Infrared fluorescent automated detection of thirteen short tandem repeat polymorphisms and one gender-determining system of the CODIS core system

Abstract

We used an infrared (IR) automated fluorescence monolaser sequencer for the analysis of 13 autosomal short tandem repeat (STR) systems (TPOX, D3S1358, FGA, CSF1PO, D5S818, D7S820, D8S1179, TH01, vWA, D13S317, D16S359, D18S51, D21S11) and the X-Y homologous gene amelogenin system. These two systems represent the core of the combined DNA index systems (CODIS). Four independent multiplex reactions, based on the polymerase chain reaction (PCR) technique and on the direct labeling of the forward primer of every primer pair, with a new molecule (IRDye800), were set up, permitting the exact characterization of the alleles by comparison with ladders of specific sequenced alleles. This is the first report of the whole analysis of the STRs of the CODIS core using an IR automated DNA sequencer. The protocol was used to solve paternity/maternity tests and for population studies. The electrophoretic system also proved useful for the correct typing of those loci differing in size by only 2 bp. A sensibility study demonstrated that the test can detect an average of 10 pg of undegraded human DNA. We also performed a preliminary study analyzing some forensic samples and mixed stains, which suggested the usefulness of using this analytical system for human identification as well as for forensic purposes.

Prolonged human/sheep cellular chimerism following transplantation of human hemopoietic stem cellsinto the ewe celomic cavity

“…Single positive expected 242 bp PCR bands were sequenced (Big Dye, Terminator V 2.0 Cycle Sequencing Ready Reaction Kit, Applied Biosystem, Foster City, CA, USA) to confirm the human HLA-DQα1 DNA sequence. DNA was also tested for inherited DNA polymorphisms by the presence of 6 microsatellites (short tandem repeats, STR, from CODIS, a method largely employed for paternity testing and noninvasive prenatal diagnostic applications (Ricci et al, 2000;Cha et al, 2005)), for HSC traceability and for excluding sample or operational contaminations.…”

Introduction of the DNase in forensic analysis

“…Determination of the best conditions of decontamination by DNAase was performed using a home-made STR amplification system [4]. Initially, to simulate DNA contamination, 0.9 Al of genomic DNA corresponding to 1 ng of genomic DNA, was added into PCR sterile tubes containing 5.8 Al of the QIAGENR Multiplex PCR Kit (Qiagen, Germany).…”

“…5.8 Al of primers were added to the mix just after DNase I inactivation. Finally, PCR reaction was performed using 40 cycles of amplification and amplified products were analysed by electrophoresis in a polyacrylamide gel as described [4]. Activity was considered effective when no amplification products were present; inactivation was considered effective when primer fluorescence was observed in the polyacrylamide gel.…”

Trends in DNA forensic analysis

“…Most fluorophores used in commercial kits absorb in the UV range. However, a successful result was achieved with infrared (IR) fluorescence technology [13]. All thirteen CODIS core loci were analyzed by the IR-based sequencer (LI-COR Biotechnology, Lincoln, NE, USA).…”