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  pro-metaphase  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 Designation Constitution Number of chromosomes Monoploid n ABC 3 Diploid 2n AABBCC 6 Triploid 3n AAABBBCCC 9 Tetraploid 4n AAAABBBBCCCC 12 Monosomic 2n − 1 ABBCC 5 AABCC 5 AABBC 5 Trisomic 2n + 1 AAABBCC 7 AABBBCC 7 AABBCCC 7

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

Banding techniques G-banding Q-banding R-banding T-banding C-banding 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)