Download PCR - AREA

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.