Introduction to Recombinant DNA Technology PDF

STBP1013 Fundamentals of Molecular Biology Recombinant Introduction to Recombinant DNA Technology 1 2 Recombinant...

STBP1013 Fundamentals of Molecular Biology Recombinant Introduction to Recombinant DNA Technology 1 2 Recombinant Recombinant DNA A recombinant organism A form of artificial DNA that is created by combining – an organism that contains a different combination two or more sequences that would not normally of alleles from either of its parents occur together Recombinant virus Involves cloning and creation of chimeric genes – a virus formed by recombining genetic material Recombinant DNA – a form of artificial DNA 3 4 Recombinant DNA technology Recombinant DNA technology Joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry 5 6 1  Recombinant DNA technology Recombinant DNA technology Since the focus of all genetics is the gene, the A technology which allows DNA to be produced via fundamental goal of laboratory geneticists is to isolate, artificial means characterise and manipulate genes The procedure has been used to change DNA in living Although it is relatively easy to isolate a sample of DNA organisms and may have even more practical uses in from a collection of cells, finding a specific gene within the future this DNA sample can be compared to finding a needle in a haystack 7 8 Recombinant DNA technology Recombinant DNA technology Researchers at UC San Francisco and Stanford used Stanley N. Cohen restriction enzymes to cut DNA from different species at Researcher at Stanford University Was amongst the first to produce a recombinant DNA molecule specific sites, and then fused the cut strands from the Together with Herbert Boyer, applied for and were granted the different species back together first patent on recombinant DNA technology First recombinant DNA was produced Herbert Boyer – 1972 Researcher at University of California San Francisco Was amongst the first to produce a recombinant DNA molecule First publications on recombinant DNA Together with Stanley N. Cohen, applied for and were granted – 1972 and 1973 the first patent on recombinant DNA technology First patent on recombinant DNA technology Paul Berg – 1980 (Stanley N. Cohen & Herbert Boyer) Researcher at Stanford University Was amongst the first to produce a recombinant DNA molecule Was awarded a Nobel Prize in Chemistry in 1980 "for his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant DNA" 9 10 Important events in recombinant DNA technology Recombinant DNA technology Works by taking DNA from two different sources and 1972 DNA ligase joins two fragments, creating first recombinant combining that DNA into a single molecule DNA molecules That alone, however, will not do much 1973 Potential hazards of recombinant DNA technology raise Only becomes useful when that artificially-created DNA concerns is reproduced 1974 World moratorium on some recombinant experiments This is known as DNA cloning 1975 Development of experimental guidelines for recombinant DNA technology discussed at the Asilomar Conference (International Conference on Recombinant DNA Molecules) 1978 First human hormone (insulin) produced by recombinant DNA technology 1982 First animal vaccine produced by recombinant DNA technology 11 12 2  Recombinant DNA technology Bacterial DNA Construction of recombinant DNA involves the insertion of a foreign DNA fragment into a plasmid Plasmid vector In the example below, the gene indicated by the white color is inactivated upon insertion of the foreign DNA fragment Chromosome: Most bacteria have one circular DNA chromosome ranging in size from 1,000 to 8,000 kilobase pairs Plasmid: Extra chromosomal genetic element also made of a circular DNA molecule Bacterial genome: The collection of all of the genes present on the bacteria’s chromosome or its extra chromosomal genetic elements 13 14 Plasmid Plasmid Plasmids can replicate autonomously within a host Plasmids frequently carry genes conferring resistance to antibiotics such as tetracycline, ampicillin, or kanamycin The expression of these marker genes can be used to distinguish between host cells that carry the vectors and those that do not 15 16 Recombinant DNA technology Recombinant DNA Recombinant DNA molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms Recombinant DNA is possible because DNA molecules from all organisms share the same chemical structure They differ only in the nucleotide sequence within that identical overall structure 17 18 3  Gene cloning Recombinant DNA A clone is a collection of molecules or cells, all Recombinant DNA is the general name for taking a identical to an original molecule or cell piece of one DNA, combining it with another strand of To "clone a gene" is to make many copies of it - for DNA example, by replicating it in a culture of bacteria Recombinant DNA molecules are sometimes called Cloned gene can be a normal