What is the rationale for studying GMO? Gene Cloning

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1 Genetically modified organisms (GMO) Lectures covering the basic methods, applications and ethics of gene cloning and expression in microbes, plants and animals. 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., 1999) What is the rationale for studying GMO? Gene Cloning Clone = a collection of organisms or DNA molecules that are all identical to each other because they result from replication or multiplication of the same original organisms or DNA Why clone a gene? Steps involved in cloning a gene 1. Extraction of DNA or nucleic acids of interest Nucleic acids may be extracted from cells, tissues or recovered from environmental samples by a variety of methods. Environmental samples often require extensive purification to remove proteins, humic acid and other compounds that will interfere with analysis. Nucleic acids must first be released from the cells of interest and then purified to remove contaminants Cell lysis enzymatic treatment - lysozyme, zymolase, proteinase K chemical disruption - SDS, phenol mechanical disruption - bead beater, grinding, freeze/thaw 1

2 Purification may involve a number of steps including cesium chloride ultracentrifugation hydroxyapatite or affinity chromatography phenol chloroform extractions ethanol or isopropanol precipitation Two basic approaches for recovery of nucleic acids from environmental samples 1. Isolation of microbial cells, cell lysis and nucleic acid purification e.g., water and air samples collection of cells by filtration 2. Direct lysis of microbial cells in environmental matrix and nucleic acid purification Both DNA and RNA may be used in cloning however RNA must first be converted to DNA through the use of reverse transcriptase to form complementary DNA (cdna or copied DNA). Recall that eukaryotic organisms may have introns in their genes. This complicates the cloning of complete gene sequences. Why? Post-transcriptional modification removes introns and produces the mature mrna, which is translated to produce the polypeptide How can we distinguish between the different types of RNA? Eukaryotic modification of RNA after transcription Alteration of transcript ends - 5 guanosine triphosphate cap - 3 poly(a) tail These modifications function to protect the mrna from degradation and 5 cap helps ribosomes attach. RNA splicing much of the transcript may be non-coding or not translated Have regions of coding (exons) interspersed with non-coding regions (introns or intervening sequences). Introns are excised from the primary transcript before it is exported from the nucleus. This process maybe mediated by spliceosome complexes and ribozymes. Functional role of introns Regulate gene activity or expression Production of different proteins from the same gene Evolution of new genes 2

3 2. Insertion of DNA into a suitable cloning vector A. Restriction Digestion - Restriction endonucleases Type II cut at a specific palindromic sequence of bases (i.e., reads the same on both strands whether it is read in the 5 3 or 3 5 direction.) Restriction enzyme recognition sequences may be 4, 6, 8 or more bps long Restriction enzymes generate one of two types of DNA fragments i) sticky ends EcoRI G AATTC; PstI CTGCA G easier to clone with but may not always have a useful site in the appropriate location ii) blunt ends SmaI CCC GGG do not have to have compatible ends but more difficult to clone with B. Ligation DNA ligase joins the 3 -OH to the 5 -PO 4 reforming the phosphodiester bonds C. Cloning vector Molecular vehicle for carrying and replicating introduced DNA fragments. Examples of Vectors plasmid phage and viruses YACs - yeast artificial chromosomes - linear plasmids Vector features cloning sites markers - in the case of plasmids to identify cells carrying the vector Ampicillin resistance (bla) is the most common selectable marker replication functions insertional inactivation - identification of recombinant molecules 3

4 3. Introduction of recombinant molecules into host cells 4. Screening or Detection of Recombinant Molecules Depending on the type of cloning this process may be very simple and involve techniques as simple as plasmid extraction, restriction digestion and agarose gel electrophoresis. On the othehand the cloning approach (library screening) may be creating a scenario not much different than looking for 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) or some other fragment library you need some way to identify the genes that you are after. Gene Probes - use one gene sequence to probe a library for similar sequences PCR amplification Expression of the gene product - phenotype - e.g., enzymes Reporter genes - encode easily detectable traits such as an enzyme activity. Promoters can be cloned using this technology 4

5 Polymerase Chain Reaction (PCR) in vitro replication of define sequences of DNA Ingredients for PCR reaction Buffer containing Mg ++ Enzyme Substrate Template Primers Nucleotides Repeated cycling of the following steps in an automated thermal cycler Denaturation Annealing Extension 5