Transcript Name Designation Constitution Number of chromosomes

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)