PCR: P olymerase C hain R eaction

1. PCR AACTCTGGCGTGTCGATCAAACTGTTTGATGAG AACTCTGGCGTGTCGATCAAACTGTTTGATGAG AACTCTGGCGTGTCGATCAAACTGTTTGATGAG AACTCTGGCGTGTCGATCAAACTGTTTGATGAG AACTCTGGCGTGTCGATCAAACTGTTTGATGAG AACTCTGGCGTGTCGATCAAACTGTTTGATGAG AACTCTGGCGTGTCGATCAAACTGTTTGATGAG PCR: Polymerase Chain Reaction Enzymatic reaction used to copy (“amplify”) a specific region of DNA (“target sequence”) Target sequence Template DNA molecule (only one strand is represented)

2. target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target target PCR PCR is specific and extremely sensitive Single copy of target sequence within the template DNA molecule Billions of copies easily analyzable

3. Real-time PCR: the instrument Optical system

4. Real-Time PCR: automation and portability http://www.idahotech.com • DNA extraction • Plate setup • Amplification • Data analysis • Portability • Stress-resistance

5. Many commercial kits, protocols and robots available Nucleic acids extraction from environmental matrices • Protocol • Sample enrichment (centrifugation, filtration, IMS..) • Cell disruption (bead beating, sonication, freeze-thawing, boiling, chemical lysis...) • Matrix nature (PCR inhibitors..) • Species representation (dominant species..) • Costs, time, automation

6. Flow cytometry

7. Flow cytometry • Flow→ (cells in) motion • Cyto-→ cell • -Metry→ measure “Measuring properties of cells while in a fluid stream” High-throughput single-cell analysis

8. Detector Laser beam Data analysis stream of fluid • Cells are conveyed into a hydrodynamically-focused stream of fluid… • The stream of fluid is hit by a laser beam • Cells flowing “one-by-one”, within the stream can… • Scatter the light, in many different ways • Give off fluorescence… • Detectors pick up the signals and convert them to digitized values… • Specialized software analyses data and provides results Principles Flow cytometer Sample

9. www.cytobuoy.com 1. fluorescence detector Laser beam 2. forward scatter detector Data analysis 3. side scatter detector Light scattering and fluorescence detectors Forward Scatter (FSC) detectors Side Scatter (SSC) detectors Fluorescence detectors Sample

10. oligonucleotide probe ATCGATCTAGCG TAGCTAGATCGC Target sequence Forward scatter (FSC), side scatter (SSC), fluorescence • FSC→cell size, refractive index, membrane permeability, cell surface • SSC→ cell complexity (granules, cell inclusions, organelles…) • FLUORESCENCE • Endogenous (i.e. auto-fluorescence) • cellular pigments • Exogenous • fluorescent molecular probes • Specific staining • conjugated to antibodies (various specific targets) • oligonucleotide probes (specific sequences of DNA) • Non-specific staining • various dyes (carbohydrates, lipids, proteins, nucleic acids…)

11. Lymphocytes Granulocytes Monocytes RBCs, debris, dead cells FSC vs. SSC A correlated measurement between FSC and SSC allows differentiation of cell types within a heterogeneous cell population Cell size Cell complexity SSC FSC Marvin J, Flow Cytometry Facility, Northwestern University (WEB)

12. Auto-fluorescence of photosynthetic plankton can be exploited to characterise abundance and community structure Plot http://www.cytobuoy.com/

13. Scatter plot of chlorophyll fluorescencevs. side scatter. • Prochlorococcus (in red), • Synechococcus (in orange), • Picoeukaryotes (in green) • can be distinguished by auto-fluorescence. • Plotting the same data as a scatter plot of • chlorophyll fluorescence vs. phycoerythrin fluorescence • permits the cyanobacteria Synechococcus to be distinguished easily from the other picophytoplankton (populations color coded as in top panel)

15. Examples of applications and measurable parameters Measurable parameters • Size • Shape • Surface texture • Viability • DNA content (cell cycle, cell kinetics, proliferation…) • Cell surface antigens • Intracellular antigens • Nuclear antigens • RNA • Cell pigments (chlorophyll, phycoerythrin) • Volume and morphological complexity • Chromosome analysis and sorting • Protein expression and localization • Transgenic products in vivo • Enzymatic activity • Membrane fluidity • Apoptosis • Electropermeabilization of cells • Multidrug resistance (MDR) in cancer cells • Others Applications • Molecular biology • Pathology • Immunology • Plant biology • Environmantal biology • Aquatic ecology • Others

16. Flow cytometer: the instrument

17. Hand-held flow cytometer for in-situ operations submersible moored www.cytobuoy.com

18. Cell sorting • Flow cytometers can be equipped with cell sorting devices • Users define sorting criteria based on cellular properties 3,000-100,000 cells/sec can be sorted and collected for further analyses www.cytopeia.com

19. Data analysis: “gating” • It allows to isolate a sub-population of cells, on a plot • It allows the analysis of specific parameters associated to only that subpopulation of cells Gating can be coupled with cell sorting to collect a very specific sub-population of cells!