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DNA Technology Recombinant DNA Technology Chapter 4 Recombinant DNA Technology Recombinant DNA Technology Generation of recombinant DNA molecule Cloning vector-insert DNA construct (DNA construct) Cut DNA from a donor organism Cloned DNA, insert DNA, target DNA, foreign DNA Ligation to a cloning vector DNA Transformation Introduction and maintain the DNA construct within a host cell Selection of transformed cells Production of the foreign protein in the host (optional) Recombinant DNA Technology DNA Cloning Preparation of Insert DNA Genomic cDNA PCR DNA Joining to a vector Ligation Recombination E. coli transformation and selection 1. Cutting DNA Molecules Cutting DNA Molecules Mechanical shearing Discovery of endonuclease Meselson and Yuan, 1968, from E. coli Smith and Wilcox, 1970 from Haemophilus influenzae Werner Arber, Daniel Nathans and Hamilton Smith :Nobel Prize for physiology or Medicine, 1978 Restriction and Modification Bacterial protection fd system against Invasion of foreign E. coli K DNA Phage fd K fd K Modified during growth in E.coli K E. coli K E. coli B Restricted in growth in E.coli B Restriction and Modification Restriction: Endonuclease Modification: Methylase Types of Endonuclease System Recognition Cleavage System Enzyme Subunits sites sites 3 (Recognition, Type I 1 Methylation, No feature Up to 1 kb away Cleavage) 2 (Cleavage, Type II Symmetrical Recognition site Modification) 2 (Recognition and Type III 1 Modification, Symmetrical 24-26 bp away Cleavage) Up to 20 bp 2 (Cleavage, Type IIs Asymmetrical away Modification) Advantage of Type II systems Separate restriction and modification : Cleavage without modification No cofactors necessary for restriction activity Recognize defined sequence (symmetrical, palindromic sequence) Cut within the recognition sequence Nomenclature Sma I 1st enzyme Serratia marcescencs : species Hind III 3rd enzyme Strain d Haemophilus influenzae : species Cleavage by EcoRI Recognition site: GAATTC Symmetrical staggered cleavage 5’ overhang, protruding ends, sticky ends 5’ phosphate and 3’ hydroxyl group Cleavage by HindII Recognition site: GTTAAC Blund-end cleavage Restriction Patterns Cohesive ends 5’ overhang: Major, EcoRI, BamHI, etc. 5’ G/AATTC 3’ 5’ G AATTC 3’ 3’ CTTAA/G 5’ 3’ CTTAA G 5’ 3’ overhang: PstI, KpnI 5’ CTGCA/G 3’ 5’ CTGCA G 3’ 3’ G/ACGTC 3’ G ACGTC 5’ Blunt ends SmaI, EcoRV 5’ CCC/GGG 3’ 5’ CCC GGG 3’ 3’ GGG/CCC 5’ 3’ GGG CCC 5 Recognition Sequences >5000 enzymes http://rebase.neb.com/rebase/rebase.html 4-Base Cutters DpnI/Sau3AI, AluI 6-Base Cutters EcoRI, BglII, PvuII 8-Base Cutters NotI, SbfI Restriction Enzymes Isoschizomer Enzymes that recognize the same target DNA sequence and cleave it in the same way e.g. SphI and BbuI (CGTAC/G) Neoschizomer Enzymes that recognizes the same target DNA sequence but cleave at different points e.g. SmaI (CCC/GGG) and XmaI (C/CCGGG) Isocaudomers Enzymes that produce the same nucleotide extensions but have different recognition sites e.g. BamHI (G/GATCC) and Sau3AI (/GATC) Methylases in E. coli Frequency Methylase Recognition Sequence (if 50% GC) dam GAmTC (N6) 256 bp dcm CmCA/TGG (C5) 512 bp M.EcoKI AAmCGTGC GCAmCGTT (N6) 8 kb Restriction enzymes have different preference for methylated DNA e.g. MboI (GATC), DpnI (GAmTC), Sau3AI (GAmTC, GATC) EcoRII (CCA/TGG), BstNI (CmCA/TGG, CCA/TGG) Reduced transformation efficiency of methylated DNA to other species: use dam-, dcm- strain for DNA preparation 2. Separation of DNA Molecules Gel Electrophoresis . Electrophoresis A technique used to separate macromolecules (proteins and nucleic acids) that differ in size, charge or conformation Migration of molecules in an electric field DNA (negative charge): migrate toward positive pole Protein: migrate either positive or negative pole according to their charge SDS PAGE: proteins are treated with sodium dodecyl sulfate (SDS) Similar charge to mass ratio Migration according to the molecular weight Types of Gel Agarose Polysaccharide extracted from seaweed 0.5 to 2% Used for DNA and RNA Large range of separation (0.1 to 50 kb DNA) Low resolving power Polyacrylamide Cross-linked polymer of acrylamide 3.5 to 20%. Used for DNA, RNA, and protein Small range of separation (<500 bp DNA) High resolving power Inhibition of polymerization process by oxygen Neurotoxin