Predictive Models as Screening Tools for DNA Recovery from Baked and Burned Porcine Bones

Research Article

Austin J Forensic Sci Criminol. 2015;2(3): 1029.

Predictive Models as Screening Tools for DNA Recovery from Baked and Burned Porcine Bones

Velzen IV², Shaw M¹, Raveendran M² and J. Gonzalez-Rodriguez²*

¹University of Lincoln, School of Life Sciences, Brayford Pool, United Kingdom

²University of Lincoln, School of Chemistry, Brayford Pool, United Kingdom

*Corresponding author: J. Gonzalez-Rodriguez, University of Lincoln, School of Chemistry, Brayford Pool, Lincoln, United Kingdom

Received: June 01, 2015; Accepted: June 25, 2015; Published: June 30, 2015


Burnt bones and skeletal remnants continue to challenge the proficiency of forensic investigations in human individualization and identification. The various natural disasters and human inflicted crimes involving fire leave the forensic investigators with very little to work on. Thus, demand for practical studies to obtain useful facts for improvisation of current techniques and to overcome the short comings is a prerequisite. In this study Design of Experiments (DOE) as an investigative and screening tool to relate the different variables (burning temperature, time, thickness of flesh, presence of accelerants) involved in the burning process and to detect the probability of obtaining successful DNA identification from burnt bones is proposed. We show that high temperature and large base pair PCR primer have a significant effect on DNA retrieval and amplification. The baking study provides reproducible DNA identification with maximum retrieval temperature of 320°C for the smallest (106bp) amplicon. The study involving accelerants demonstrates that those with high specific heat capacity decrease DNA recovery, hence suggesting probable damage to DNA. Through this study the positive effect of presence of flesh for DNA recovery was also verified with a maximum DNA recovery temperature of 500°C. Utilizing all these information through DOE, predictive models were also created with regression equations to calculate positive DNA amplification and to predict the different variables respective to the burning process. These models created using porcine bones could be related for real scenarios and with more data procurement it could be used effectively in forensic investigations.

Keywords: Design of Experiments; DNA recovery; Forensic investigation; Burnt bones


Forensic investigations involving identification and individualization of burnt human bones remain a challenge. Human bodies subjected to extreme temperatures as a result of accidents, mass disasters [1, 2, 3, 4, 5], death by fire [6, 7, 8, 9] and in some cases as an act to conceal crime [10, 11, 12] often leave behind only burnt bones or fragments of bones and dental remnants at the crime scene. In these cases, with loss of skin and tissue, visual identification and fingerprinting methodologies are not possible. Occasionally, even dental identification becomes impossible or difficult due to lack of records. Histological studies may shed light on the origin of skeletal remains but DNA analysis is needed for singular identification. Thus, under these circumstances genetic finger typing becomes a much-expected choice, but the absence of flesh or proper substrate for DNA makes extraction problematic. In severely burnt/charred bones there is a high likelihood of degraded DNA template, which makes successful amplification difficult, reducing the probability of identification.

Studies in this area have led to the development of modified and improved DNA isolation procedures [13] to overcome problems and achieve effective DNA extraction. Yet identifying severely burnt bones through DNA methods remains problematic as the process of burning changes the bone both physically and chemically [14] degrading the DNA. To understand the different complications, research concerning the histology of burnt bones, elemental analysis of burnt bones, and relation between burning temperature and changes in bone has been conducted [15, 16, 17]. The above studies provide valuable information regarding the burning temperature, indication about the tests to be performed and even estimations on how the burnt bones had been treated. Reports investigating the probability of successful DNA retrieval from bones subjected to high temperatures seem to show that the positive DNA results are a factor of both temperature and duration of burning.

While some studies show a positive DNA extraction and amplification even at high burning temperatures [18], most studies were only able to yield positive results at lower temperatures [19, 20]. Studies also indicate mitochondrial DNA (mtDNA) to be a better choice than nuclear DNA [18] as mtDNA is available in greater numbers and thus there is a higher chance to achieve positive extraction and amplification. Contradicting results reporting the lack of amplifiable DNA (even mtDNA) at all temperatures [21] could be due to the different DNA extraction procedures used or burning temperatures and procedures. Nevertheless it is of evidence through these studies that the probability of successful DNA extraction and amplification is higher in bones burnt at a lower temperature for a short period, and the difficulty increases with increasing burning period and temperature.

From the above practical conclusions, the possibility of various factors affecting the probability of DNA extraction, amplification and individual identification from burnt bones should be recognized. While studies on burning temperature and burning time as variables are available and acknowledged, as stated above, and since the already available experimental studies involve burning bones in an electrical or industrial furnace, a study concentrating on several variables like effect of ignitable liquids, presence of flesh, effect of baking including burning temperature and time is required. As the majority of crimes related to burning victims (accidental fire, arson cases, etc.) contains use of accelerants or fuels [22], the present study providing information linking ignitable liquids-burning temperature/time and DNA retrieval is needed to accept or reject any theories concerning inhibitory effect of ignitable liquids on DNA extraction and amplification (if any).

Previous works [23, 19] list histological studies and analytical techniques as approaches to evaluate the potential of DNA recovery. These procedures provide us an indication if the bone samples are capable of a subsequent positive result, thus acting as a diagnostic tool. These tests not only save money and energy wasted on timeconsuming procedures but also suggest useful information regarding temperature and time the bones were burnt, which is relevant in a forensic investigation. With regard to this fact, the use of statistics to build a model, by experimental design (DOE), to be used as a diagnostic tool was attempted in this study.

