The most used method during further processing of biological material is
centrifugation – the speeding up of solid particles sedimentation in a liquid environment,
caused by increased gravitation induced by rotation. Particles sink to the bottom, depending
on the properties of the substance (size, form, density) and the environment (density,
viscosity). A part of centrifuges with higher rotations have a cooling system (protecting the
biological material). A vacuum system is used mainly during ultracentrifugation, where it is
necessary to prevent rubbing of the rotor to air and an unwanted increase in temperature –
during high rotations. The space where the rotor is located is frozen, for the same reason.
A rotor is a symmetrical entity made of metallic alloy, which turns around a central axis
inside the centrifuge. It can be angular (for higher rotations) or swinging one. At a certain
distance from the middle are located the centrifugal tubes containing samples. The tubes
number is usually even and they are always placed into the opposite way positioned openings
in the rotor. The necessary parameters (time, temperature, number of rounds/min.) are set on
the control panel of the centrifuge.
9.7 Methods of the nucleic acids isolation
Most common sources of DNA to molecular-genetic examination are live cells –
peripheral (venous) blood lymphocytes, buccal mucosa, hair roots, skin, chorionic villi, or
bacteria. For analysis it is also possible to use degraded DNA, optionally a DNA obtained
from old tissue fixed in formalin or from paraffin blocs.
Isolation of nucleic acids belongs to the most common techniques used in a laboratory of
molecular biology. The prerequisite of a successful molecular analysis of a human DNA is the
obtaining of a sufficiently clean highly molecular weight DNA. The main problem of nucleic
acids isolation is the removal of proteins and polysaccharides so, that the primary and
secondary structure of DNA or RNA remains unchanged. Reason is, that “free” nucleic acids
do not exist in living organisms (except tRNA), because they are always in complexes with
supporting and regulatory proteins. To suppress devastating activity of endonucleases,
inhibitors (polyamines as spermidine and putrescine) and anticolaguants (e.g. EDTA) are used.
The classical methods of DNA isolation is phenol-chloroform extraction from cell
lysate (sodium sulfate/proteinase K) and the salting method. The method for DNA isolation
consists of the following steps:
• lysis of cells – by the disturbance of the cytoplasmic membrane (cell wall in bacteria) the
content of the cell is loosened;
• inhibition of nucleases – by the removal of RNA with the helps of RNases (during RNA
isolation DNases is used);
• breakup of the nucleoprotein complex– for the break up of the bonds between nucleic
acids and proteins, organic reagents are used (phenol, chlorophorm, urea etc.);
• the removal of proteins and precipitation of the DNA – proteins are removed by
centrifugation. DNA molecules remain in the solution, they are precipitated by salts and
alcohol. DNA is “fished-out”, left to dry, and dissolves in re-distilled water, – result is
formation of so called DNA sample.
Currently – in molecular genetic are preferred kits for isolation DNA or RNA. The
DNA isolation kit is a rapid and effective method for isolation of high molecular weight
genomic DNA from bacteria, plant, yeast, blood cells and other mammalian cells and tissues
of all types. The kit uses RNase mix to eliminate RNA immediately after lysis, and protease
mix to rapidly degrade cellular proteins. This is followed by a proprietary salting out
technique that precludes the need for phenol, chloroform or other organic extractions.
The concentration and purity of the isolated DNA are ascertained by electrophoresis or
spectrophotometry. The principle of electrophoretic determination is that the well isolated
high-molecular DNA is gathered in a gel on one place – close to the start. If disintegration
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