Recombinant DNA Technology - GenBio2 Lesson 1 PDF

Recombinant DNA Technology  Reference Books 1. Hortons’ Principles of Biochemistry 4th edition 2. Molecular Biology, Robert Weaver 2nd edition 3. Harpers’ Biochemistry 26th edition 4. Styers’ Biochemistry Objectives: At the end of the session, the learners should be able to:...

Recombinant DNA Technology  Reference Books 1. Hortons’ Principles of Biochemistry 4th edition 2. Molecular Biology, Robert Weaver 2nd edition 3. Harpers’ Biochemistry 26th edition 4. Styers’ Biochemistry Objectives: At the end of the session, the learners should be able to: 1. Outline the processes involved in genetic engineering - STEM_BIO11/12-IIIa-b-6 2. Discuss the applications of recombinant DNA - STEM_BIO11/12-IIIa-b-7 (15 mins) Film Viewing https://www.youtube.com/watch?v=ZW9zPdb_Bs0 Buzz Session 1. What is rDNA? What is its common term? 2. How does it occur and what is the process? 3. Advantages of rDNA? Disadvantages?  Recombinant DNA and Gene Cloning Recombinant DNA (rDNA) is a form of artificial DNA that is created by combining two or more sequences that would not normally occur together through the process of gene splicing. Recombinant DNA technology is a technology which allows DNA to be produced via artificial means. The procedure has been used to change DNA in living organisms and may have even more practical uses in the future. Chinese Dragon 龙  The dragon is a mythical creature that can fly and walk. Dragon can change its form and has divine powers to summon wind and rain. The dragons are said to be made up of many different types of animals of the Earth.  The Nine parts of A Chinese Dragon Dragon is an imagination creature, which has deer's antlers, camel's head, hare's eye, snake's neck, carp's scales, eagle's claws, tiger's paws; and ox's ears.  Recombinant DNA: Cloning and Creation of Chimeric Genes Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists named Boyer and Cohen in 1973.  Stanley N. Cohen , who received the Nobel Prize in Medicine in 1986 for his work on discoveries of growth factors. Stanley N. Cohen (1935–) (top) and Herbert Boyer (1936–) (bottom), who constructed the first recombinant DNA using bacterial DNA and plasmids. What is Recombinant DNA Technology? Recombinant DNA technology is a technology which allows DNA to be produced via artificial means. The procedure has been used to change DNA in living organisms and may have even more practical uses in the future. It is an area of medical science that is just beginning to be researched in a concerted effort.  Recombinant DNA technology works by taking DNA from two different sources and combining that DNA into a single molecule. That alone, however, will not do much. Recombinant DNA technology only becomes useful when that artificially-created DNA is reproduced. This is known as DNA cloning. Brief Introduction Recombinant DNA Technology 1. The basic concepts for recombinant DNA technology 2. The basic procedures of recombinant DNA technology 3. Application of recombinant DNA technology  The basic concepts for recombinant DNA technology  In the early 1970s, technologies for the laboratory manipulation of nucleic acids emerged. In turn, these technologies led to the construction of DNA molecules composed of nucleotide sequences taken from different sources. The products of these innovations, recombinant DNA molecules, opened exciting new avenues of investigation in molecular biology and genetics, and a new field was born— recombinant DNA technology.  Concept of Recombinant DNA Recombinant DNA is a molecule that combines DNA from two sources. Also known as gene cloning. Creates a new combination of genetic material – Human gene for insulin was placed in bacteria – The bacteria are recombinant organisms and produce insulin in large quantities for diabetics – Genetically engineered drug in 1986 Genetically modified organisms are possible because of the universal nature of the genetic code!  Genetic engineering is the application of this technology to the manipulation of genes. These advances were made possible by methods for amplification of any particular DNA segment( how? ), regardless of source, within bacterial host cells. Or, in the language of recombinant DNA technology, the cloning of virtually any DNA sequence became feasible.  Recombinant technology begins with the isolation of a gene of interest (target gene). The target gene is then inserted into the plasmid or phage (vector) to form replicon. The replicon is then introduced into host cells to cloned and either express the protein or not. The cloned replicon is referred to as recombinant DNA. The procedure is called recombinant DNA technology. Cloning is necessary to produce numerous copies of the DNA since the initial supply is inadequate to insert into host cells.  