1	Practical approach of hybridisation and related techniques
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6	DNA STRUCTURE
7	Components of DNA Deoxyribose Bases: adenine guanine cytosine thymidine
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10	Double Helix DNA Back-bone= DEOXYRIBOSE SUGAR Base pairing Adenine - Thymidine Guanine-Cytosine
11	DNA STRUCTURE
12	BASICS DNA = AMPHOPHILIC MOLECULE HYDROPHILIC : SUGAR BACKBONE HYDROPHOBIC: BASES
13	Components of RNA Ribose Bases: adenine guanine cytosine uracil
14	CLASSICAL TECHNIQUES SOUTHERN BLOTTING NORTHERN BLOTTING DOT BLOT TECHNIQUE
15	Hybridisation Basic principle Complementarity of both strands based on hydrogen bounds between bases
16	"5 AATGGCCCAAAATGCATTAGCT 3" 5 AATGGCCCAAAATGCATTAGCT 3 3’ GTTTTACGTAA 5’
17	GoldenOFormula Tm= 81.5 + 16.6 logM + 0.41(%G+C)- - 500/ L - 0.62 (% formamide)
18	Southern Blotting GENOMIC DNA RESTRICTION ENZYMES ELECTROPHORESIS BLOTTING NYLON MEMBRANE HYBRIDIZATION RADIOACTIVE PROBE
19	Northern Blotting PURIFIED RNA ELECTROPHORESIS BLOTTING NYLON MEMBRANE HYBRIDIZATION RADIOACTIVE PROBE
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21	Dot-Blot Technique GENOMIC DNA NYLON MEMBRANE DNA PROBES
22	IN SITU Morphological technique Chromosomes Whole cells Tissue: frozen, PET
23	Applications in pathologyO Detection of viruses Detection of chromosomal aberrations Detection of gene transcripts (mRNA)
24	Principles of hybridization PRETREATMENT HYBRIDIZATION POST-HYBRIDIZATION WASHING VISUALIZATION
25	PRETREATMENT Paraffin embedded tissue Frozen tissue sections Coated slides: silanized Proteolytic enzymes
26	Hybridization Sodium Saline Citrate = SSC FORMAMIDE PROBE DEXTRANSULPHATE
27	Probes RNA probes: detection of both RNA/DNA DNA probes: idem
28	DNA Probes WHOLE GENOMIC PROBES OLIGONUCLEOTIDE PROBES Cosmid probes PNA PROBES
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30	Post-Hybridisation washing Low stringency: high salt-low formamide High stringency: low salt-high formamide
31	Visualisation Radioactive Fluorescence: FITC, TRITC, … Immunohistochemistry: biotine, digoxygenine
32	PCR Reaction Mixture DNA or cDNA Taq-polymerase MgCl₂ Primer
33	PCR-PRIMERS Synthetic oligonucleotides 20- 30 bp %GC
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35	PCR Reaction cycle Denaturation: 95°C Annealing: 50-60°C Elongation: 72°C
36	PCR-PRINCIPLE
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43	PCR-Controls Known positive control Known negative control with DNA negative control without DNA quality control of DNA: genomic sequence
44	Paraffin embedded tissue Formalin fixation: breaks in DNA strand DNA degeneration Maximal amplification: 150-200 bp DNA inhibitors DNA extraction
45	PCR TECHNOLOGY Classical gelelectrophoresis Sequencing of PCR product Enzyme-immunoassay (EIA) Linearized Probe assay (LIPA) Real time PCR with quantification
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