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
Blunt ends and sticky ends “B| unt ends e.g. Sma I, Hae I, any other blunt-cutter CCCGGG GCC GGGCCC CCGG CCC CC CCCCC -GGG GG - GGGGG
Blunt ends and sticky ends • “Blunt ends” • e.g. Sma I, Hae III, any other blunt-cutter CCCGGG GGGCCC GGCC CCGG CCC GGG CC GG + CCCCC GGGGG
Plasmid: Cloning Vectors Origin of replication Selectable marker Cloning sites(unique) Shuttle vectors grow in more than one host cell
Plasmid: Cloning Vectors • Origin of replication • Selectable marker • Cloning sites (unique) • Shuttle vectors • grow in more than one host cell
Using plasmid vectors (a)Human DNA Plasmid vectors EcoRI site EcoRI site Q Origin of replication a are cut with Ecai ectors DNA and plasmid v Gene for ampicillin resistance Cleaved fragments and vectors are combined in the presence of ligase. Ligase (b)Recombinant plasmids are added to a population of E. coli cells Host chromosome 9os E coli plated onto medium containing ampicillin Cells containing recombinant plasmids are able to grow
Phage and cosmid vectors Foreign DNa Phage n chromosome fragment tithe tr gt fo eiazypna faplmcn 2 lika phag Incorporate into virus capsules 入R Infect E. coli host cells One bacterial cell Lawn of e. coli cells infected with recombinant viral dna Clear plaques contain many copies of single recombinant phage
Phage and cosmid vectors Phage A DNA Plasmid Incorporate into virus capsules Restriction cos sites amp site Or g n o 息息 replication Infect E. coli host cells Cleave dNA and plasmid with restriction enzyme. Splice cos DNA into plasmid cos sites F Cosmid vector Select for ampicillin resistance by growing infected cells on medium Linearize cosmid at restriction site containing ampicillin Combine with genomic DNA and ligate Genomic dna strands Surviving cells contain recombinant plasmids which may be cloned. Incorporate into virus capsules
