Paternity analysis in special fatherless cases without direct testing of alleged father

The ability to establish the biological father, in which the father of a person is not available, named "reverse paternity determination", is based on the determination of STR alleles in mother and her child, other children and brothers of the alleged father, and deduction of genetic constitution of the father by the basis of genetic laws. Presented herein are two cases of reverse paternity determinations. Mother and parenthood DNA was isolated from 300 microl of fresh blood collected into tubes containing the EDTA, using Wizard Genomic DNA purification kit (Promega). DNA amplification was performed using GenePrint STR System (CTT, FFv, and Silver STR III multiplex) (Promega). Amplification results were read on 0.4 mm thick sequencing size polyacrilamide gel (4% for CTT and FFv, and 6% for Silver STR III). Gels were silver stained using Silver Sequence DNA Staining Reagents (Promega). Possible alleles of the alleged father for the loci CSF1PO, TPOX, TH01, F13A01, FESFPS, vWA, D16S539, D7S820, D13S317, LPL, F13B, and HPRTB were established by the selection from alleles determined in mother and parenthood, as to as obligate alleles with which the alleged father contributes to the genetic constitution of investigated child. Probability of paternity was calculated using population data on STR allele frequencies for Romanian population established by our laboratory. These are the first cases of litigious reverse paternity determinations in Romania.