Some other terms are also in common use to describe genetic engineering. Gene manipulation Recombinant DNA technology Gene cloning (Molecular cloning) Genetic modification  Cloning——In classical biology, a clone is a population of identical organisms derived from a single parental organism. For example, the members of a colony of bacterial cells that arise from a single cell on a petri plate are clones. Molecular biology has borrowed the term to mean a collection of molecules or cells all identical to an original molecule or cell.  Recombinant DNA technology——A series of procedures used to join together (recombine) DNA segments. A recombinant DNA molecule is constructed (recombined) from segments from 2 or more different DNA molecules. Under certain conditions, a recombinant DNA molecule can enter a cell and replicate there, autonomously (on its own) or after it has become integrated into a chromosome. How recombinant technology works These steps include isolating of the target gene and the vector, specific cutting of DNA at defined sites, joining or splicing of DNA fragments, transforming of replicon to host cell, cloning, selecting of the positive cells containing recombinant DNA, and either express or not in the end. Six steps of Recombinant DNA 1. Isolating (vector and target gene) 2. Cutting (Cleavage) 3. Joining (Ligation) 4. Transforming 5. Cloning 6. Selecting (Screening) Recombinant DNA Technology 1. The basic concepts for recombinant DNA technology 2. The basic procedures of recombinant DNA technology 3. Application of recombinant DNA technology  The basic procedures of recombinant DNA technology  DNA molecules that are constructed with DNA from different sources are called recombinant DNA molecules. Recombinant DNA molecules are created in nature more often than in the laboratory; – for example, every time a bacteria phage or eukaryotic virus infects its host cell and integrates its DNA into the host genome, a recombinant is created. – Occasionally, these viruses pick up a fragment of host DNA when they excise from their host’s genome; these naturally occurring recombinant DNA molecules have been used to study some genes.  Six basic steps are common to most recombinant DNA experiments 1. Isolation and purification of DNA. Both vector and target DNA molecules can be prepared by a variety of routine methods, which are not discussed here. In some cases, the target DNA is synthesized in vitro. 2. Cleavage of DNA at particular sequences. As we will see, cleaving DNA to generate fragments of defined length, or with specific endpoints, is crucial to recombinant DNA technology. The DNA fragment of interest is called insert DNA. In the laboratory, DNA is usually cleaved by treating it with commercially produced nucleases and restriction endonucleases. 3. Ligation of DNA fragments. A recombinant DNA molecule is usually formed by cleaving the DNA of interest to yield insert DNA and then ligating the insert DNA to vector DNA (recombinant DNA or chimeric DNA). DNA fragments are typically joined using DNA ligase (also commercially produced). – T4 DNA Ligase 4. Introduction of recombinant DNA into compatible host cells. In order to be propagated, the recombinant DNA molecule (insert DNA joined to vector DNA) must be introduced into a compatible host cell where it can replicate. The direct uptake of foreign DNA by a host cell is called genetic transformation (or transformation). Recombinant DNA can also be packaged into virus particles and transferred to host cells by transfection. 5. Replication and expression of recombinant DNA in host cells. Cloning vectors allow insert DNA to be replicated and, in some cases, expressed in a host cell. The ability to clone and express DNA efficiently depends on the choice of appropriate vectors and hosts. 6. Identification of host cells that contain recombinant DNA of interest. Vectors usually contain easily scored genetic markers, or genes, that allow the selection of host cells that have taken up foreign DNA. The identification of a particular DNA fragment usually involves an additional step—screening a large number of recombinant DNA clones. This is almost always the most difficult step. DNA cloning in a plasmid vector permits amplification of a DNA fragment.  First step: Isolating DNA 1. Vector 2. Target gene  How to get a target genes? 1. Genomic DNA 2. Artificial synthesis 3. PCR amplification 4. RT-PCR  Polymerase chain reaction (PCR) A technique called the polymerase chain reaction (PCR) has revolutionized recombinant DNA technology. It can amplify DNA from as little material as a single cell and from very old tissue such as that isolated from Egyptian mummies, a frozen mammoth, and insects trapped in ancient amber. method is used to amplify DNA sequences The polymerase chain reaction (PCR) can quickly clone a small Initial sample of DNA in a DNA segment test tube Number of DNA molecules PCR primers  RT-PCR Reverse transcription polymerase chain reaction (RT-PCR) is a variant of polymerase chain reaction (PCR. In RT-PCR, however, an RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional. – Template:RNA – Products: cDNA  Vectors- Cloning Vehicles Cloning vectors can be plasmids, bacteriophage, viruses, or even small artificial chromosomes. Most vectors contain sequences that allow them to be replicated autonomously within a compatible host cell, whereas a minority carry sequences that facilitate integration into the host genome.  