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DNA DNA mRNA 5’ process 3’ ribosome mature mRNA mRNA cap 5’ 3’ proteins tail proteins Prokaryote Eukaryote Technique involving the insertion of a fragment of foreign DNA into a vector capable of replicating autonomously in a host cell (usually Escherichia coli). Growing the host cell allows the production of multiple copies of the inserted DNA for use in a variety of purposes. Foreign DNA Host organism Vector DNA for cloning Means of inserting foreign DNA into the vector Method of placing the in vitro modified DNA into the host cell Methods for selecting and/or screening cells that carry the inserted foreign DNA Restriction Endonucleases Type I – Long recognition sequences and random to cleavage Type II – Tetra, Penta and Hexa recognition sequences Type III – Similar to type I but specific to clevages Polymerases DNA Polymerase – catalyzes the polymerization of deoxyribonucleotides along the template strand DNA-dependent RNA Polymerase Nucleases Enzymes capable of cleaving the phosphodiester bonds between nucleotide subunits of nucleic acids Other Modifying Enzymes Ligases forms phosphodiester bonds to join two pieces of DNA utilizes ATP in the presence of Mg++ T4 DNA ligase for “blunt”or “cohesive” ends E.coli DNA ligase for “blunt” ends Kinases transfers phosphate groups from donor molecules phosphorylase Phosphatases catalyzes the removal of 5’-phosphate residues Foreign DNA Genomic DNA Complementary DNA (cDNA) Synthetic DNA Chemical Synthesis Polymerase Chain Reaction (PCR) Vector DNA DNA molecule that functions as a “molecular carrier” that carry the DNA of interest into the host cell & facilitates its replication. Plasmids – used in cloning small segments of DNA (10-15 kb) Bacteriophage – used in cloning larger segments of DNA (~20 kb) Cosmids – plasmids containing DNA sequences (cos) from bacteriophage λ used to clone larger fragments of up to 45 Kb • small circular dsDNA that autonomously replicates apart from the chromosome of the host cell • “molecular parasites” • carry one or more genes some of which confer resistance to certain antibiotics • origin of replication (ORI) --- a region of DNA that allows multiplication of the plasmid within the host • plasmid replication: stringent or relaxed Desirable properties of plasmids: small size known DNA sequence high copy number a selectable marker a second selectable gene large number of unique restriction sites • viruses that infect bacteria • known dsDNA sequence of ~ 50 kb • linear double-stranded molecule with single-stranded complementary ends • cohesive termini (cos region) http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/lambdaMap.gif Desirable properties of λ phage: • can accept large pieces of foreign DNA • tremendous improvement over the years • can be reconstituted in vitro • modified plasmids containing cos sequences • carry an ORI & an antibiotic resistance marker • can accommodate ~35 to 45 kb of foreign DNA • can be propagated as plasmids • can be introduced into host by standard procedures • chief technical problems occur when used for library construction ? Cloning or Expression ? Single or Library gene construct ? Size of foreign gene ? Restriction size ? Regulatory sequences ? Signal sequences ? Tag sequences http://www-micro.msb.le.ac.uk/109/GeneticEngineering1.gif http://www-micro.msb.le.ac.uk/109/GeneticEngineering1.gif Means of inserting foreign DNA into the vector Ligation of the DNA into the linearized vector http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/ligation.gif Requirements for a ligation reaction: • two or more fragments of DNA (blunt/cohesive) ng of vector x kb size of insert x insert:vector molar ratio = ng of insert kb size of vector • buffer containing ATP • T4 DNA ligase Method of placing the in vitro modified DNA into the host cell Transformation into the host cell • bacterial cells take up naked DNA molecules • cells are made “competent” • cells treated with ice-cold CaCl2 then heat-shocked • efficiency of 107 to 108 transformed colonies/μg DNA • maximum transformation frequency of 10-3 Electroporation of the DNA into the host cell http://bme.pe.u-tokyo.ac.jp/research/ep/img/electroporation.jpg • “electric field-mediated membrane permeabilization” • high strength electric field in the presence of DNA • protocols differ for various species • efficiencies of 109 per μg DNA (3 kb) & 106 (136 kb) Conjugation • natural transmission from donor to recipient • host cell that is not readily transformed • form cell to cell junctions Transfection of the DNA • DNA is packaged in vitro into phage particles • phages are allowed to infect bacterial cells • term also used in DNA transfer to eukaryotic cells • DNA is transiently expressed Escherichai coli Well Characterized genetic system Easily to be manipulated for maximum gene expression Release of exdotoxin Excrete very few proteins Bacillus subtilis Secretes many proteins Non- pathogenic strian Transcribe only genes from closed relative genus Vector instability Streptomeces sp. Already being used at large scale Non- pathogenic strian Transcribe genes from wide range Less understood about genetic system Methods for selecting and/or screening cells that carry the inserted foreign DNA Selection refers to application of conditions that favors the growth of cells or phages that carry the vector or vector and insert. • antibiotic resistance • nutrient requirements • plaque formation Screening allows all cells to grow, but tests the resulting clones for the presence of the insert in the vector. • restriction analysis • antibiotic resistance/sensitivity • nutrient requirements • plaque type • alpha complementation • specific (PCR, hybridization, product assay) • vector carrying lacZ gene – encodes for N-terminus of β-galactosidase (α-fragment) • host chromosome carrying C-terminal of β-galactosidase (ω-fragment) • IPTG (isopropyl-[beta]-D-thiogalactopyranoside) – inducer • X-Gal – substrate for the enzyme • cloning sites within the LacZ gene • disruption of gene by insertion of the foreign DNA • blue – functional protein • white – non-functional protein http://www.eppendorfna.com/applications/images/gel_cleanup1.jpg • vector carrying two or more antibiotic resistance genes • cloning sites within the one of antibiotic resistance gene • disruption of gene by insertion of the foreign DNA • Southern hybridization – Detection of DNA by DNA probe • Nortern hybridization – Detection of RNA by DNA probe • Western hybridization – Detection of Protein by antibody probe Isolation of desired genes DNA vector isolation Restriction enzyme digestion Restriction enzyme digestion Digested DNA fragment Digested DNA vector Ligation Recombinant plasmid Transformation into host cells Selection/Screening of recombinant clones Maxam-Gilbert's Method: Chemical Destroy → Cleavage method 32P ATCGATCG AT 32P ATCGATCGAT 32P ATCG ATCGAT Non-radioactive Specific Reaction to G (invisible) Destroy → Cleavage Sanger's Method: Biosynthetic method Keep on going Terminator ATCGA A,T,C,G A TAGCTAGCTA 32P ATCG or Producing various fragments TAGCTAGCTA STOP Terminated Template ATCG 32P A TAGCTAGCTA DNA fragments having a difference of one nucleotide can be separated Polyacrylamide Gel Electrophoresis on gel electrophoresis T A G C 32P ATCGATCGAT 32P ATCGATCGA 32P ATCGATCG 32P ATCGATC 32P ATCGAT 32P ATCGA G 32P ATCG C 32P ATC T 32P AT 32P A A But these bands can’t tell us If those band with the same the identity of the terminal terminal nucleotide can be nucleotides grouped, then it is possible to read the whole sequence Open reading frame Aminoacid sequences Hydrophobic profile Homology with other proteins Inverted repeated sequences Search for promoter region(s) or transcription site Genetically engineered E.coli to produce antigen for develop to vaccine or diagnostic kit test Genetically engineered E.coli to produce antibody for therapeutics or diagnostic kit test Genetically engineered E.coli to produce hormones for therapetics ?
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