[The method of DNA isolation can affect the rate of preferential amplification of alleles by the polymerase chain reaction].
Today, polymerase chain reaction is a common part of approaches serving for identification of individuals in legal medicine. This method is easily practicable, however attention must be paid to 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. from one cell) the polymerase chain reaction preferably amplifies only one of two in the template DNA present 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 on different fabrics was isolated by two different methods (by treatment with proteinase K and boiling and by treatment with Chelex 100). The obtained DNA samples were amplified using primers, they are complementary to the amelogenin gene sequences. The system is suitable for sex determination, because amplification of the X-chromosomal sequence provides a fragment in length of 632 bp, amplification of the Y-chromosomal one a fragment in length of 443 bp. The isolation method based on proteinase K led in 17.38% of samples to the very intensive preferential amplification of the longer allele, and therefore to a false result. The isolation method based on Chelex 100 provided in all cases correct results with clearly recognizable preferential amplification of the shorter allele. The reported results accentuate the meaning of choice of the appropriate isolation method, the need of accurate PCR optimization, and the careful interpretations of its outputs.
Korabecná
M
M
Biologický ústav LF UK, Plzen.
D000483:Alleles; D053523:Amelogenin; D001801:Blood Stains; D004247:DNA / Q000302:isolation & purification*; D003746:Dental Enamel Proteins / Q000235:genetics; D005554:Forensic Medicine; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction; D012732:Sex Determination Analysis
D053523:Amelogenin; D003746:Dental Enamel Proteins; D004247:DNA