Desenvolvimento de um sistema multiplex mini-STR de cromossomo y e análise das frequências haplotípicas na população da cidade de Manaus

Abstract

Methodologies for molecular analysis population and forensic studies, have been developed based on microsatellites or STRs. The application of the markers, enhance the PCR amplification system, utilizing only one pair of primers (monoplex), or several pairs (multiplex). For human identification by using DNA techniques, the first multiplex system were stabilished with STRs on autossomal chromosomes followed by using of reduced-size STRs (Mini-STRs). Then to the Y chromosome, have been developed the Y-STRs, mini–YSTRs and more recently the rapidly mutating (RM)-YSTRs. However, there are no studies using STRs on Y chromosome for the population of Manaus city, the objective of this study was developed a multiplex system Mini-YSTR to evaluate the amplification efficiency of degraded forensic DNA samples. In addtion, to determine haplotype frequency of the population in Manaus, a commercial Kit PowerPlex ® Y23 System (Promega) was used. Therefore, was developed a multiplex system denominated Mini YSTR09, combining nine mini-YSTR markers and (RM)-YSTR (Chapter I). For validation, tests to evaluate sensbilty, tests of detection sample containing mixtures and tests of degraded forensic DNA samples (98 samples of bones human) were conducted, by SWGDAM procedures.The multiplex was able to amplified 09 (100%) in a DNA mixture system and in 64,4% of degraded forensic DNA samples. For the population studies (chapter II), 214 blood samples were collected of male people, not correlated genetically. All de samples were extracted by using commercial kits and the amplified PCR products (31 Y-STRs in three dferent groups) and they are subjected to capillary electrophoresis in ABI 3500, automatic DNA sequence and analyzing in Genemapper v.4.1 software. 204 DNA samples were typed with the commercial kit (group I),189 with the Mini YSTR09 (group II) and 168 with the set (Mini YSTR09 more commercial Kit PowerPlex ® Y23 System),(group III). Results demonstrated the existing unique haplotypes in group I, II e III of the 202, 181 e 168, respectively.The greatest genetic diversity was found in locos DYS626, DYS570, DYS576, DYS447 (group I); DYS385, DYS570, DYS458, DYS481 e DYS456 (group II) e DYS385, DYS461, DYS626, DYS458, DYS576, DYS447 e DYS456 (group III).The locos DYS447, is was most polymorphic of group I and III. Therefore, the use of multiplex Mini YSTR09 developed made possible the forensic DNA samples amplification and for population analysis, provided a higher capacity of discrimination, when it was used in addition with the commercial kit PowerPlex ® Y23 System, highlighting the locos DYS447, DYS570, DYS576, DYS626.