The major aim of this study is to design a predictive model that could be used for calculating burning time, burning temperature and also work as a diagnostic tool to ascertain a probability of recovering DNA from burnt bones. Analysis of burnt and baked bones with ignitable liquids, temperature, time and flesh as variables was performed to study the associations between them. The data collected from this study and the designed model would serve as an indicative tool to assess burnt bones for various forensic purposes. The recurrent encounters of burnt bones in forensics and the constant attempts by the suspect to mislead and conceal evidence [24, 25, 26] necessitates more experimental work to be done in this field. This will bring about reliable methods and useful data to diminish limitations and overcome drawbacks that are encountered with present procedures. Thus this project proposes the use of statistics to study the association between different variables involved in burning bones and depicts a basic model as a screening tool, alike which further models could be designed for various other requirements.

Methods and Materials

Bone samples preparation

Porcine bones selected for this experiment were acquired from a butchers shop, Lincoln, UK. Similar sized phalangeal and rib bones (Figure 1) were used in this study. The bones were divided into three groups and labeled BRF (burned with flesh), BR (burned with ignitable liquids) and Baked without flesh (BK) for ease of reference and separate temperature studies were generated for each group. Group BR and BK bones were de-fleshed and cleaned before further analysis. Group BKF consisted of bones with flesh of different thickness (5mm, 10mm, and 15mm). The bones were burnt with and without using ignitable fluids in a crucible inside a chemical hood/ gas chamber, and the oven set at different temperatures was used for baking.

Experimental Conditions

Burning with ignitable liquids (BR): De-fleshed phalangeal and rib bones were burnt with white spirit, petrol and ethanol at different temperatures and then tested for retrievable DNA. The bones were weighed and 0.5ml/g to 2ml/g accelerant with an increase of 0.5ml was added for the burning procedure. Maximum burning time and temperature association of different ignitable liquids used were also studied by burning the accelerants on their own in a crucible. The maximum time taken to self extinguish and the related maximum temperatures reached were recorded using a stop clock and an infrared thermometer. Temperature studies relating to the particular accelerant used, accelerant volume, burning time and the probability of obtaining a positive DNA amplification were also implemented.

Baking without flesh (BK): Bones were baked at temperatures between 50ºC to 400ºC for 10 to 40 minutes and DNA was extracted using either Chelex or Phenol chloroform method. Two separate DOE models were built for both the extraction procedures. The baking studies in the oven were done to maintain a constant and nonfluctuating temperature, thus establishing an accurate temperature model. The main aim of the study was to chart the effect of baking on DNA recovery. The use of two different extraction procedures would shed light on the limits of the method in relation to the maximum burning temperature and time till which DNA extraction is possible. This study could also postulate evidence on the more capable DNA extraction procedure of the two.

Burning with flesh (BRF): The phalangeal and rib bones with flesh of various thicknesses (5 to 15 mm) were first burnt without any temperature or time constraint to gain an overview of temperatures and time. The uncontrolled experimental conditions would also provide information about how fleshed bones burn and how the other variables have an effect on it. The fleshed bones were then subjected to different sets of temperature and time period ranging between 100ºC to 500ºC and 5 to 30 minutes. The effect of thickness of flesh on extracting DNA and its relationship with burning time and temperature was established. All the temperature models built were designed using the statistical Design of experiments (DOE) technique as explained below, using the stat graphics software.

Design of experiments

Design of Experiments (DOE) is a systemic statistical way of designing a model that delivers the most information out of a fewest number of experiments by creating an equal data. It has three main phases, creating an experiment, analyzing the results and further experimentation.

The parameters considered for this study were burning temperature, burning time, effect of ignitable liquids (IL) and volume of IL and the presence of flesh. Two major designs one for studying the relationship between baking temperature, time and its effect of obtaining DNA and one for studying the influence of flesh in obtaining DNA at different burning temperatures and time was performed. The effect of different type of ignitable liquids on DNA extraction was also tested as a univariate function. The bones were heat-treated (burned or baked) to a set of temperatures for appropriate time periods as predicted by statistical Design of Experiments (DOE) and different models were developed to test the parameters considered in this study.

DNA analysis – chelex and phenol chloroform method

Bone samples taken from each group were grounded finely using pestle and mortar and DNA was extracted from 0.25 grams of ground bones using Chelex and Phenol Chloroform [Phenol: Chloroform: Isoamyl Alcohol 25:24:1, pH 8] methods as stated elsewhere [27, 28]. The two methods were compared to figure out the most effective and plausible way of extracting usable DNA from burnt bones. The DNA pellets were suspended in 50μl TE buffer and stored at -20 degree Celsius. The concentration and purity of the extracted DNA was analyzed using a thermo scientific Nanodrop UV spectrophotometer.

DNA amplification and detection

Three forward and reverse primer pairs (Table 1) targeting different sized fragments (106bp, 199 bp, 411 bp) of 12s rRNA gene of Sus scrofa were designed and purchased from Sigma Oligo. Polymerase chain reaction (PCR) was performed to amplify the indicated fragments in 0.2ml PCR tubes. The primers were optimised to a concentration of 10 pmol to obtain clear and reproducible bands. The different annealing temperatures for each primer set were also optimised. Refer supplementary data, Table 10, for cycling parameters- temperatures and times, for each primer set.