Download Restriction Endonucleases and Plasmids

Restriction Endonucleases,
Plasmids, and
Transformation
Restriction Endonucleases
• Enzymes purified from bacteria
• Protect bacterial cell from viruses by
degrading viral DNA
• Each endonuclease recognizes a specific
sequence of 4-8 nucleotides
• Cleave between deoxyribose and
phosphate
• Bacterial DNA is protected from
degradation by methylating an A or C
Types of Restriction Endonucleases
• Some produce staggered cuts, producing sticky
ends
• Sticky ends allow any two DNA fragments to be
easily joined together as long as those fragments
are generated by the same endonuclease (Result:
recombinant DNA)
• 5’ overhangs
• 3’ overhangs
• Some restriction endonucleases produce
blunt ends
• Notice that base pair sequences are often
palindromic (nucleotide sequence is the
same if the sequence is turned by 180
degrees around the center)
Plasmids
• Naturally occurring, circular,
extrachromosomal DNA found in bacteria
• Carry genes for metabolic activities that
are advantageous to the host bacterium
– E.g., resistance to antibiotics, heavy metals,
bacteriophages
• Cloning vectors
– Must have an origin of replication (ori)
– Must have a gene for an antibiotic resistance
Restriction Maps
• Show restriction endonuclease cleavage
sites
• Procedure:
– Digest plasmid with restriction endonucleases
with individual enzymes and with paired
enzymes
– Run digests out on an agarose gel
– Use a marker to find out the size of the
fragments
Transformation
• Occurs when a cell takes up and express
a new piece of genetic material
• New trait is observed (e.g., antibiotic
resistance; frost, pest, or drought
resistance; green fluorescence)
http://www.phschool.com/science/biology_place/labbench/lab6/trananim.html
Bacterial cells are permeable to Cl-1, but not Ca2+.
Ca2+ makes plasmid +vely charged.
Cl1- enters cell with an influx of water.
Entry of plasmid is further facilitated by
heat shocking the cells at 42oC, by making the
membrane more porous.
Competent cells