The armed conflict that Colombia has lived through for more than 40 years has forced Colombian national forensic laboratories to be global leaders in adopting DNA genotyping technologies and systems that allow analysts to efficiently solve the complex cases they face. The majority of these cases involve bone samples from human remains, many of which have been found in caves throughout the country. Due to the topographical layout of Colombia, local climates can vary dramatically, particularly temperature and relative humidity. These factors and a complex soil diversity often make DNA isolation from bone samples difficult. Colombian national forensic laboratories have a large number of cases that are very complex due to the specific soil conditions where the samples were found. Such conditions hinder effective identification of the remains.
The technical staff of Biomol Latinoamerica, Inc., worked with forensic analysts from the forensic DNA laboratory at the National Institute of Legal Medicine and Forensic Sciences in Cali to genotype difficult samples using the new PowerPlex® 16 HS System. Ten samples from ten different cases were used. DNA was purified from four bones, four teeth, a piece of muscle and one postmortem blood stain. Samples were quantitated by real-time PCR using the Plexor® HY System, which confirmed the presence of inhibitors in many samples. Previously, we were unsuccessful in our attempts to type these samples using other amplification kits from Applied Biosystems and Promega. None of these samples yielded complete STR profiles. However, the amplification conditions and sensitivity of the PowerPlex® 16 HS System allowed us to obtain full profiles for eight of these ten samples.
“Using the PowerPlex® 16 HS System, we were able to obtain more full profiles from DNA isolated from long bones and teeth than with other commercially available STR amplification systems.”
Methods and Materials
Cleaning and digestion of bone samples and teeth: After we evaluated storage conditions and reviewed internal processes, all samples were individually cleaned as follows: Samples with traces of tissue and soil were disinfected using water, gauze and brushes soaked in a Decol solution (5–10% sodium hypochloride). These were further cleaned with a knife, incubated in a 10% SDS solution in a 37°C water bath overnight and dried at 60°C. Samples previously washed and prepared by the forensic anthropology group had the periosteum and bone marrow removed in a hood using a rotary power tool. A piece of each bone sample (4–5g) was crushed into a fine powder. Teeth were prepared using a Retsch® MM301 Ball Mill. Each crushed sample was decalcified in 0.5M EDTA (pH 8.0) and digested in 8ml of EDTA, 80µl of Tween® 20 and 500µl of proteinase K (10mg/ml) overnight at 56°C.
Preparation and digestion of tissue samples: A 0.5 × 0.5cm piece of internal vascular muscle was cut with a scalpel, washed with distilled water, ground and digested in 1.5ml of digestion buffer (10mM Tris HCl, 10mM EDTA, 100mM NaCl, 20% SDS and 40mM DTT) with 250µl proteinase K (10mg/ml). Samples were incubated at 56°C overnight.
Preparation of a postmortem blood sample: A 0.5 × 0.5cm fragment of the victim’s clothing was cut and washed in 1ml of sterilized water. DNA was extracted using Chelex® resin.
Organic extraction and purification: DNA was isolated from bone and teeth samples using phenol:chloroform extraction. DNA was extracted from tissues samples using phenol:chloroform:isoamyl alcohol extraction. DNA purification was subsequently carried out with an Amicon Ultra-4 100k centrifugal filter unit (Millipore), and DNA was washed six times with water to remove contaminants and inhibitors.
Quantitation, amplification, genotyping and analysis: Samples were quantitated by real-time PCR using the Plexor® HY System (Cat.# DC1001) on a Stratagene MX3005P® qPCR instrument. For each sample, 1–2ng of DNA was amplified using Identifiler® (Applied Biosystem) and PowerPlex® 16 Systems with an Eppendorf thermocycler (Merck). Only 0.5ng of DNA was amplified using the PowerPlex® 16 HS System (Cat.# DC2101). Amplified products were analyzed by capillary electrophoresis on an ABI PRISM® 3130 Genetic Analyzer (Applied Biosystems) using GeneMapper® ID software, version 3.2.
DNA purification following quantitation: Samples were resuspended in 30–50µl of sterile, deionized water before amplification. For samples that contained inhibitors, as detected by Plexor® HY quantitation, an additional drying process at 60°C was required. These samples were divided into two aliquots, one of which was archived. The second aliquot was dried at 60°C to concentrate the DNA and eliminate volatile inhibitors, resuspended in 5–10µl of sterile, deionized water, allowed to incubate at room temperature for 1 hour to solubilize the DNA, then vortexed.
The results of DNA quantitation and amplification are summarized in Table 1.
Table 1. Results of DNA Quantitation Using the Plexor® HY System and Amplification Using the PowerPlex® 16 HS System.
In eight of ten samples, improved STR amplification was observed with the PowerPlex® 16 HS System in terms of the number of amplified alleles, peak height, allele specificity and reduced allele dropout when compared to profiles initially obtained using Identifiler® and PowerPlex® 16 Systems (Figure 1). The results for these latter two kits were as follows:
- Only one sample, the postmortem blood stain, showed a clear profile.
- No profile was obtained for one of the samples (bone).
- The remaining six samples showed allele dropout, low peak heights and poor reproducibility (data not shown).
In contrast, the results obtained with the PowerPlex® 16 HS System were as follows:
- Full profiles were obtained for six samples (muscle, two teeth, blood stain and two bone samples).
- Partial profiles were obtained for two samples (bone and tooth, samples 4 and 5, respectively).
Two samples (one bone and one tooth, samples 8 and 9, respectively) could not be amplified with any of the STR systems tested.
Analysis and Conclusions
One of the most relevant aspects of this work was the improved specificity and sensitivity of STR amplifications using the PowerPlex® 16 HS System. This was reflected in an increased number of alleles obtained, higher peak heights, a reduction in allele dropout and lower background.
Moreover, it is important to note that an additional DNA purification step that involved drying and resuspending the DNA reduced or eliminated inhibitors and other contaminants in the samples, as did a cleanup protocol conducted prior to DNA extraction.
One additional observation is that the complexity of a sample does not depend upon the sample type (bone, soft tissue, teeth or blood) but rather on the extent of DNA degradation and possible presence of inhibitors.
For bodies undergoing decomposition or several years after burial, DNA from dental tissue (molars) and long bones, such as the femur or tibia, tends to be better preserved. This finding, together with appropriate protocols for collecting and preserving samples, must be considered when conducting genetic analyses.