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Transcript
Northern blotting • Variant of Southern in which the target is RNA in stead of DNA • Study of expression pattern of a cloned gene in several tissues • No restriction enzymes necessary Northern blotting Cytogenetics • Technique to visualize the chromosomes • Chromosomes are only visible in dividing cells (in the M-phase): stimulate T-cells isolated from blood in culture (e.g. with phytohemaglutinin) or fetal cells. • Make cells swell with hypotonic saline • M-phase is short (see figure), low mitotic index • Mitotic index ↑ by blocking spindle with colcemid • Other techniques include thymidine starvation, and release = synchronized cycling optimization prometaphase less condensed than metaphase Cytogenetics • Classification of chromosomes according to their size and position of the centromere, numbering from long to short NO UNAMBIGUOUSLY identification • Introduction of banding techniques: allowed for identification of each chromosome and the positions of deletions etc on the chromosomes • Several banding techniques available, give information about the structural organization (resolution 1-10 Mb) • Karyotype 46XX or 46XY and abnormalities (overheads) • Down-syndrome trisomy Cytogenetics Name DesignationConstitution Number of chromosomes Monoploid n ABC 3 Diploid 2n AABBCC 6 Triploid 3n AAABBBCCC 9 Tetraploid 4n AAAABBBBCCCC 12 ABBCC 5 AABCC 5 AABBC 5 AAABBCC 7 AABBBCC 7 AABBCCC 7 Monosomic 2n − 1 Trisomic 2n + 1 Banding-paterns • Banding patterns are caused by differences in binding of the dye due to differences in the scaffold loop structure next slide • Scaffold attachment regions (SARs) • More SARs per length unit in G bands than in R bands G bands have smaller loops • G-banding Banding techniques • trypsin digestion Giemsa stain G-bands dark, pale bands G negative (see figure) • Q-banding • Fluorescent AT-rich DNA binder (Quinacrine, DAPI, Hoechst 33258) UV-fluorescence Q-bands same regions as G-bands • R-banding • Reverse G-bands heat treatment denatured AT-regions from Giemsa stain. Same pattern is obtained by GC-specific dyes • T-banding • Subset of R bands in proximity of the telomers, T-bands are the most intense R-bands extreme heat treatment followed by Giemsa stain • C-banding • Staining of the centromers denature with barium hydroxide followed byGiemsa stain Karyogram In situ hybridization • Chromosome in situ hybridisation • Tissue in situ hybridization Chromosome in situ hybridization • Method to “map” genes and other DNA-sequences hybridize labeled DNA probe with denatured chromosomes in situ • Metaphase or prometaphase microscope slide preparation (see cytogenetics), treat with Rnase and proteinase K purified chromosomal DNA denature with formamide probe • chromosome banding is performed before or after hybridization • FISH fluorescence label direct or indirect • For good signal strength long probes are used (40kb) necessity for “blocking” of repeat sequences by suppression hybridization Chromosome in situ hybridization • Detection with fluorescence microscopy • Metaphase spreads double hybridization spots (sister chromatids, see cycle) • Resolution about 1 Megabase Chromosome in situ hybridization 17 17 3 3 Tissue in-situ hybridization • In this procedure a labeled probe is hybridized agianst RNA in tissue sections • Hybridization mix contains 50% formamide (lower hybridisation temp) • Single stranded probes complementary RNA probes antisense riboprobes gene in reverse orientation in cloning vector • Radioactive or non-isotopic labels • Fluorescent microscopic detection • Commercial kits available for eg cytomegalovirus, Epstein-Barr virus • Same precautions and steps as with in-situ PCR • see transparancies Immunological techniques Serological techniques • Serological techniques are all techniques using antibodies in fact most immunological techniques used in diagnosis • A selection of the most used techniques in medical diagnosis will be discussed Bloodgrouping using the gel-technique Monoclonal antibodies for bloodgroup A antigen bound on gelmatrix Bloedgroup: A RhD neg Latex agglutination • Polystyrene latex micro-particles coated with viral or other antigens • Mix with serum of patient • When Ab for the antigen are present the particles will agglutinate visibly • This is a very common technique: simple and quick • Many commercial kits available: rubella, toxoplasmosis, cytomegolovirus... Latex agglutination Hemaglutination • Variant of the latex agglutination technique • Red bloodcells are particles • Determine bloodgroup by antigens already present on the cells • Red bloodcells can also be coated with other antigens (see TPHA test for syphillis)