
-Can not fit as much new foreign DNA in
In vitro packaging systems
M13 , a single stranded filamentous phage
How M13 infects and reproduces
Cloning using cosmids
Measurement of viral growth
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.