copy of a gene (“wild chimeric DNA, because they are usually made of type”) material from two different species, like the mythical Cloned gene can be an altered version of a gene chimera (“mutant”) Recombinant DNA technology uses: Recombinant DNA technology makes manipulating – restriction nuclease/endonuclease/enzyme that genes possible cuts DNA and leads to the production of blunt and sticky/cohesive ends – DNA ligase that joins 2 pieces of DNA 19 20 Restriction nuclease/endonuclease/enzyme * Blunt ends Cleaves DNA at a specific nucleotide sequence Example: Produces a specific DNA molecule that can be cloned – HaeIII recognises the restriction site: Cleavage of double-stranded DNA produces: GGCC CCGG – cleavage of double-stranded DNA produces: 5'NNNNNGGCCNNNNN3' 3'NNNNNCCGGNNNNN5' – DNA separates to form 2 fragments: 5'NNNNNGG pCCNNNNN3' 3'NNNNNCCp GGNNNNN5' 21 22 * Examples of restriction Cohesive ends nucleases/endonucleases/enzymes Example: – EcoRI recognises the restriction site: GAATTC CTTAAG – cleavage of double-stranded DNA produces: 5'NNNNNGAATTCNNNNN3' 3'NNNNNCTTAAGNNNNN5' – DNA separates to form 2 fragments: 5'NNNNNG AATTCNNNNN3' 3'NNNNNCTTAA GNNNNN5' 23 24 4  DNA ligase DNA ligase activity at blunt ends Synthesises the joining of 2 pieces of DNA 5'… pCpGpAO OH pC pG pT pA … 3' Catalyses the formation of phosphodiester bonds 3'…GpCpTp OHG pC pA pT p … 5‘ Uses – joins compatible ends of double-stranded DNA i.e. DNA ligase – joins blunt ends of double-stranded DNA – joins cohesive ends of double-stranded DNA 5'… pCpGpApCpGpTpA … 3' 3'…GpCpTpGpCpApTp … 5' 25 26 DNA ligase activity at cohesive ends DNA ligase covalently joins two DNA strands 5'… pApC pGOH pA pA pT pT pC pG pT… 3' 5' 3' 3'…TpGpC pTpTpApAp OHG pC pA p … 5' Restriction endonuclease DNA ligase Ligase 5'… pApC pGpApApTpTpC pGpT… 3' 3'…TpGpC pTpTpApApGpC pA p … 5' 3' 5' nick Restriction endonuclease Ligase G 27 28 Recombinant DNA Recombinant DNA The DNA sequences used in the construction of Genetic recombination is a normal biological process recombinant DNA molecules can originate from any that results in the remixing of existing DNA sequences species in essentially all organisms – For example, plant DNA may be joined to bacterial Recombinant DNA results from artificial methods in DNA, or human DNA may be joined with fungal the test tube DNA In addition, DNA sequences that do not occur anywhere in nature may be created by the chemical synthesis of DNA, and incorporated into recombinant molecules Using recombinant DNA technology and synthetic DNA, literally any DNA sequence may be created and introduced into any of a very wide range of living organisms 29 30 5  Recombinant DNA Cloning vector Laboratory processes can be used to create Formation of recombinant DNA requires a cloning recombinant DNA vector, a DNA molecule that replicates within a living Two widely used methods to direct the replication of cell any specific DNA sequence chosen by the Vectors are generally derived from plasmids or experimentalist are: viruses; – molecular cloning – represent relatively small segments of DNA that – polymerase chain reaction (PCR) contain necessary genetic signals for replication The fundamental difference between the two methods is – additional elements for convenience in that molecular cloning involves replication of the inserting foreign DNA DNA within a living cell, while PCR replicates DNA in identifying cells that contain recombinant DNA the test tube, free of living cells expressing the foreign DNA in living cells vs in test tube 31 32 * Vectors used in cloning experiments Example Type Description Cloning vector pBluescript Plasmid A type of vector used to clone small segments of DNA and propagate them in E. coli The choice of vector for molecular cloning depends on: YEp24 Plasmid This plasmid is an example of a shuttle vector, which can replicate – the choice of host organism in two different host species i.e. E. coli and Saccharomyces cerevisiae – the size of the DNA to be cloned (DNA insert) It carries an origin of replication for both species – whether and how the foreign DNA is to be λgt11 Viral This vector is derived from the bacteriophage λ expressed It contains a promoter from the lac operon When fragments of DNA are cloned next to this promoter, the DNA The DNA segments can be combined by using a is expressed in E. coli variety of methods, such as restriction enzyme - This is an example of an expression vector, a vector designed to clone the coding sequence of genes so they are transcribed and ligase cloning translated correctly SV40 Viral This virus naturally infects mammalian cells Genetically altered derivatives of the SV40 viral DNA are used as vectors for the cloning and expression of genes in mammalian cells that are grown in the laboratory Baculovirus Viral This virus naturally infects insect cells Unlike, many other types of cells, insect cells often