Recombinant DNA Technology Common General Cloning Strategy Target DNA from donor organism extracted, cut with restriction endonuclease and ligated into a cloning vector cut with compatible restriction endonuclease Recombinant construct transferred into host cell Host cells which do not take up construct are eliminated by selection protocol Host cell library screened to identify desired clone if necessary
Cloning Into Bacterial Cells Restriction Endonuclease DNA Ligase transformation
DNA Cleavage By Restriction Endonucleases (2)
Recognition Sequences of Restriction Endonucleases
Restricti Mapping Restriction Endonuclease Cleavage Sites on Mappin g
DNA fragment sizes (in kilobase pairs) after single and double restriction endonucleases digestions of a plasmid
Restriction Endonuclease Cleavage Map Created from single and multiple enzyme digestions Useful markers for noting gene locations and subcloning strategies
Enzymes Used In Recombinant DNA Protocols
Annealing of Complementary Sticky Ends
T4 DNA Ligase Action
Early Cloning Vectors pbr322 Plasmid Small independent replicon with selectable markers and useful cloning sites
Cloning DNA Into a Plasmid Vector Restriction endonuclease cleave vector/target Phosphatase vector Ligate target into vector Transform into host cells
Other Plasmid Cloning Vectors Now too many to count Many specialized for expression, etc. puc series Multiple cloning sites Improved reporter/selection genes
Multiple Cloning Sites Synthetic oligonucleotide construction Polymer of cutting sites Can be included in reporter gene coding sequence (e.g. lacz )
Antibiotics Commonly Used as Selective Agents
Creating and Screening a Recombinant DNA Library A library is a collection of subdivided portions of a larger genetic element or genome Commonly created by partial digestion of genomic DNA with restriction endonuclease and cloning the fragments into vectors (plasmid, phage, etc.) Resultant transformed collection of cells is called a library
Partial Restriction Endonuclease Digestion of DNAs
Partial Digestion Profile Collect fragments of a given target size after digestions for different times or using different restriction endonuclease concentrations Size fractionate and combine fractions of desired target size
Genome Sizes of Various Organisms The number and size of library clones required to be screened to find a single copy gene varies according to the genome size of the organism to be studied
Screening by Hybridization Probes: DNA or RNA 100+ bp in size good Sequence match >80% best Stringency conditions
Production of Labeled Probes Random Primer Method
Three Activities of E. coli DNAP I (1) (1) Polymerization of dntps at the 3 end of the growing chain (1) 5 exonuclease removes nucleotides from 5 end of chain immediately upstream of growing chain (2) 3 exonuclease removes unpaired nucleotides from 3 end of growing chain
Three Activities of E. coli DNAP I (2) (2) (3) Note that the 5 exonuclease is used in nick translation and the 3 exonuclease activity is used for the proofreading function
Screening Colonies by Hybridization Nucleic acid probe Cells transferred to nylon membrane and lysed DNA binds to membrane, is denatured and probe hybridized Bound probe detected by autoradiography after washing membrane
Screening by Immunological Assay
Screening by Functional Complementation Requires strain unable to produce desired product/function Cloned DNAs must be in expression vector or include elements required for expression Select for restoration of lost function
Isolation of Poly(adenylated) mrnas Matrix
cdna Synthesis Oligo(dT) primer Reverse transcriptase Klenow/DNAP I RNase H Degrades RNA of DNA:RNA hybrid S1 nuclease Degrades ss nucleic acids (unpaired loop)
Enriching for Full adapter Length cdnas (1) Primer has adapter (RE cutting sequence) Ribose ends of mrna are biotinylated RNase I degrades ss RNA Only full length cdna is still attached to a biotinylated mrna (biotin still on 5 end) Capture full length copies
Enriching for Full Length cdnas (2) RNase H degrades mrna Add poly(g) to cdna Primer/Adapter with oligo (C) DNAP I (Klenow) Restriction endonucleases Cut Vector DNA Ligase Transform
Inert Capacities Common Vector Systems
Bacteriophage Lambda Life Cycle Lysogenic phage Lysogeny vs. lytic cycle Chromosome about 50 kb Protein coat for efficient delivery into cells (E. coli) Packages DNA 38-52 kb with cos sites at each end DNA Replication is by rolling circle mechanism
Packaging of λ Chromosomes Natural DNA is concatemer with cos sites separated by about 50 kb (from rolling circle replication DNA is cleaved at cos and inserted into capsid
Mature λ Phage DNA packaged in protein coat Looks much like a lunar lander (actually has six tail fibers)
Phage λ cloning vector Internal segment deleted (now requires helper phage to replicate) Has cos sites intact Target DNA inserted between the two λ arms (up to about 20 kb) DNA packaged in vitro Recombinant phage infect E. coli cells
Cosmid Cloning System λ cos sites inserted into a small plasmid Target DNA ligated between two cosmid DNA molecules Recombinant DNA packaged and E. coli Infected as before Can clone DNAs up to 45 kb
High-Capacity Bacterial Vector Systems 100-300 kb target size P1 bacterial systems F plasmid systems BACs (bacterial artificial chromosomes
Genetic Transformation of Prokaryotes Chemical transformation Usually involves CaCl 2 and heat shock Transformation frequency about 1/1000 Electroporation Electric field mediated membrane permeabilization 10-100 times more efficient that chemical approach Much better for large plasmids (100+ kb)
Electroporation Cells suspended in DNA solution in cuvette between two electrodes High voltage electric field pulses administered DNA migrates through HVEF induced openings in cells
Conjugation Natural system of transmitting plasmids from one cell/strain to another Requires specific DNA sequences on transferred plasmid and certain proteins which can be provided in trans Plasmids of >10 6 bp can be transferred in this manner Can be interspecies Tripartite mating and multiple selection
Tripartite Mating P. putida difficult to transform Transform mobilizable recombinant plasmid into E.coli Make culture with P. putida (wt), recombinant E. coli (auxotroph) and E. coli (aux) with conjugative mobilizable plasmid Recombinant plasmid transferred to P. putida