DNA analysis

Everything said above concerning mutations has great importance if mutation occurs in gametal cells – spermatozoon or ovule, thus diversifying genetic pool of the species directly. But if mutation occurs in non-gametal cell, it will apply to the genome of the very limited number of cells that are direct descendants of mutated cell. That’s why it will be true to say that nothing may modify one’s genetic code in the course of the individual’s life. Hence it is possible to make for the every person the chart of some regions of non-coding DNA that display high levels of polymorphism.

These sequences will be unique to every single person and therefore records of these sequences may be used as genomic passports for the people. Modern methods of detection are so powerful that DNA from only a several tens of cells is required to make a successful DNA analysis. These cells even may be dead or dried or frozen. The only requirement is that DNA of these cells was intact. But this analysis is not an instant procedure. It may be divided in several stages.

The initial stage is the extraction of DNA and its purification from other organic materials. If there is a lot of substrate and, though, plenty of DNA, it is possible to proceed directly to the step of performing analyzes. But if there is only small crop of dried blood available, or the volume of template material is otherwise limited, there would not be enough DNA to perform the fingerprinting analysis. This made the need for the second stage – multiplication of the selected areas of DNA that will be analyzed subsequently.

Multiplication is achieved by the means of PCR (stands for polymerase chain reaction) – an enzymatic reaction that runs in repeated cycles of DNA synthesis and produces billions of copies of the required area of DNA after thirty of forty cycles only. This reaction takes no more than three hours in general, but to perform it successully special equipment is required, that sometimes may be very expencive, and, like  the extraction phase, all the preparations to the PCR should be performed by the highly qualified staff in the specialized “clear” laboratory department.

PCR is a highly sensitive method and hence false positive results in case of contmination with the foreign DNA are quite possible, as well as the false negatives if key steps of the reaction are performed incorrectly. This ensures the need for positive and negative controls of each step to be included into the reaction, which, in turn, raises slightly the price of the analysis which is already high enough.

But, despite all the difficulties and expenciveness, PCR-based methods are relatively fast and much more sensitive and have significantly less strict demands to quantity and quality of template DNA than the other detection method – RFLP – that will be discussed below. After the PCR is performed, the results of the reaction (amplicons) are separated on an agarose or polyacrilamide electrophoretic gel. After the electrophoresis all the amplicons are divided according to their size.

Considering high polymorphism of amplified regions, gel pattern is even more unique than casual fingerprint. Gel with amplicon bands may be stained and photographed for the latter storage and comparison. Another DNA fingerprint method that is now almost entirely replaced by PCR is the method of RFLP, or restriction fragments length polymorphism. Described in brief the method comprises four steps: the DNA is cut into small pieces by the special restriction enzymes. These restriction fragments are subjected to gel electrophoresis to distribute them across the gel.

After the electrophoresis the DNA fragments are transferred onto the nitrocellulose or nylon membrane to form a “blot”. This blot later is treated with a probe DNA that is complementary to the sertain sequence that may occur in some of the restriction fragment. (Riley, 2005) And, finally, the sizes of the fragments recognized by the probe are measured and the result is compared with the databases of already performed studies. Using the same enzyme each time it is possible to create such databases in a relatively short period of time.