Biotechnology and Recombinant DNA

Transcription

1 Biotechnology and Recombinant DNA Recombinant DNA Technology Development of this technology was initiated in the early 1970s following the discovery of restriction enzymes. In 1972 Jackson, Symons and Berg generated recombinant DNA molecules Plasmid vectors that carry foreign DNA fragments were developed in 1973 Southern developed the Southern blot procedure in 1975 Stanley Cohen and Herbert Boyer patent filed in 1978 entitled Biologically Functional Molecular Chimeras This technology has lead to the development of a number of important tools for the study of life (e.g., Gene cloning (recombination), Hybridization analysis, Nucleotide sequence analysis, mutagenesis, gene expression, DNA fingerprinting). This technology relies on several basic principles DNA as the hereditary material of all living cells - Conserved mechanisms for DNA replication Universality of the Genetic Code What does this technology allow us to do and how does it do it? Biotechnology "The industrial use of living organisms or their components to improve human health and food production" (Campbell et al., 1999) "Those processes in which living organisms are manipulated, particularly at the molecular genetic level, to form useful products" (Prescott et al., 2008) the use of microorganisms, cells or cell components to make a product. (Tortora et al., 2010) I. Tools of Biotechnology 1. Selection Humans use artificial selection to select desire breeds of animals or strains of plants to cultivate. Similarly, microbiologists can select specific strains of bacteria or fungi that produce a desired product (e.g., antibiotic, enzyme, fermentation end-product) 2. Mutation Biologists use random mutagenesis to create new strains of a particular microorganism with enhanced characteristics (e.g., increased penicillin production) Site directed mutagenesis can be used to introduce specific changes into a particular gene 1

2 Repeated rounds of mutagenesis and selection have been used to produce novel industrial strains with improved characteristics (e.g., production of an antibiotic, enzyme, amino acid, ) 3. Restriction Enzymes How do prokaryotic cells deal with foreign DNA? One mechanism common to prokaryotic cells is the production of restriction endonucleases (restriction enzymes). Discovered by Arber and Smith in the late 1960s. Most common kind of restriction enzymes - type II enzymes that recognize very specific DNA sequences and cause double stranded breaks in the DNA. >3000 restriction enzymes have been identified from >200 species. Most of these recognition sites show a two-fold symmetry around a given point (palindrome - reads the same from the left or right). Recognition sequence length varies - 4, 6, 8 cutters. Named after the producing organisms (Sau3AI is from Staphylococcus aureus) Sau3AI GATC BamHI PstI NotI G GATCC CTGCA G GC GGCCGC Restriction produces overhanging (sticky ends) or blunt ends EcoRI G AATTC SmaI CCC GGG Modification enzymes Host must protect its own DNA modifying enzymes or methylases Methylases methylate (-CH 3 ) specific bases within the restriction enzyme recognition site (usually A or C) CH 3 BamHI methylase GGATCC 2

3 Restriction enzymes and methylases are part of restriction-modification system (RMS) One cell type may contain more than one RMS. Different strains of the same species may contain different RMS Restriction enzymes have been extensively characterized and are an integral part of recombinant DNA technology. Used to characterize DNA - restriction enzyme analysis (physical map). When cut by a restriction enzyme, any particular fragment will yield a limited number of pieces of DNA. These pieces of DNA can be resolved by gel electrophoresis. Used in cloning of DNA fragments Web tools can be used to determine restriction sites in known nucleotide sequences (e.g. NEBcutter at 4. Hybridization (Fig 9.16) Recall that dsdna can be denatured into two complementary strands and the ssdna molecules can form hybrids with other complementary DNA or RNA molecules Small ssdna molecules can be labeled (i.e., with isotopes 35 P and 32 S, fluorescent dye molecules ) and used to detect the presence of complementary sequences. These small labeled ssdna molecules are called probes. Southern blot Northern blot 5. Sequencing and synthesis of DNA i) Nucleotide sequence analysis Two methods were developed that, using a specific fragment of DNA, generate 4 pools of labeled random DNA fragments (i.e., radioisotopes); each pool contains fragments of DNA ending at only one of the four bases. The pools of DNA fragments are resolved by gel electrophoresis and the sequence is read directly from the gels. a) Maxim and Gilbert technique creates the pools of fragments chemically and is not used anymore 3