Agarose Gel Electrophoresis Migration of DNA in Agarose Gel Molecular weight of DNA Conformation of DNA Supercoil > Linear> Nicked circle Agarose Concentration Higher concentration : better separation of smaller DNAs low concentrations : better resolution of larger DNAs Migration distance Log10 MW Migration of DNA in Agarose Gel (2) Voltage High voltage Lower resolution of large DNA For the resolution of DNA larger than 2 kb <5 volts/cm (between two electrode) Electrophoresis buffer TAE (Tris-acetate-EDTA), TBE (Tris-borate-EDTA) Provide ions to support conductivity Establish pH Ethidium Bromide A fluorescent dye that intercalates between bases of nucleic acids Restriction Mapping of DNA Cut DNA with various endonuclease Determination of the sizes of the restriction fragments by gel electrophoresis 3. Enzymes for Recombinant DNA Technology DNA 5’ End labeling 1 Calf intestine alkaline phosphatase Dephosphorylation of 5’ end T4 polynucleotide kinase Addition of radioisotope-labeled g32P-ATP g-phosphate from g- 32P ATP DNA 5’ End labeling 2 Filling in reaction with Klenow fragment a32P-dATP Enzymes Used for Recombinant DNA Technology Alkaline phosphatase DNaseI Digestion of dsDNA E. coli exonulcease III Digestion from recessive or blunt 3’ OH ends Klenow fragment E.coli DNA polymerase I with polymerase and 3’ exonuclease activity Mung bean nuclease Digestion of ssDNA and RNA Enzymes Used for Recombinant DNA Technology Poly(A) polymerase Addition of AMP to the 3’ end of mRNA Reverse transcriptase Synthesis of DNA from RNA RNaseH Degrades the RNA strand from a DNA-RNA hybrid S1 nuclease Digestion of ssDNA 4. Joining DNA Molecules Cloning DNA Annealing of cohesive ends by base-pairing Generation of nick Variations on Cutting and Joining DNA Compatible cohesive ends 5’ A/CCGGT 3’ AgeI 3’ TGGCC/A 5’ + + 5’ A/CCGGT 3’ 5’ C/CCGGG 3’ AgeI AvaI 3’ TGGCC/A 5’ 3’ GGGCC/C5’ 5’ A/CCGGT 3’ 5’ A/CCGGT 3 5’ A/CCGGG 3’ 5’ C/CCGGT 3’ 3’ TGGCC/A 5’ 3’ TGGCC/A 5 3’ TGGCC/C 5’ 3’ GGGCC/A5’ Blunt ends 5’ CCC/GGG 3’ 5’ GAT/ATC 3’ SmaI EcoRV 3’ GGG/CCC 5’ + 3’ CTA/TAG5 5’ CCC/ATC 3’ 5’ GAT/GGG 3’ 3’ GGG/TAG 5’ 3’ CTA/CCC5’ DNA ligase Formation of phosphodiester bonds between 3’ OH and 5’ phosphate T4 DNA ligase E. coli ligase Ligation Conditions Temperature Consider enzyme activity and base pairing of cohesive termini Cohesive ends: 4-15oC: ensure base pairing Blunt ends: 18oC, use 10 to 100 times higher concentration of T4 DNA ligase DNA concentration Dilute concentration favors circulization of linear fragment Insert : Vector = 2 : 1 molar ratio Phosphatase treatment Prevention of self ligation of vector Ligation Strategy Linker linker Blunt ends DNA containing restriction enzyme site RE Adaptor Chemically synthesized DNA with cohesive ends adaptor Cloning of PCR Products Pfu polymerase Blunt end ligation Taq polymerase: Blunt end ligation after filling in with Klenow Use T/A cloning with a vector containing 3’ T Addition of restriction enzyme sites at the end of primers Add additional 3-4 nucleotide for efficient cleavage R1 PCR 5’ 3’ 3’ 5’ R2 R1 R2 Efficiency of Enzyme Digestion % Cleavage Enzyme Oligo Sequence Chain length 2 hr 20 hr CGGATCCG 8 10 25 BamHI CGGGATCCCG 10 >90 >90 CGCGGATCCGCG 12 >90 >90 GGAATTCC 8 >90 >90 EcoRI CGGAATTCCG 10 >90 >90 CCGGAATTCCGG 12 >90 >90 CAAGCTTG 8 0 0 HindIII CCAAGCTTGG 10 0 0 CCCAAGCTTGGG 12 10 75 CCCGGG 6 0 10 CCCCGGGG 8 0 10 Sma I CCCCCGGGGG 10 10 50 TCCCCCGGGGGA 12 >90 >90 CTCTAGAG 8 0 0 GCTCTAGAGC 10 >90 >90 Xba I TGCTCTAGAGCA 12 75 >90 CTAGTCTAGACTAG 14 75 >90 Cloning Using in vitro Recombination Vector and insert with recognition sites for site- specific recombinase l integrase Flp recombinase Recombinase Recombination of Phage l in E. coli Int: integrase IHF: integration host Phage l factor Xis : Excisonase E. coli Lysogen Recombination of Phage l in E. coli 7 bp core region is responsible for specificity Invitrogen Cloning Using Recombination ccdB : Encoding toxin counter selection marker gene Generated by PCR attL1 attL2 ccdB Entry Clone attP1 attP2 attB1 gene attB2 attR1 ccdB attR2 Donor KanR + Vector + KanR BP Clonase™ II Int, IHF 90-99% correct clones on Kan plates gene attB1 attB2 ccdB Expression Clone attR1 attR2 Destination AmpR + Vector + AmpR LR Clonase™ II Int, IHF, Xis 90-99% correct clones on Amp plates Invitrogen.
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