Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Recombinant DNA Technology
Document related concepts
DNA barcoding wikipedia , lookup
DNA sequencing wikipedia , lookup
Transcriptional regulation wikipedia , lookup
Promoter (genetics) wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Molecular evolution wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Non-coding DNA wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Community fingerprinting wikipedia , lookup
DNA vaccination wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
Restriction enzyme wikipedia , lookup
DNA supercoil wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Transcript
Plasmids are excellent cloning vectors Figure 5.21 Figure 11.11 Ampicillin resistance Order of restriction enzyme cut sites in polylinker lacZʹ′ ApoI - EcoRI BanII - SacI Acc651 - KpnI AvaI - BsoBI SmaI - XmaI BamHI XbaI AccI - HincII - SalI BspMI - BfuAI SbfI PstI SphI HindIII Polylinker pUC19 2686 base pairs lacI Origin of DNA replication Figure 11.12 lacZʹ′ AmpR Foreign DNA Vector Digestion with restriction enzyme Opened vector Recyclized vector without insert Join with DNA ligase Vector plus foreign DNA insert Transform into Escherichia coli and select on ampicillin plates containing Xgal Transformants blue (β-galactosidase active) Transformants white (β-galactosidase inactive) Figure 11.18 Capsid genes J att int xis N cos Replaceable region QSR cos Wild-type lambda Charon 4A β-Gal gene Another substitution (replacement vector) Charon 16 β-Gal gene Another substitution (insertional vector) Figure 11.19 Replaceable region R L Digestion with restriction enzymes cos L R Foreign DNA R Ligation with foreign DNA L Hybrid DNA Packaging cloned DNA into phage head L R Phage assembly Infective lambda virion Figure 11.13 Bacteria Escherichia coli Bacillus subtilis Eukaryote Saccharomyces cerevisiae Well-developed genetics Many strains available Best known bacterium Easily transformed Nonpathogenic Naturally secretes proteins Endospore formation simplifies culture Well-developed genetics Nonpathogenic Can process mRNA and proteins Easy to grow Potentially pathogenic Periplasm traps proteins Genetically unstable Genetics less developed than in E. coli Plasmids unstable Will not replicate most bacterial plasmids Advantages Disadvantages Figure 11.15 oriC Ampicillin resistance t/pa ESM Promoter oriY t/pa CEN Promoter Polylinker (cloning site) Figure 11.20 BamHI SmaI EcoRI KpnI XbaI SalI PstI HindIII Polylinker lacP lacZʹ′ M13 genomic DNA Phage in clear plaques have cloned DNA Phage in blue plaques do not have cloned DNA X-‐gal Cloning Results Three major types of restric8on endonucleases Restric8on endonucleases produce blunt or s8cky ends Type II enzymes cut at palindromic sequences Restric8on endonucleases cleave DNA at specific sites Restric8on maps Figure 5.18ab Photo courtesy of FOTODYNE Incorporated. Construc8ng a restric8on map Figure 5.19ab Figure 11.5 Foreign DNA Cut with restriction enzyme Sticky ends Add vector cut with same restriction enzyme Vector Add DNA ligase to form recombinant molecules Cloned DNA Introduction of recombinant vector into a host Figure 11.9 cDNA synthesis by reverse transcriptase Figure 5.45 Figure 11.14 After gas release Before gas release Plunger Helium gas Gas vent Disc Microprojectiles with transfecting nucleic acid Fine screen Rough screen Target tissue Figure 11.6 Transformant colonies growing on agar surface Replica-plate onto membrane filter Lyse bacteria and denature DNA; add RNA or DNA probe (radioactive); wash out unbound radioactivity Partially lyse cells; add specific antibody; add agent to detect bound antibody in radiolabeled form Autoradiograph to detect radioactivity X-ray film Positive colonies Polymerase chain reac8on (PCR) to amplify DNA Figure 5.31 Southern bloHng is used to detect specific DNA fragments Figure 5.22
