Direct PCR amplification of STR loci using samples that have undergone human hemoglobin identification test

Objective: Forensic DNA assay is mainly used in individual identification, but sometimes it is necessary to confirm the source of the sample that has been identified by DNA assay. Sample quantity is always a problem. A very small sample may not be sufficient to support both confirmatory and DNA assays. We used two commercial kits; OC-Hemocatch and Hexagon OBTI, to identify human blood, and attempted to use the samples remaining in the test kits to amplify STR loci by direct PCR. Methods: Human bloodstains prepared on sterilized gauze and stored for one to 25 years were tested. After performing human blood identification using both human blood identification kits, the cotton yarn remaining in the sample well of the kit was used for amplification of STR polymorphisms. The cotton yarn measuring 0.3 cm was placed into a 0.2 ml PCR tube, and PCR solution was added to the tube. PCR amplification was performed using the multiplex STR kit AmpFℓ STR Identifiler. Results: For samples that were identified to be human bloodstains by the two kits, the yarns in the sample wells of the kits were recovered and directly amplified for STR typing without DNA extraction. All of 15 STR loci and the amelogenin locus were analyzed. The entire amelogenin locus was detected from all tested samples, and almost all the STR loci were amplified from bloodstain samples stored up to 15 years. Conclusions: Detection of human bloodstain in a forensic setting is commonly associated with individual identification. When a bloodstain evidence is identified as of human origin, then it is usually further analyzed by forensic DNA testing. The OC-Hemocatch and Hexagon OBTI human blood identification kits are quick, sensitive and easy to perform, and are especially effective for a small amount of sample in forensic casework. Since a small piece of bloodstain sample can be used for human blood test followed by STR typing for individual identification without DNA extraction, this method is considered useful in crime scene investigation.