express large amounts of proteins that are encoded by cloned genes When researchers want to make a large amount of a protein, they can clone the gene that encodes the protein into baculovirus and then purify the protein from insect cells 33 34 restriction sites restriction site genomic DNA Steps in standard cloning protocols Digestion cloning vector 1. Choice of host organism and cloning vector DNA cloning DNA fragment Digestion 2. Preparation of vector DNA Dephosphorylation 3. Preparation of DNA to be cloned (DNA insert) Ligation 4. Creation of recombinant DNA host cell 5. Introduction of recombinant DNA into the host recombinant organism (transformation) molecule 6. Selection of organisms containing recombinant DNA Transformation 7. Screening for clones with desired DNA inserts and biological properties Replicate inside host cell cell recombinant colony molecule inside host cell many copies of Recombinant the recombinant molecule molecule analysis 35 36 6  Selection of recombinant colonies Expression of recombinant DNA 1. Non-transformed: Expression of recombinant DNA within living cells can – cannot grow on ampicillin or result in recombinant proteins tetracycline When recombinant DNA encoding a protein is 2. Transformed: introduced into a host organism, the recombinant – only transformed colonies can grow in protein is not necessarily produced ampicillin or tetracycline containing medium Expression of foreign proteins requires the use of a. Transformed with non-recombinant or specialised expression vectors and often unaltered vector: necessitates significant restructure by foreign coding – can grow in both ampicillin and sequence tetracycline containing medium b. Transformed with recombinant vector carrying gene of interest: – can grow only in ampicillin medium but cannot grow in tetracycline medium due to insertional inactivation – recombinant colonies can be easily selected from the master plate 37 38 Expression of recombinant DNA Expression of recombinant DNA Following transformation into the host organism, the Specific changes to the host organism may be made foreign DNA contained within the recombinant DNA to improve expression of the foreign gene construct may or may not be expressed In addition, changes may be needed to the coding That is, the DNA may simply be replicated without sequences as well expression, or it may be transcribed and translated O – To optimise translation so that a recombinant protein is produced ② – To make the protein soluble Generally speaking, expression of a foreign gene requires restructuring the gene to include sequences ③ – To direct the recombinant protein to the proper cellular or extracellular location that are required for producing an mRNA molecule that can be used by the host's translational apparatus ⑪ – To stabilise the protein from degradation (e.g. promoter, translational initiation signal and transcriptional terminator) Opan suka petik sayur 39 40 Expression of recombinant DNA Polymerase chain reaction In most cases, organisms containing recombinant DNA A technique called the polymerase chain reaction (PCR) have apparently normal phenotypes has revolutionised recombinant DNA technology That is, their appearance, behaviour and metabolism It can amplify DNA from as little material as a single are usually unchanged, and the only way to cell and from very old tissue such as that isolated from demonstrate the presence of recombinant sequences is Egyptian mummies, a frozen mammoth, and insects to examine the DNA itself, e.g. using a polymerase trapped in ancient amber (fossilised tree resin) chain reaction (PCR) method If the recombinant DNA sequence encode a gene that is expressed, then the presence of RNA and/or protein products of the recombinant gene can be detected e.g. using RT-PCR or western hybridisation methods 41 42 7  Insertional inactivation Insertional inactivation In some cases, recombinant DNA can have deleterious Researchers can "knock out" genes to determine their effects even if it is not expressed biological function and importance One mechanism by which this happens is insertional Another mechanism by which recombinant DNA inactivation, a technique used in recombinant DNA insertion into chromosomal DNA can affect gene engineering where a plasmid is used to disable expression is by inappropriate activation of previously expression of a gene unexpressed host cell genes The inactivation of a gene can occur by inserting a – e.g. when a recombinant DNA fragment containing fragment of DNA into the middle of its coding an active promoter becomes located next to a sequence previously silent host cell gene, or when a host cell gene that functions to restrain gene expression undergoes insertional inactivation by recombinant DNA 43 44 * DNA sequencing History of DNA sequencing Genetic information is contained in DNA by a linear order of nucleotide bases Determining this order is called DNA sequencing Rapid and efficient methods for DNA sequencing were first devised in the mid 1970s, following the introduction of recombinant DNA technology Messing, J. & Llaca V. (1998) PNAS 95: 2017-2020 45 46 Chain termination method (Sanger) Automated DNA sequencing with fluorescently labeled dideoxynucleotides (A) The chain termination reactions are carried out in a single tube, with each dideoxynucleotide labelled with a different fluorescent label (B) The printout from an automated sequencer Sanger, F., Nicklen, S. & Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA 74: 5463–5467. Brown, T.A. Genomes 2n d Ed.