4 b) Sanger dideoxy method creates the pools enzymatically (i.e., uses DNA polymerase) and has been adapted to automated sequencing methods. A copy of the DNA fragment is made using DNA polymerase. A DNA primer is used to initiate synthesis (i.e., the sequencing reaction). The polymerase reaction generates random fragments of DNA with known nucleotides at the ends, by incorporating dideoxy analogs of the dntps used as substrates. The dideoxy analogs (ddatp, ddctp, ddgtp, dttp) are chain termination reagents and separate reactions are set up for each ddntp. Thus each reaction generates pools of fragments ending at a particular base. Fragments of varying lengths are obtained The pools of fragments are separated by electrophoresis. One lane for each reaction. The fragments have been labeled (e.g., isotope or fluorescent dyes for automated sequencing) for detection and reading of sequence. ii) DNA synthesis Automated DNA synthesizers can make ssdna oligonucleotides several to 100 nt in length 6. Polymerase Chain reaction in vitro replication of define sequences of DNA Developed by Kary Mullis (1984; Nobel prize in 1992) Repeated cycling of the following steps in an automated thermal cycler i) Denaturation (94 98 C) ii) Annealing (temperature depends upon t m of primers) iii) Extension (elongation - usually at 72 C) Ingredients for PCR reaction Buffer containing Mg ++ Enzyme Taq DNA polymerase Substrate Template Primers Nucleotides 4

5 Real-time PCR Newly synthesized DNA is tagged with fluorescent dye and the levels of fluorescence are measured by the real-time instrument quantitative PCR 7. Gene Cloning What is gene cloning? Why clone a gene? A basic concept in recombinant DNA technology is that of gene cloning. This involves in vitro recombination followed by replication of recombinant DNA. We need some way of reproducing these hybrid molecules in such a way as we can produce enough of them to study. Steps involved in cloning a gene i. Extraction of DNA or nucleic acids of interest Both DNA and RNA may be used RNA must first be converted to DNA through the use of reverse transcriptase to form complementary DNA (cdna or copy DNA). Recall that eukaryotic organisms may have introns in their genes. This complicates the cloning of complete gene sequences. Post-transcriptional modification removes introns and produces the mature mrna that is translated to produce the polypeptide How do we distinguish between the different types of RNA? Just want mrna recall Poly A tail DNA may come from a variety of sources, including genomic DNA, cdna or PCR products or it may be synthesized. Synthetic genes may be made nowadays for as little as $0.75/bp Cloning may involve introducing a single gene fragment into a suitable cloning vector. It may be more complicated and involve the creation of a genomic library. A genomic library is a collection of clones that is large enough to ensure that at least one clone exists for every gene in the organism. 5

6 ii. Insertion of DNA fragments into a suitable cloning vector. This is often accomplished in the following two steps. a) Restriction digestion using restriction endonucleases o sticky ends EcoRI G AATTC - easier to clone with but may not always have a useful site in the appropriate location o blunt ends SmaI CCC GGG - do not have to have compatible ends but more difficult to clone with b) Ligation T4 DNA ligase joins the 3 -OH to the 5 -PO 4 reforming the phosphodiester bonds Cloning vector vehicle for carrying and replicating introduced DNA fragments. A variety of vectors may be used including plasmid most common cloning vectors phage/viruses Cosmid BACs bacterial artificial chromosomes/yacs yeast artificial chromosomes linear plasmids Vector features 1. Origin of replication (ori) and other replication functions for stable maintenance in host cells. Shuttle vector: can replicate in several different species (e.g., E. coli and Saccharomyces cerevisiae 2. Selectable markers to identify cells carrying the vector Ampicillin resistance (bla) is the most common selectable marker on plasmid vectors Other antibiotic resistance genes have been used as well as auxotrophic markers 3. cloning sites unique restriction sites multiple cloning sites (MCS). Vectors often have features that allow one to identify recombinant vectors by a process known as insertional inactivation 4. Most cloning vectors are also small in size. For plasmids, phage vectors and cosmids, there are limits on how large a fragment can be cloned. 6

