Metoda izolace DNA může ovlivnit stupeňpreferenční amplifikace alel polymerázovouřetězovou reakcí

The Method of DNA Isolation Can Affect the Rate of PreferentialAmplification of Alleles by the Polymerase Chain Reaction

Today, polymerase chain reaction is a common part of approaches serving for identification ofindividuals in legal medicine. This method is easily practicable, however attention must be paidto the optimization of reaction conditions and to the interpretation of results. From the literature,such cases are known, in which during amplification of extremely small amount of DNA (e.g. fromone cell) the polymerase chain reaction preferably amplifies only one of two in the template DNApresent alleles. If the amplified fragments differ in length, the shorter one is amplified preferably,and it may be cause of false results. In the presented study, DNA from 23 stains of male blood ondifferent fabrics was isolated by two different methods (by treatment with proteinase K andboiling and by treatment with Chelex 100). The obtained DNA samples were amplified usingprimers, they are complementary to the amelogenin gene sequences. The system is suitable forsex determination, because amplification of the X-chromosomal sequence provides a fragment inlength of 632 bp, amplification of the Y-chromosomal one a fragment in length of 443 bp. Theisolation method based on proteinase K led in 17.38 % of samples to the very intensive preferentialamplification of the longer allele, and therefore to a false result. The isolation method based onChelex 100 provided in all cases correct results with clearly recognizable preferential amplificati-on of the shorter allele. The reported results accentuate the meaning of choice of the appropriateisolation method, the need of accurate PCR optimization, and the careful interpretations of itsoutputs.

Key words:
polymerase chain reaction (PCR) - isolation of DNA - identification of individuals -preferential amplification