1. 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

2. Northern blotting

3. 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

6. 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

7. 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

8. 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

10. 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

11. Karyogram

13. In situ hybridization Chromosome in situ hybridisation
Tissue in situ hybridization

14. 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

15. Chromosome in situ hybridization
Detection with fluorescence microscopy
Metaphase spreads  double hybridization spots (sister chromatids, see cycle)
Resolution about 1 Megabase

16. Chromosome in situ hybridization17 17 3 3

17. 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

18. Immunological techniques

19. 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

20. Bloodgrouping using the gel-technique
Monoclonal antibodies for bloodgroup A antigen bound on gelmatrix
Bloedgroup: A RhD neg

21. 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...

22. Latex agglutination

23. 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)