© BIOS Scientific Publishers Ltd, 2002 47 48 8  Application of recombinant DNA technology Applications of DNA sequencing Forensics – Identification of particular individual as every individual has unique DNA sequence – Used to identify criminals by finding proof from the crime scene, to determine the paternity of a child, and to distinguish endangered and protected species Medicine – Used to detect genes that are associated with heredity and acquired conditions/diseases 49 50 * Field Application Importance Recombinant proteins Basic biology DNA sequencing Answer questions about gene structure, gene Recombinant insulin function, relatedness of genes and organisms Medicine Directed mutagenesis Answers questions about gene function Therapeutic proteins Make human proteins for treating diseases e.g. diabetes, pituitary dwarfism, hemophilia Gene therapy Treats genetic disease e.g. cystic fibrosis Recombinant chymosin Improved vaccines Produces more effective vaccines with fewer side Recombinant human growth hormone effects Diagnosis Allows rapid, accurate diagnosis of infection and other diseases Veterinary medicine Better diagnosis, prevention and treatment of diseases Industry Altering Improved production of antibiotics, amino acids, microorganism vitamins and enzymes Also improved waste disposal, including persistent toxic chemical Agriculture Altering plants More rapid breeding of disease-resistant and improved plants e.g. tomatoes that stay fresh- tasting longer Altering farm animals More rapid development of superior breed Criminal DNA fingerprinting Can determine if a biological sample such as investigation blood, semen or tissue is from a particular person Recombinant coagulation factor VIII 51 52 Recombinant chymosin Zebra fish (Denio rerio) Chymosin is an enzyme required to manufacture cheese Scientists at the National University of It was the first genetically engineered food additive used Singapore were working with a gene commercially that encodes the green fluorescent protein (GFP), originally extracted Traditionally, processors obtained chymosin from rennet, a from a jellyfish, that naturally preparation derived from the fourth stomach of milk-fed calves produced bright green fluorescence Scientists engineered a non-pathogenic strain (K-12) of E. coli They inserted the gene into a bacteria for large-scale laboratory production of the enzyme zebrafish embryo, allowing it to integrate into the zebrafish's This microbiologically produced recombinant enzyme, identical genome, which caused the fish to be structurally to the calf derived enzyme, costs less and is brightly fluorescent under both produced in abundant quantities natural white light and ultraviolet light Today about 60% of U.S. hard cheese is made with genetically Their goal was to develop a fish that engineered chymosin could detect pollution by selectively In 1990, FDA granted chymosin "generally-recognized-as-safe" fluorescing in the presence of (GRAS) status based on data showing that the enzyme was environmental toxins safe 53 54 9  Golden rice Production of gene of interest (GOI) in transgenic sheep A variety of rice (Oryza sativa) produced through genetic engineering to biosynthesise beta-carotene, a precursor of vitamin A, in the edible parts of rice 55 56 Making a transgenic, RoundupTM-tolerant tobacco plant by introducing a COVID-19 Vaccine Development Platforms modified form of the bacterial gene for the enzyme 5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS) that is resistant to the herbicide 57 58 COVID-19 Vaccine Development Platforms COVID-19 Vaccine Development 59 60 10  45 Recombinant DNA technology 30 25 Recombinant DNA is widely used in biotechnology, medicine and research Today, recombinant proteins and other products that result from the use of recombinant DNA technology are commonly found in essentially every western pharmacy, doctor's or veterinarian's office, medical testing laboratory, and biological research laboratory In addition, organisms that have been manipulated using recombinant DNA technology, as well as products derived from those organisms, have found their way into many farms, supermarkets, home medicine cabinets, and even pet shops, such as those that sell GloFish and other genetically modified animals 61 11