CLONING VECTORS

A "cloning Vector" is any biochemical vehicle that facilitates insertion of a DNA sequence into an appropriate host.

Specific Cloning Vectors include:

• 1.Plasmids
• 2.Bacteriophage
• 3.Cosmids
• 4.Yeast artificial chromosomes (YACs)
• 5.Bacterial artificial chromosomes (BACs)
• l
• Deftn: “Amplify”- to increase the number of (inserted DNA segments).

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Vector 1: Plasmids

• lSmall, circular DNA molecules that replicate separately from the host chromosome (Xm)
• lIn nature, usually 5,000 – 400,000 bp in length
• lPlasmids can be introduced into cells via “Transformation”

What is Transformation?"

• lA method of placing “new” DNA into host cells
• lIncubate cells + DNA in CaCl2 at 0ºC
• lApply heat shock (raise temp to 37-43ºC)
• lA few cells in the host bacteria population take up the plasmid DNA
• lMust have a method of isolating the few transformed cells
• lAn inexpensive way of amplifying DNA

 

Characteristics of a “Useful" Plasmid  (i.e. pbr322)

• 1.Makes at least 10-20 copies per cell
• 2.2 genes that provide resistance to antibiotics present
• 3.Has several recognition sequences for different R.E. so it can be easily cut and foreign DNA inserted
• 4.Small size – easier to enter cells (bigger plasmids have reduced transformation efficiency)

http://athena.uwindsor.ca/units/biochem/web/protein.nsf/18e8732806421826852569830050331b/7a371e9af805f74e85256a4f00538021/$FILE/11

 

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 Vector 2: Bacteriophage λ (Phage)

• A bacteriophage is a virus capable of replicating in a bacterial (host) cell – example: “Lambda”
• •Can clone larger DNA fragments than plasmid vectors
• •1/3 of phage genome (DNA) is non-essential (therefore can be replaced with foreign DNA)
• •Size specific – only DNA ~ 40-50 kbp will be inserted into phage vector

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Vectors 3: Cosmids

• •Overcome insert size limit of plasmid and bacteriophage vectors (15-20 KB) •
• Cosmid vectors are plasmid / bacteriophage hybrids •
• Contain cohesive site (cos) of phage (required for in vitro packaging)
• •Accept inserts of 35 to 45 kb

 

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Different Cloning Vectors for Different Applications

 

Different types of cloning vector can transfer did sizes of DNA fragments:

 

Standard high copy number plasmid   <10 kbp (inserted sequence size in kbp)

Bacteriophage l insertion <10 kbp

Bacteriophage l replacement  9-23 kbp

Cosmid 30-44 kbp

Bacteriophage P1  70-100 kbp

PAC (P1 Artificial Chromosome)  130-150 kbp

BAC (Bacterial Artificial Chromosome)  < 300

YAC (Yeast Artificial Chromosome)  0.2-2000 kbp

http://www.biotechlearn.org.nz/var/biotechlearn/storage/images/themes/from_genes_to_genomes/images/dna_cloning_overview/4211-1-eng-AU/dna_cloning_overview_large.jpg

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