A Study on Nucleic Acid Degradation in Drying and Dried Bloodstains as a Means to Determine the Time since Deposition

Abstract

Forensic evidence often plays a crucial role in linking a suspect or victim to a crime. DNA evidence can provide the gender of the victim or offender and can also indicate the strength of the DNA evidence in favour of a probable source. Forensic investigators will often identify and secure body fluids such as blood, semen and saliva stains as a source of evidence. However, it is not always clear if the evidence relates to the particular incident under investigation or another unrelated event. Therefore, determining the time since deposition of the sample can clarify the circumstances of the investigation. Bloodstains are the most plentiful forms of evidence at a crime scene, and presently there is no method available that can accurately determine when they were deposited. Previous studies have shown that it is possible to correlate ribonucleic acid degradation to bloodstain age, but this correlation was only apparent in stains that were more than six months old; recent bloodstains could not be distinguished from six month old bloodstains. The aim of this study was to determine whether it is possible to use the degradation profile of nucleic acids to provide a better estimate to when a bloodstain was deposited. The main hypothesis was that as a bloodstain ages, nucleic acids are degraded and become fragmented. By examining the degradation of beta hemoglobin (HBB) DNA, Glucose-6-phosphate dehydrogenase (G6PD) mRNA and 18S rRNA in aged bloodstains by capillary electrophoresis, these fragments could be used to determine bloodstain age. In order to test this hypothesis, a real-time quantitative PCR method to standardise the input amount of cDNA analysed was developed as previous studies have not taken sample variation into account. The pattern of degradation of these nucleic acids was then investigated as bloodstains transitioned from wet to dry when exposed to different storage conditions to determine the directionality of degradation, whether it was from the 5’ end, 3’ end or random. This was achieved by developing a multiplex PCR assay with primers and probes specific to different regions of the HBB DNA, G6PD mRNA and 18S rRNA sequences. Regression analysis was performed on the peak heights of these marker sequences from aged bloodstains deposited on cotton cloth to determine which fragments of which markers were statistically relevant to age prediction. The result was an age prediction equation which used the peak heights of the G6PD 5’ medium, 3’ long and 3’ medium fragments and the central long fragment of 18S rRNA. The equation was evaluated using blind samples and on bloodstains found on common forensic type substrates such as carpet and articles of clothing stored under different conditions. The study found that the prediction equation was able to discern age differences between recent and old stains on the substrates tested. However, the equation was more accurate at predicting the age of bloodstains found on cotton cloth stored at room temperature with exposure to sun. The method can be used to age bloodstains that are days, weeks and even months old.