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Restriction Enzymes
DNA-cutting enzymes which recognizes one or a few target sequences
Endonucleases
break the sugar phosphate backbone at internal sites
Restriction Enzymes
endonucleases that recognize specific base sequences and break or restrict the DNA polymer at the sugar-phosphate backbone
Genetic Engineering
stimulated by the discovery of deoxyriboendonucleases or endonucleases
BamHI
isolated from Bacillus amyloliquefaciens H
HindIII
isolated from Haemophilus influenzae Rd
SmaI
isolated from Serratia marcescens
DNA recombination
combination of two genes in a single host
DNA Cloning
set of procedures that uses living cells to make many identical copies of a DNA fragment
Plasmid
circular form of dsDNA which is a good template to introduce genes
pBR322
antimicrobial resistance gene
EcoR1
restriction enzyme that cuts pBR322
Type I Restriction Enzymes
have both nuclease and methylase activity in a single enzyme. Bind to host-specific DNA that contains methylated adenines
Type II Restriction Enzymes
used most frequently in the laboratory; do not have inherent methylation activity in the same molecule as the nuclease activity
Type II Restriction Enzymes
palindromic/bilateral symmetry
Prokaryotes
Type II Restriction Enzymes have been found in almost all
Overhangs
cuts the DNA duplex with a staggered separation at the recognition site; produces sticky ends
Blunt
cuts the DNA duplex at the same place on both strands
Type III Restriction Enzymes
resemble type I enzymes in their ability to methylate and restrict (cut) DNA; do not always able to provide complete restriction enzyme digestion
Type III Restriction Enzymes
adenine methylation occurs on only one strand.
Type IV Restriction Enzymes
can only cleave methylated DNA and sequence specificity is weak
Type V Restriction Enzymes
Requires guide DNA to target specific sequences and it is these that are being modified or used in genome engineering methods such as TALENS or CRISPR-Cas9
CRISPR
Clustered Regularly Interspaced Short Palindromic Repeats
restriction endonucleases
provide a convenient tool for molecular characterization of DNA
Restriction map
DNA is exposed to several restriction enzymes separately and then in particular combinations
pattern of fragments
produced by restriction enzyme digestion that can be used to identify that DNA and monitor changes in size,structure or DNA sequence
Restriction Fragment Length Polymorphisms (RFLPs)
resulting differences in the size or number of restriction fragments; used for the clinical analysis of structural changes in chromosomes associated with disease