All cloning vectors have in common at least one unique cloning site, a sequence that can be cut by a restriction endonuclease to allow site-specific insertion of foreign DNA. The most useful vectors have several restriction sites grouped together in a multiple cloning site (MCS) called a polylinker.  Types of vector 1. Plasmid Vectors 2. Bacteriophage Vectors 3. Virus vectors  Types of blotting techniques Southern blotting Southern blotting techniques is the first nucleic acid blotting procedure developed in 1975 by Southern. Southern blotting is the techniques for the specific identification of DNA molecules. Northern blotting Northern blotting is the techniques for the specific identification of RNA molecules. Western blotting Western blotting involves the identification of proteins. Antigen + antibody  Expression of Proteins Using Recombinant DNA Technology Cloned or amplified DNA can be purified and sequenced, used to produce RNA and protein, or introduced into organisms with the goal of changing their phenotype. One of the reasons recombinant DNA technology has had such a large impact on biochemistry is that it has overcome many of the difficulties inherent in purifying low-abundance proteins and determining their amino acid sequences.  Recombinant DNA technology allows the protein to be purified without further characterization. Purification begins with overproduction of the protein in a cell containing an expression vector. – Prokaryotic Expression Vectors – Eukaryotic Expression Vectors  Recombinant DNA Technology 1. The basic concepts for recombinant DNA technology 2. The basic procedures of recombinant DNA technology 3. Application of recombinant DNA technology Applications of Recombinant DNA Technology 1. Analysis of Gene Structure and Expression 2. Pharmaceutical Products – Drugs – Vaccines 3. Genetically modified organisms (GMO) – Transgenic plants – Transgenic animal 4. Application in medicine 5. ……  Analysis of Gene Structure and Expression Using specialized recombinant DNA techniques, researchers have determined vast amounts of DNA sequence including the entire genomic sequence of humans and many key experimental organisms. This enormous volume of data, which is growing at a rapid pace, has been stored and organized in two primary data banks: the GenBank at the National Institutes of Health, Bethesda, Maryland, and the EMBL Sequence Data Base at the European Molecular Biology Laboratory in Heidelberg, Germany.  Pharmaceutical Products Some pharmaceutical applications of DNA technology: Large-scale production of human hormones and other proteins with therapeutic uses Production of safer vaccines A number of therapeutic gene products —insulin, the interleukins, interferons, growth hormones, erythropoietin, and coagulation factor VIII—are now produced commercially from cloned genes Pharmaceutical companies already are producing molecules made by recombinant DNA to treat human diseases. Recombinant bacteria are used in the production of human growth hormone and human insulin Use recombinant cells to mass produce proteins – Bacteria – Yeast – Mammalian  Growth hormone Insulin deficiency – Hormone required to – Faulty pituitary and properly process sugars regulation and fats – Had to rely on cadaver – Treat diabetes source – Now easily produced by – Now easily produced by bacteria bacteria Subunit Herpes Vaccine Not always used for good... High doses of HGH can cause permanent side effects – As adults normal growth has stopped so excessive GH can thicken bones and enlarge organs Genetically modified organisms (GMO) Use of recombinant plasmids in agriculture – plants with genetically desirable traits herbicide or pesticide resistant corn & soybean – Decreases chemical insecticide use – Increases production “Golden rice” with beta-carotene – Required to make vitamin A, which in deficiency causes blindness  Crops have been developed that are better tasting, stay fresh longer, and are protected from disease and insect infestations. “Golden rice” has been genetically modified to contain beta-carotene  Genetic Engineering of Plants Plants have been bred for millennia to enhance certain desirable characteristics in important food crops. Transgenic plants. The luciferase gene from a firefly is transformed into tobacco plant using the Ti plasmid. Watering the plant with a solution of luciferin (the substrate for firefly luciferase) results in the generation of light by all plant tissues. Insect-resistant tomato plants The plant on the left contains a gene that encodes a bacterial protein that is toxic to certain insects that feed on tomato plants. The plant on the right is a wild-type plant. Only the plant on the left is able to grow when exposed to the insects.  