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18.04.2016 AREA Conference Advancing Research in Agricultural and Food Sciences Through the Use of DNA-Based Technologies Dr Dimitrije Krstic Overview • Brief reflection on the history of molecular biology and development of DNA-based methods • Reference to molecular biology technologies being applied in agricultural and food sciences • Discussion of future challenges and applications of molecular tools to agricultural and food sciences • Reference to DNA-related training activities of AREA research groups Brief History of Molecular Biology 1869 Johann Friedrich Miescher discovered DNA and named it nuclein 1881 Edward Zacharias showed chromosomes are composed of nuclein. 1899 Richard Altmann renamed nuclein to nucleic acid. 1911 Thomas Hunt Morgan discovers genes on chromosomes are the discrete units of heredity Johann Miescher Thomas Morgan Brief History of Molecular Biology 1941 – George Beadle and Edward Tatum identify that genes make proteins 1950 – Edwin Chargaff find Cytosine complements Guanine and Adenine complements Thymine (C:G, A:T) Edwin Chargaff George Beadle Edward Tatum 1952-1953 James D. Watson and Francis H. C. Crick deduced the double helical structure of DNA Central Dogma of Molecular Biology Crick’s comment on the word dogma: “As it turned out, the use of the word dogma caused almost more trouble than it was worth.” Structure Expression Localization Polymerase Chain Reaction (PCR) 1983 Kary Mullis developed a PCR technique In recognition of his improvement of the polymerase chain reaction (PCR) technique He was awarded by a Nobel Prize in 1993. Baby Blue 1971 Kjell Kleppe and co-workers in the laboratory of H. Gobind Khorana described the method in Journal of Molecular Biology Kary Mullis Lessons for Life Kary Mullis In his Nobel Prize lecture, he remarked that the success didn't make up for his girlfriend breaking up with him shortly before. Married and remarried 4 times. 1985 Reported being visited by Martians PCR technique: from 1983 to 2016 • Allele-specific PCR: • Assembly PCR • Multiplex ligation-dependent probe amplification (MLPA) • Asymmetric PCR: • Multiplex-PCR: • Dial-out PCR: • Nanoparticle-Assisted PCR (nanoPCR): • Digital PCR (dPCR) • Nested PCR: • Helicase-dependent amplification: • Overlap-extension PCR or Splicing by overlap extension (SOEing) • Hot start PCR: • PAN-AC • In silico PCR • quantitative PCR (qPCR): • Intersequence-specific PCR (ISSR) • Reverse Transcription PCR (RT-PCR) • Inverse PCR: • Solid Phase PCR: • Ligation-mediated PCR: • Suicide PCR: • Methylation-specific PCR (MSP): • Thermal asymmetric interlaced PCR (TAIL-PCR) • Miniprimer PCR • Touchdown PCR • Miniprimer PCR: • Universal Fast Walking: PCR application in Agriculture and Food Sciences Product development: • Gene discovery and cloning • Vector construction • Identification of transformants • Screening and characterization • Seed quality control PCR application in Agriculture and Food Sciences GMO testing: • PCR testing for unapproved events – qualitative PCR • PCR testing for GM content – quantitative PCR • PCR testing for a presence of high-value commodity e.g. soybean with altered oil profile PCR application in Agriculture and Food Sciences Cultivar Identification: • It became possible to differentiate 60 Japanese dominant rice cultivars from each other using DNA extracted and purified from rice grains. PCR application in Agriculture and Food Sciences Gene expression changes as a result of: • Environmental and growth conditions • Food and/or treatment • Breeding schemes to improve specific characteristics • Pests Quantification of the pathogen concentration in a sample Hybridization Techniques Dot-Blot Hybridization (DNA) Southern blot - DNA Developed in 1975 by Sir Edwin Southern Northern blot - RNA DOT BLOT ELISA AREA Hybridization Techniques Fluorescent In-Situ Hybridization (FISH) DNA Chip (Microarrays) Sequencing Techniques and Genomics 1977, Frederick Sanger 1977, Walter Gilbert 2.7 billion dollars up to 2003 Cover page, Nature 10 million dollars per genome ~5,000 dollars per genome Genomics and Bioinformatics • Phylogenetic analysis • Information on natural diversity • Understanding of evolutionary forces shaping species natural population structure • Molecular epidemiology: etiology, distribution and prevention of disease • Identification and (re)classification of pathogen microorganisms • Dr Smilja Teodorovic (recruited through AREA project) - valuable help with bioinformatics AREA training in DNA-based technologies DNA and RNA extraction, q(RT)-PCR, gene expression, sequencing FOODBIOCHEM, Pavlicevic Milica University of Parma, Department of Life Science, Italy FOODTECH , Nemanja Mirković University of Parma, Department of Life Science, Italy FRUITBREED, Mirjana Djokic School of Agriculture, Policy and Development, University of Reading, UK WEEDSCI , Markola Saulic School of Agriculture, Policy and Development, University of Reading, UK PLANTPHYS, Mrs. Ivana Petrovic Institute of Agronomic Research (INRA), Avignon, France AQUACARP, dr Zorka Dulic Nofima Research Institute, As, Norway AREA training in DNA-based technologies qPCR, molecular cloning, FISH MICROBECOL, dr Igor Kljujev Helmholtz Zentrum Munchen, AMP Research Unit Microbe-Plant Interaction, Germany Dot-blot and Northern-blot, molecular cloning, qRT-PCR MICRODIAG, dr Ivana Stanković and dr Ana Vučurović, Department of Biology and Plant Pathology, Faculty of Sciences and Biotechnology, University of Bari, Italy PCR-based marker methods to establish phylogenetic relations, population variability and patterns of variability BIODIV, dr Ivan Šoštarić School of Biological Sciences, University of Reading, UK FRUITBREED, dr Zorica Rankovic-Vasic School of Agriculture, University of Reading, UK AREA training • Collaborations between AREA groups: AREA beer & chips • Strengthening collaborations between EU host groups and AREA research groups • Collaboration with and training of regional partners Regional collaborations: - Institute Sinisa Stankovic, Belgrade - University of Zagreb and Osjek, Croatia - University of Banja Luka, BIH Future scientific challenges for AREA groups • Training at the next level, continuous development of expertize • Designing research for publication in journals with higher impact factor • Increasing number of citations in future, and hence visibility of Faculty of Agriculture • Establishing a strong scientific network through collaborations, joint grant submission (e.g. H2020), student exchange.