Desenvolvimento e aplicação de polimorfismos de inserção: deleção do cromossomo x em genética forense

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

Nowadays the human genetic identification its based on the utilization of a set of autosomal markers, the microsatellites, which fulfils the majority of the forensic genetic labs requirements. However, there are some situations when that the usual autosomal microsatellites dont always offer good results. Those situations include cases where the genetic sample of the alleged father is not available and cases where the DNA sample presents itself degraded or in low quantities, rendering the utilization of commercial systems to the amplification of autosomal markers useless. With the aim of assisting the resolution of these problematic situations, this study aimed the use of X-chromosome markers, allowing to improve kinship studies, more specifically, scenarios using degraded or low quantity DNA samples. The markers chosen were insertion/deletion (indel) polymorphisms from XChromosome, ranging from 2-11 base pairs. Primers were designed for 17 X-chromosome indels, in order to generate Amplicons ranging from 70 to 170 base pairs. Only eight of these 17 indels allowed their use in multiplex reaction. The X-chromosome based multiplex system has shown to be highly sensitive in their amplifications, being able to amplify as low as 0.1 ng of DNA in a reaction volume of 12.5 μl, through sensitivity studies. To test the amplification efficiency of the multiplex, artificially and controlled degraded samples using DNAse I enzyme were used. From these artificially degraded samples, a new amplification protocol based on the increase from 30 cycles to 34 cycles was used. This technique proved to be really effective. Also, degraded samples were amplified using whole genome amplification prior to the PCR reaction for comparison sake that proved ineffective. A Brazilian population study was held on 200 samples from the five geopolitical Brazilian regions in order to establish the frequency of X-Chromosome indels. Population comparison indicated genetic difference between regions (FST= 0.021 P<0.05). Along with that, some kinship case studies, which were already been analyzed through the use of autosomal marker, were conducted using this novel 8 X-chromosome indel marker multiplex set. This novel multiplex system based on X-chromosome indels markers has proven to be useful and its usage on kinship cases as a complementary tool has been approved, and, in addition, it has proven to be successful in the amplification of degraded samples as well as in low quantity DNA samples along with other technique, Low Copy Number.

ASSUNTO(S)

mini-indel. x-chromosome cromossomos x genética forense polimorfismo low copy number dna. forensic science mini-indel genetica casos de parentesco biotecnologia kinship testing

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