Recombinant DNA TechnologyAnas Bahnassi PhD RPh CDE CDM
The Role of Recombinant DNA Technology in Biotechnology• Recombinant DNA Technology – Intentional modification of organisms’ genomes for practical purposes – Three goals • Eliminate undesirable phenotypic traits • Combine beneficial traits of two or more organisms • Create organisms that synthesize products humans need
Bacterial cell DNA containing gene of interest Bacterial Plasmid chromosome Isolate plasmid. Gene of interest Enzymatically cleave DNA into fragments. Isolate fragment with the gene of interest.Overview of Insert gene into plasmid.recombinant Insert plasmid and gene into DNA bacterium. technology Culture bacteria. Harvest copies of gene to insert into Harvest proteins coded by gene plants or animals Eliminate Create Produce vaccines, undesirable beneficial antibiotics, phenotypic combination hormones, or traits of traits enzymes
The Tools of Recombinant DNA Technology• Mutagens – Physical and chemical agents that produce mutations – Scientists utilize mutagens to • Create changes in microbes’ genomes to change phenotypes • Select for and culture cells with beneficial characteristics – Mutated genes alone can be isolated
The Tools of Recombinant DNA Technology• The Use of Reverse Transcriptase to Synthesize cDNA – Isolated from retroviruses – Uses RNA template to transcribe molecule of cDNA – Easier to isolate mRNA molecule for desired protein first – mRNA of eukaryotes has introns removed • Allows cloning in prokaryotic cells
The Tools of Recombinant DNA Technology• Synthetic Nucleic Acids – Molecules of DNA and RNA produced in cell-free solutions – Uses of synthetic nucleic acids • Elucidating the genetic code • Creating genes for specific proteins • Synthesizing DNA and RNA probes to locate specific sequences of nucleotides • Synthesizing antisense nucleic acid molecules
The Tools of Recombinant DNA Technology• Restriction Enzymes – Bacterial enzymes that cut DNA molecules only at restriction sites – Categorized into two groups based on type of cut • Cuts with sticky ends • Cuts with blunt ends
Origin and function• Bacterial origin = enzymes that cleave foreign DNA• Named after the organism from which they were derived – EcoRI from Escherichia coli – BamHI from Bacillus amyloliquefaciens• Protect bacteria from bacteriophage infection – Restricts viral replication• Bacterium protects it’s own DNA by methylating those specific sequence motifs
Availability• Over 200 enzymes identified, many available commercially from biotechnology companies
Classes• Type I – Cuts the DNA on both strands but at a non- specific location at varying distances from the particular sequence that is recognized by the restriction enzyme – Therefore random/imprecise cuts – Not very useful for rDNA applications
Classes• Type II – Cuts both strands of DNA within the particular sequence recognized by the restriction enzyme – Used widely for molecular biology procedures – DNA sequence = symmetrical
Classes• Reads the same in the 5’ 3’ direction on both strands = Palindromic Sequence• Some enzymes generate “blunt ends” (cut in middle)• Others generate “sticky ends” (staggered cuts) – H-bonding possible with complementary tails – DNA ligase covalently links the two fragments together by forming phosphodiester bonds of the phosphate-sugar backbones
The Tools of Recombinant DNA Technology• Vectors – Nucleic acid molecules that deliver a gene into a cell – Useful properties • Small enough to manipulate in a lab • Survive inside cells • Contain recognizable genetic marker • Ensure genetic expression of gene – Include viral genomes, transposons, and plasmids
mRNA for human growth hormone (HGH) Antibiotic Restriction resistance site gene Reverse transcription cDNA for HGH Plasmid (vector) Restriction Restriction enzyme enzyme Sticky ends Gene for human growth hormoneProducing arecombinant Ligase vector Recombinant plasmid Introduce recombinant plasmid into bacteria. Bacterial Recombinant chromosome plasmid Inoculate bacteria on media containing antibiotic. Bacteria containing the plasmid with HGH gene survive because they also have resistance gene.
Requirements of a vector to serve as a carrier molecule• The choice of a vector depends on the design of the experimental system and how the cloned gene will be screened or utilized subsequently• Most vectors contain a prokaryotic origin of replication allowing maintenance in bacterial cells.
Requirements of a vector to serve as a carrier molecule• Some vectors contain an additional eukaryotic origin of replication allowing autonomous, episomal replication in eukaryotic cells.• Multiple unique cloning sites are often included for versatility and easier library construction.
Requirements of a vector to serve as a carrier molecule• Antibiotic resistance genes and/or other selectable markers enable identification of cells that have acquired the vector construct.• Some vectors contain inducible or tissue-specific promoters permitting controlled expression of introduced genes in transfected cells or transgenic animals.
Requirements of a vector to serve as a carrier molecule• Modern vectors contain multi-functional elements designed to permit a combination of cloning, DNA sequencing, in vitro mutagenesis and transcription and episomal replication.
Choice of vector• Depends on nature of protocol or experiment• Type of host cell to accommodate rDNA – Prokaryotic – Eukaryotic
Plasmid vector• Covalently closed, circular, double stranded DNA molecules that occur naturally and replicate extrachromosomally in bacteria• Many confer drug resistance to bacterial strains• Origin of replication present (ORI)
The Tools of Recombinant DNA Technology• Gene Libraries – A collection of bacterial or phage clones • Each clone in library often contains one gene of an organism’s genome – Library may contain all genes of a single chromosome – Library may contain set of cDNA complementary to mRNA
Genome Isolate genome or organism. Generate fragments using restriction enzymes.Production of a Insert each fragment into a vector. gene library- overview Introduce vectors into cells. Culture recombinant cells; descendants are clones.
Definition of recombinant DNA• Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other• DNA fragments are usually derived from different biological sources
Steps involved in isolating a particular DNA fragment from a complex mixture of DNA fragments or molecules1. DNA molecules are digested with enzymes called restriction endonucleases which reduces the size of the fragments Renders them more manageable for cloning purposes
Steps involved in isolating a particular DNA fragment from a complex mixture of DNA fragments or molecules2. These products of digestion are inserted into a DNA molecule called a vector Enables desired fragment to be replicated in cell culture to very high levels in a given cell (copy #)
Steps involved in isolating a particular DNA fragment from a complex mixture of DNA fragments or molecules3. Introduction of recombinant DNA molecule into an appropriate host cell – Transformation or transfection – Each cell receiving rDNA = CLONE – May have thousands of copies of rDNA molecules/cell after DNA replication – As host cell divides, rDNA partitioned into daughter cells
Steps involved in isolating a particular DNA fragment from a complex mixture of DNA fragments or molecules4. Population of cells of a given clone is expanded, and therefore so is the rDNA. – Amplification – DNA can be extracted, purified and used for molecular analyses • Investigate organization of genes • Structure/function • Activation • Processing – Gene product encoded by that rDNA can be characterized or modified through mutational experiments