FISH well. FISH probes can be purchased commercially.

FISH
is a technique used to identify the presence of a single nucleic acid sequence
(often specific to a particular chromosome) through hybridization of fluorescently
labeled DNA probes to denatured chromosomal DNA in cytological material.
Interphase nuclei are hybridized with the FISH probe, though metaphase spreads
can be used as well. FISH probes can be purchased commercially. By using FISH, a
copy of aberrations numbers as well as specific cytogenetic abnormalities can
be sketched and enumerated because it can easily detected chromosomal micro-deletion,
amplification, and subsequently translocation. Compare with cytogenetic
metaphase karyotype analysis, FISH is less time consuming detect and monitor
the specific therapy with regards to the gene abnormalities.

Flow cytometer:

The flow cell is where the cellular
interrogation takes place, the objective being very simple: to allow the cell
of interest to pass through the laser (or lasers) interrogation point and then
for the product of that interrogation to be displayed as physical and
fluorescent properties of that cell.

Major application
where low
cytometry is used include the diagnosis and subclassification of acute
leukaemia and chronic lymphoproliferative disorders, including chronic
lymphocytic leukaemia and non-Hodgkin lymphoma, HIV monitoring and DNA
analysis.

Microarray-based
Comparative Genomic Hybridization

Array CGH is based on the same
principles as metaphase CGH, a technique that has been extensively used for the
genomic characterization of a number of solid tumours. Both techniques allow
the study of DNA copy-number alterations genome-wide, except that the targets
for hybridization are mapped clones in the aCGH technique instead of chromosomes
as in metaphase CGH. Propidium iodide, phycoerythrin and fluorescein are the
common dyes to used although many other dyes are available. Furthermore, the
frequency of the recurrent amplification and the specific tumour subgroup where
it is most prevalent can also be established in this manner. By interrogating
the genome to identify critical molecular drivers in cancer, aCGH offers the
means to identify the therapeutic target and hence the appropriate biomarker
assay as well in the process of drug development.

Tissue in
situ hybridization (ISH)

The basic requirements of a probe for
use in tissue in situ hybridization are that it is complementary to the target
nucleic acid sequence and can be labelled in such a way as to allow microscopic
visualization of the hybrid formed. The different types of probe include the
following. Double-stranded DNA probes are most commonly used to detect DNA
targets. They are generated by the cloning and amplification of specific
sequences of DNA or cDNA, derived by reverse transcription of mRNA employing
vectors (bacterial plasmids and cosmids). Single-stranded RNA probes, so-called
riboprobes, are most commonly used to detect RNA in tissue sections. Riboprobes
are usually generated by in vitro transcription from plasmids containing the
sequence of interest. The plasmids are designed with promoter sites for RNA
polymerases (e.g. T3, T7 and SP6) and can produce probes complementary to the
target RNA sequence (antisense) or identical to the target sequence (sense). Tissue ISH is used to detect nucleic
acid sequences in a wide variety of solid neoplastic and infectious conditions
and is becoming a crucial theranostic tool, helping to guide therapy by
identifying relevant targets for new drugs in the field of pharmacogenomics. The deciphering of the human genome,
in combination with recent developments in nucleic acid-based testing, has
positioned tissue ISH as a central tool in diagnosis and predictive therapy.
Molecular diagnostics including tissue ISH are widely used in the areas of
inherited genetic disorders and infectious diseases, as well as haematologic
and solid tumours. In addition, tissue ISH has a role to play in guiding
appropriate therapy.