7 iii. Introduction of recombinant molecules into host cells Escherichia coli and Saccharomyces cerevisiae are the most common prokaryotic and eukaryotic cloning hosts Transformation is the most common method for introducing foreign DNA into host cells. One can also use conjugal and viral systems. Transformation uptake of naked DNA directly from the environment DNase sensitive DNA is originally derived from donor cell and taken up by recipient which is then called the transformant Cornerstone technique of molecular biology Most bacterial cells will not take up DNA efficiently unless they are exposed to special chemical or electrical treatments. However naturally transformable bacteria can take up DNA from their environment without special treatment a) Natural competence The naturally transformable bacteria take up DNA when they are in the state of natural competence A number of Gram positive and Gram negative bacteria are capable of natural competence Examples Bacillus subtilis Haemophilus influenzae Streptococcus pneumoniae (Griffith and Avery, McCarty, MacLeod, 1944) Most naturally transformable bacteria can take up DNA only late in their growth cycle - usually stationary phase A number of genes are involved; B. subtilis - com genes b) Artificially induced competence Most bacteria are not naturally transformable, al least not at easily detected levels. However this does not preclude them from transformation They can be artificially induced to a state of competence Methods of Induced Competence Chemical induction CaCl 2 - E. coli Polyethylene glycol - Bacillus thuringiensis Electroporation formation of transient membrane pores through the use of high voltage fields 7

8 protocols for many prokaryotic and eukaryotic organisms Biolistic approach i.e., gene gun Microscopic particles of gold or tungsten are coated with DNA and propelled by a blast of helium into cells protocols for many eukaryotic organisms Microinjection Small drawn glass micropipette are used to inject DNA solutions through the plasma membrane of animals cells Agrobacterium tumefaciens mediated plant transformation This bacterium naturally infects plants and introduces foreign DNA into the plant cell nucleus resulting in neoplastic growth (produces a plant gall i.e., tumour) and the abnormal production of amino acid derivatives known as opines. The bacterium is capable of metabolizing opines as a source of carbon and nitrogen. The genetic information necessary for the transfer of the bacterial DNA into the plant cell is encoded on the Ti (tumour inducing) plasmid. Only a small portion of the Ti plasmid (T-DNA) is transferred into the plant cell. This system has been well characterized and is now used to introduce foreign DNA into plants as well as some animal cells. iv. Screening or Detection of Recombinant Molecules May be creating a scenario not much different than the proverbial needle in the haystack. This technology is only useful if you can recover the desired molecules. If you have made a gene library (collection of all genes in an organism) you want some way to identify the genes that you are after. Gene Probes - use one gene sequence to probe a library for similar sequences Expression of the gene product - phenotype - e.g., enzymes PCR amplification Reporter genes - encode easily detectable traits such as an enzyme activity. Promoters can be cloned using this technology v. Gene Expression It is possible to use the above techniques to introduce foreign genes into almost any organism. It is also possible to introduce foreign gene constructs that are expressed by the host, thereby making a desired gene product. Expression vectors are specialized vectors that have been designed to promote gene expression in a particular host. These vectors contain the appropriate regulatory sequences (e.g., promoter, operator, terminators) that can be used to drive expression of a coding sequence of interest. 8

9 II. Application of Recombinant DNA Technology Genetic analysis Gene Mapping Gene Cloning Systematics Genome Sequencing Projects Gene Cloning Fingerprinting Mutagensis random directed site-directed Gene silencing RNA interference (RNAi) small interfering RNAs (sirna) are introduced into a cell where the sirna bind to mrna causing the enzymatic destruction of the mrna thereby silencing the expression of the gene Products Pharmaceuticals insulin, human growth hormone, vaccines Medical intervention gene therapy Enzymes - fibrolytic enzymes, restriction enzymes... Substrates - cellulose, dextrans Food products and additives - Flavr saver tomato, cheese (rennet); Herbicide tolerant Canola (glyphosate resistance); Insect resistant corn, Soy bean, cotton and potatoes Industrial products - solvents, ethanol... Cloned livestock Nanotechnology Forensic Medicine Ethics and safety of genetically modified organisms? 9