Restriction Enzymes
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Restriction enzymes are enzymes that cut DNA at certain sequences. They were originally purified from bacteria. In bacteria, restriction enzymes are a defence against viruses and other cellular invaders, but scientists have taken advantage of their DNA-cutting abilities.
Each restriction enzyme recognizes a different sequence within DNA. For example, EcoRI, a common restriction enzyme, recognizes the nucleotide sequence GAATTC. In the DNA strand below, the enzyme would first find the sequence GAATTC, then cut the strand after the G. Note that the sequence GAATTC, read 5’ to 3’ on one strand, corresponds to a sequence of CTTAAG on the other strand.
The DNA sequence has been fragmented into three pieces. If a strand of DNA has 10 sites for cutting, there will be 11 smaller pieces of DNA after it has been digested by a restriction enzyme. Note that EcoR1 will cut after a G only when the following sequence is AATTC.
There are many different kinds of restriction enzymes, and they all recognize different sequences. A scientist will choose a certain restriction enzyme depending on where they want their DNA cut. Some examples of restriction enzymes are EcoR1 (as discussed above), HindIII, BamH1, TaqI, NotI, MstII and AluI. Most restriction enzymes leave overhangs, which are also called sticky ends. These can be use to join two pieces of foreign DNA. A few restriction enzymes, like AluI, leave blunt ends, where there is no overhang of unpaired nucleotides.
Each restriction enzyme recognizes a different sequence within DNA. For example, EcoRI, a common restriction enzyme, recognizes the nucleotide sequence GAATTC. In the DNA strand below, the enzyme would first find the sequence GAATTC, then cut the strand after the G. Note that the sequence GAATTC, read 5’ to 3’ on one strand, corresponds to a sequence of CTTAAG on the other strand.
ATTACCTAGAATTCATCCGGCT - Nucleotide sequence recognized
TAATGGATCTTAAGTAGGCCGA
TAATGGATCTTAAGTAGGCCGA
ATTACCTAG AATTCATCCGGCT - Enzyme cuts DNA at the recognized location
TAATGGATCTTAA GTAGGCCGA
The result of the cut is two separate strands of DNA, with an overhanging end. If there had been another GAATTC sequence within the strand, three pieces would be made, like in the following example.
TAATGGATCTTAA GTAGGCCGA
ATTACCTAGAATTCATCCGGCTCCTTAGGGAATTCAAGGCCTTTCCGATC
TAATGGATCTTAAGTAGGCCGAGGAATCCCTTAAGTTCCGGAAAGGCTAG
TAATGGATCTTAAGTAGGCCGAGGAATCCCTTAAGTTCCGGAAAGGCTAG
ATTACCTAG AATTCATCCGGCTCCTTAGGG AATTCAAGGCCTTTCCGATC
TAATGGATCTTAA GTAGGCCGAGGAATCCCTTAA GTTCCGGAAAGGCTAG
TAATGGATCTTAA GTAGGCCGAGGAATCCCTTAA GTTCCGGAAAGGCTAG
The DNA sequence has been fragmented into three pieces. If a strand of DNA has 10 sites for cutting, there will be 11 smaller pieces of DNA after it has been digested by a restriction enzyme. Note that EcoR1 will cut after a G only when the following sequence is AATTC.
There are many different kinds of restriction enzymes, and they all recognize different sequences. A scientist will choose a certain restriction enzyme depending on where they want their DNA cut. Some examples of restriction enzymes are EcoR1 (as discussed above), HindIII, BamH1, TaqI, NotI, MstII and AluI. Most restriction enzymes leave overhangs, which are also called sticky ends. These can be use to join two pieces of foreign DNA. A few restriction enzymes, like AluI, leave blunt ends, where there is no overhang of unpaired nucleotides.