All reagents used in DNA isolation and PCR amplification were from Promega, USA.

Peripheral blood samples were collected in EDTA vacutainers. 

Genomic DNA was extracted from 15μl blood sample using DNA IQ™System according to the manufacturer’s recommendations [8]. 

PCR amplification was performed using PowerPlex®16 System, a multiplex STR kit which allows the coamplification and three-color detection of sixteen loci including D21S11 and Penta D STR markers specific for chromosome 21 [9]. Details about this loci are given in Table 1. One to five nanograms purified genomic DNA was amplified in 25μl reaction volume containing 1X Gold ST*R Buffer (1.5mM MgCl2, 200μM each dNTP), 2.5μl PowerPlex®16 10X Primer Pair Mix and 0.8U of Taq Polymerase.  The PC reaction consisted of an initial denaturation step of 1 minute at 95°C, followed by 10 cycles at 94°C for 30 sec, 60°C for 30 sec, 70°C for 45 sec, another 22 cycles at 90°C for 30 sec, 60°C for 30 sec, 70°C for 50 sec and a final extesion step of 30 minutes at 60°C. PCR amplification was performed in a MJ Research PTC 100 termal cycler.

One microliter of each PCR product was mixed with 24μl deionized formamide  (Applied Biosystems, Foster City, CA, USA) and 1μl Internal Lane Standard ILS 600 (Promega, USA). The mixture were denatured at 95°C for 3 minutes and chilled out on ice for another 3 minutes. Electrophoretic analysis was performed on the ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA) using POP4 gel [5-7] and the fluorescent intensities of the amplified products were calculated using GeneScan® Analysis Software. The sample files were analyzed using PowerTyper™16 Macro (Promega, USA).