Portuguese population and paternity investigation studies with a multiplex PCR — the AmpFlSTR® Profiler Plus™

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Abstract

A Portuguese Caucasian population of 146 unrelated individuals was studied. DNA samples were amplified by multiplex PCR for D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 using the AmpFlSTR® Profiler Plus™ PCR Amplification Kit (Perkin-Elmer). All loci met Hardy–Weinberg expectations. Forensic statistical parameters were according to those obtained by other authors. Statistical differences were observed concerning three loci when comparing the Portuguese Caucasian population and an Italian Caucasian population, although these differences mainly concern the less frequent alleles. Eighty-three paternity investigation cases were analysed. Exclusions in between three and nine loci were observed in all the 23 exclusion cases obtained. Most of the non-exclusion cases had probability of paternity >99.9%. Two cases with an isolated genetic incompatibility between the alleged father and the child were detected, which may indicate probable mutation cases. These results demonstrate that the AmpFlSTR Profiler Plus is a suitable multiplex for paternity investigation in the Portuguese population.

Introduction

Human identification is the ultimate goal for forensic genetics, either in criminal casework or in paternity testing. Short tandem repeat (STR) analysis is well established in forensic genetic laboratories.

DNA profiling is currently focused on the use of multiplex PCR which was developed including triplex, quadriplex and hexaplex systems [1], [2]. Multiplex systems are now commercially available [3] including more than six STR loci performed in one PCR reaction, giving matching probabilities in the range of more than 1 in a billion. One such system is the AmpFlSTR® Profiler Plus™ PCR Amplification Kit (PE Applied Biosystems) which co-amplifies the repeat regions of nine STR loci — D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820. A segment of the X–Y homologous gene amelogenin is also amplified.

In several European countries, DNA databases have been implemented with different multiplex systems [3], [4], [5]. These multiplex system loci form part of the 13 STR loci employed in the US National Databank CODIS (Combined DNA Index System). A Portuguese population sample was evaluated in order to obtain data concerning the application of AmpFlSTR Profiler Plus in paternity investigations and in population studies to implement a DNA database. These studies were performed according to the guidelines recommended by the DNA Commission of the International Society of Forensic Haemogenetics relating to the use of PCR-based polymorphisms [6].

Section snippets

Population sample

Samples from a total of 146 healthy unrelated Portuguese Caucasian individuals obtained from 83 paternity investigation cases requested by the Court were studied. All individuals and their parents were natives of Portugal, predominantly from the southern region.

Blood samples were collected and stored using UltraStain card (Fitzco) and DNA was extracted by the Chelex method.

Amplification and typing

DNA samples were amplified by multiplex PCR for D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820

Allele frequency estimates and forensic parameters

The observed allele frequency distributions in the Portuguese population sample for the nine loci analysed with the AmpFlSTR Profiler Plus are listed in Table 1. The genotype frequencies for these loci (data not shown) did not deviate from Hardy–Weinberg expectation based on the conventional chi-square method (P<0.01).

Statistical differences in allele frequencies were observed between this Portuguese Caucasian population and an Italian Caucasian population in D8S1179 (P<0.01), D21S11 (P<0.05)

Discussion

A Portuguese population sample was evaluated to obtain data concerning the application of AmpFlSTR Profiler Plus loci in paternity investigations and in population studies. Forensic statistical parameters such as power of discrimination, probability of exclusion and heterozygosity were according to those obtained by other authors [12]. The D5S818 locus presented the lowest forensic values, while D18S51 presented the highest forensic statistical parameters. The multiplex PCR investigated was

Acknowledgements

The authors thank Miss Isabel Lucas for excellent technical assistance.

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