Transgenic animals Green fluorescence Red fluorescence Transgenic animals A transgenic mouse Mouse on right is normal; mouse on left is transgenic animal expressing rat growth hormone Farm Animals and “Pharm” Animals Transgenic plants and animals have genes from other organisms. These transgenic sheep carry a gene for a human blood protein – This protein may help in the treatment of cystic fibrosis just a joke  Other benefits of GMOs Disease resistance There are many viruses, fungi, bacteria that cause plant diseases “Super-shrimp” Cold tolerance Antifreeze gene from cold water fish introduced to tobacco and potato plants Drought tolerance & Salinity tolerance As populations expand, potential to grow crops in otherwise inhospitable environments Where in the world? Downsides??? Introduce allergens? Pass trans-genes to wild populations? – Pollinator transfer R&D is costly – Patents to insure profits Patent infringements Lawsuits potential for capitalism to overshadow humanitarian efforts  Application in medicine Human Gene Therapy Diagnosis of genetic disorders Forensic Evidence  Human Gene Therapy Human gene therapy seeks to repair the damage caused by a genetic deficiency through introduction of a functional version of the defective gene. To achieve this end, a cloned variant of the gene must be incorporated into the organism in such a manner that it is expressed only at the proper time and only in appropriate cell types. At this time, these conditions impose serious technical and clinical difficulties.  Gene therapy is the alteration of an afflicted individual’s genes Gene therapy holds great potential for treating disorders traceable to a single defective gene Vectors are used for delivery of genes into cells Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations  Many gene therapies have received approval from the National Institutes of Health for trials in human patients, including the introduction of gene constructs into patients. Among these are constructs designed to cure ADA- SCID (severe combined immunodeficiency due to adenosine deaminase [ADA] deficiency), neuroblastoma, or cystic fibrosis, or to treat cancer through expression of the E1A and p53 tumor suppressor genes. Cloned gene Insert RNA version of normal allele into retrovirus. Viral RNA Let retrovirus infect bone marrow cells Retrovirus that have been removed from the capsid patient and cultured. Somatic cells Only! Viral DNA carrying the normal allele inserts into chromosome. Not for reproductive cells Bone marrow !! cell from patient Bone Inject engineered marrow cells into patient.  However, there are some challenging issues that need to be considered: 1. In mammalian cells, mRNA is processed before it is translated into a protein: – Introns are cut out and exons are spliced together – Bacteria can not process mRNA 2. Post-translational modifications – Enzymatic modifications of protein molecules after they are synthesized in cells – Post-translational modifications include: Disulfide bond formation (catalyzed by disulfide isomerases) and protein folding Glycosylation (addition of sugar molecules to protein backbone, catalyzed by glycosyl transferases) Proteolysis (clipping of protein molecule, e.g., processing of proinsulin to insulin) Sulfation, phosphorylation (addition of sulfate, phosphate groups) 3. Recombinant proteins are particularly susceptible to proteolytic degradation in bacteria 4. Recombinant protein may accumulate in bacteria as refractile inclusion bodies  So how can these problems be tackled? Problem: 1. mRNA processing in mammalian cells but not in bacteria Solution: Synthesize chemically gene containing only exons and insert that into vector; or, Make cDNA by reverse-transcription of processed mRNA (using the enzyme reverse transcriptase) Problem: 2. Bacteria cannot perform post-translational modifications Solution: This is a tough one! Only proteins that do not undergo extensive post-translational processing can be synthesized in bacteria Problem: 3. Recombinant proteins particularly susceptible to proteolysis Solution: Design fusion protein consisting of an endogenous bacterial protein connected to the recombinant protein through a specific amino acid sequence. Fusion protein is then specifically cleaved at the fusion site The Hope  Summary 1. Recombinant DNA technology builds on a few basic techniques: isolation of DNA, cleavage of DNA at particular sequences, ligation of DNA fragments, introduction of DNA into host cells, replication and expression of DNA, and identification of host cells that contain recombinants. 2. DNA Fragments generated by restriction endonucleases can be ligated into a wide range of cloning vectors, including: plasmids, bacteriophage, viruses, or artificial chromosomes. 3. Cells containing recombinant DNA molecules can be selected, often by the activity of a marker gene. Cells containing the desired recombinant are identified by screening. 4. The product of a gene that has been incorporated into an appropriate expression vector can be generated in prokaryotic or eukaryotic cells. Foreign genes can also be stably incorporated into the genomes of animals and plants. 5. Recombinant DNA methods allow the production of proteins for therapeutic use and the identification of individuals with genetic defects.