|a) the polymerase chain reaction
Polymerase chain reaction (PCR), is a common method of creating copies
of specific fragments of DNA. PCR rapidly amplifies a single DNA molecule
into many billions of molecules.
There are three basic steps in PCR. First, the target genetic material
must be denatured-that is, the strands of its helix must be unwound and
separated-by heating to 90-96°C.
The second step is hybridization or annealing, in which the primers bind
to their complementary bases on the now single-stranded DNA.
The third is DNA synthesis by a polymerase. Starting from the primer, the
polymerase can read a template strand and match it with complementary nucleotides
very quickly. The result is two new helixes in place of the first, each
composed of one of the original strands plus its newly assembled complementary
All PCR really requires in the way of equipment is a reaction tube, reagents,
and a source of heat. But different temperatures are optimal for each of
the three steps, so machines now control these temperature variations automatically.
it can be used to detect genetic abnormality, the presence of microbes
and cancer cells.
c) in situ hybridization.
The use of antibodies or antisera as histological tools for identifying
patterns of antigen distribution within a tissue or an organism.
An antibody (or mixture of antibodies) that binds to a specific protein
or other antigen is tagged with a fluorescent chemical or an enzyme that
can convert a substrate to a visible dye.
The tagged antibody is incubated with the tissue and after washing unbound
antibody away the bound antibody distribution is revealed by fluorescence
microscopy or incubation with a chromogenic substrate.
It is assumed that antibody distribution reflects antigen distribution.
In situ hybridization, as the name suggests, is a method of localizing,
either mRNA within the cytoplasm or DNA within the chromosomes of the nucleus,
by hybridizing the sequence of interest to a complimentary strand of a
Normal hybridization requires the isolation of DNA or RNA, separating it
on a gel, blotting it onto nitrocellulose and probing it with a complimentary
The basic principles for in situ hybridization are the same, except one
is utilizing the probe to detect specific nucleotide sequences within cells
and tissues. The sensitivity of the technique is such that threshold levels
of detection are in the region of 10-20 copies of mRNA or DNA per cell.
In situ hybridization presents a unique set of problems as the sequence
to be detected will be at a lower concentration, be masked because of associated
protein, or protected within a cell or cellular structure. Therefore, in
order to probe the tissue or cells of interest one has to increase the
permeability of the cell and the visibility of he nucleotide sequence to
the probe without destroying the structural integrity of the cell or tissue.
The tissues can be prepared in the following way:
fixed in formalin
snap frozen, and embedded in a special support medium for cryosectioning.
cells in suspension can be cytospun onto glass slides and fixed with methanol
preparation of metaphase chromosmal spreads, normally fixed with a mixture
of methanol and acetic acid.
There is essentially four types of probe used
a. Oligonucleotide probes
b. Single stranded DNA probes.
c. Double stranded DNA probes
d. RNA probes or Riboprobes