Recombinant DNA technology Restriction enzymes are used for cutting DNA at precise locations Vectors are used for the propagation of cloned DNA fragments Libraries of cloned dNA fragments allow searching for a specific DNA fragment
Recombinant DNA technology • Restriction enzymes are used for cutting DNA at precise locations • Vectors are used for the propagation of cloned DNA fragments • Libraries of cloned DNA fragments allow searching for a specific DNA fragment
Restriction Enzymes Nucleases: Exonuclease: digests from ends Endonuclease: cleaves internally Restriction Enzyme: They are synthesized by bacteria to protect themselves from invading viruses class of endonucleases which cleaves DNA molecules at specific nucleotide sequences called restriction sites 1978 Nobel Prize: Arber, Smith Nathans
Restriction Enzymes • Nucleases: Exonuclease: digests from ends Endonuclease: cleaves internally • Restriction Enzyme: They are synthesized by bacteria to protect themselves from invading viruses. • class of endonucleases which cleaves DNA molecules at specific nucleotide sequences called restriction sites. • 1978 Nobel Prize: Arber, Smith & Nathans
Naming of enzymes Over 150 different restriction endonucleases have been identified Isolated from different bacteria Recognize specific DNA sequences which are usually 4-8 bp long Escherichia coli yields several different enzymes, one is called EcoRI. The E is derived from the genus, and the second and third letters (co)# from the species name. The numbers refer to the strain in this case RY13
Naming of enzymes • Over 150 different restriction endonucleases have been identified. • Isolated from different bacteria • Recognize specific DNA sequences which are usually 4-8 bp long • Escherichia coli yields several different enzymes, one is called EcoRI. The E is derived from the genus, and the second and third letters (co) # from the species name. The numbers refer to the strain, in this case RY13
Restriction Enzyme Specificity e.g Eco RI: GAATTC Bam HI GGA“6- cutters Frequency: 4b= average of 1 cut site per 4,096 bp e.g. Sau 3A GATC “4- cutter Frequency: 44=average of 1 cut site per 256 bp e.g. Not I GCGGCCGO“8- cutter Frequency: 48=average of 1 cut site per 65, 536
Restriction Enzyme Specificity • e.g. Eco RI: GAATTC Bam HI GGATCC “6-cutters” • Frequency: 46 = average of 1 cut site per 4,096 bp • e.g. Sau 3A GATC “4-cutter” • Frequency: 44 = average of 1 cut site per 256 bp • e.g. Not I GCGGCCGC “8-cutter” • Frequency: 48 = average of 1 cut site per 65,536
Palindromes Axis of twofold rotational symmetry 5 3″ GAAT T C H I il fi Hit C下AAG 3 5 Madam 'm adam mada n’ madaM
Palindromes Madam I’m Adam madA m’I madaM