“Only Cigarette Butt is Left, DNA Fingerprinting Traps the Theft”

The examination of saliva traces left on cigarette butts as evidences are complicated due to the availability of biological material in trace amounts and its rapid degradation due to extreme effects of environmental factors. This study is aimed to assess the DNA purity and quantify the amount of DNA preserved in saliva found on cigarette butts subjected to various temperatures and humidity. Isolation of cell material from biological traces on forensic evidence is often a serious challenge to solving forensic cases. Successful isolation of high-grade DNA from cell material even in critically low quantity could be achieved in examination of traces. The proper collecting and storage of the material is very important for successful DNA typing from saliva traces and epithelial cells from the lips and oral cavity. Meeting these conditions would increase the chances for successful DNA profiling of biological traces on evidence of an earlier date. In the presented forensic cases the opportunity for solving the crime was given by the vices of the suspects. In this case one cigarette butt found at scene of crime was the only evidence to detect the accused. DNA is extracted from salivary epithelial cells adheres to the cigarette butt and profiled successfully. DNA profiles of suspected accused and the cigarette butt are matched in the source.

Keywords:Cigarette Butt; Salivary Epithelial Cells; PCR; STR Profiling

Cigarette butts are a common trace sample at crime scenes and obtaining DNA profiles from this evidence is an important capability in the forensic science repertoire. DNA extraction is a critical step in forensic analysis because quality of DNA directly affects the ability to obtain a good quality forensic profile. Cigarette butts are notably difficult samples in that they produce DNA that is contaminated with inhibitors of the polymerase chain reaction (PCR) [1]. The range of additives may lead to different levels of inhibition from different the brands, and to complicate matters, the brand is not always easily determined. Most methods in current usage solve the problem by adding a series of post-extraction steps to selectively remove inhibitors. These steps make the method complex, non-automatable, susceptible to contamination, and the many steps reduce DNA yields – a critical factor with trace samples [1,2].

Cigarette butts are one of the common carriers of saliva traces in forensic practice. Today, approximately 1.2 billion people worldwide smoke tobacco [3] and they smoke when they are nervous (i.e. when involved in a crime) [4,5]. The male and female smokers both are showing furious, annoyed rebellious impatient behavior [6]. Also in Finland about 10 town study was done by Marianna Virtanen.et.al. Where they studied the statistical of crime and the smoking behavior.Where they found that where there is strong smoking intensity and low average household income the the high local srime reate is high [7]. Cigarette butts found at crime scene may contain traces of saliva and attached mucosal epithelium cells from the lips of the smoker, which allows for DNA identification by profiling [8,9].

Saliva is evidence which can be encountered in forensic casework. Most probably the exhibits like toothpick, cigarette butt, bottle or postage stamp, all of which could contain saliva are most susceptible to damage by environmental factors like “Heat, Sunlight, Moisture, Bacteria, Mold. Direct sunlight and warmer conditions may also damage DNA [10].It has a high evidentiary value in identifying victims and suspects as well as exonerates an innocent individual [8]. A healthy adult produces 500 - 1500 mL of saliva in a day, at a rate of 0.5 mL/min. The quantity and quality of saliva produced, however, is influenced by several pathological and physiological conditions which includes, taste and smell stimulation, hereditary factors, hormonal status, and oral hygiene [11].

DNA samples recovered from a crime scene are frequently exposed to damaging environmental conditions such as light, heat and bacterial decomposition before they are collected for analysis. Hence, generating an evidentially valuable profile from these quality-compromised samples is a great Malaysian Journal of Forensic Sciences (2016) 7(1):10-16 11 challenge to the forensic scientist [12-13].

Carefully clean the platform of workstation of laminar flow with ethyl alcohol

Take approx 0.5mm sample piece and cut respective sample into small pieces and take into 2ml micro-centrifuge sample tube

To this Add 400μl Forensic Buffer +25 μl Protease K+40 μl 1mM DTT

Vertex and spin

Incubate at 56 ˚C overnight on Thermo-Shaker

To the next day, transfer the sample into the EZ1 micro-centrifuge sample tube

Set the micro-centrifuge sample tube in the EZ1 Advanced (Quiagen) magnetic bead based liquid handling system for automate DNA Isolation

Store the extracted DNA at -20 ˚C

Take 5 μl blood sample+ 97.5 μl ATL buffer+100μl AL buffer+10 μl protease K in micro-centrifuge Tube

Vortex and spin for 1 min’s

Incubate at 56 ^oC for 10 min’s on Thermo-shaker

To this add 50 μl 99% ethanol then vortex and spin

Wait for 5 min’s and transfer the supernatant into the micro-kit column

Centrifuge at 80000rpm for 1 min’s and discard the filtrate

Wash the column by adding 500 μl washing solution

Centrifuge the column at 8000rpm for 2min’s and discard the filtrate

Repeat the procedure

Spin the empty column at 10000rpm for 3min’s

Transfer the column in new Elution Tube

Finally add 100 μl Elution Buffer to the column, centrifuge at 14000rpm for 2 min’s

Remove the column and store the DNA for at -20˚C

PCR reaction Mix -10.5 ul
Primer set- 5.25 ul
Sample input -10 ul
PCR Protocol

STR genotyping is detected and analysed on 3130 Genetic Analyser (Applied Biosystems) instrument by capillary electrophoresis of single stranded amplified DNA fragments includes following steps.

Standard Mix :
1μL PCR product
0.5 μL Size standard (for GeneScan500-LIZ^®)
10-20 μLHi-Di™ formamide (PN 4311320 )

Denaturation of PCR product. (90 –95 °C, 2 –5 min)

Immediately on ice or cool to 4 °C in thermal cycler Load the mixture in auto sampler on instrument for injection.

Electrophoresis is done through fine glass capillary filled with polymeric gel. (During capillary electrophoresis, the extension products of the PCR reaction (and any other negatively charged molecules such as salt or unincorporated primers and nucleotides) enter the capillary as a result of electro kinetic injection. The extension products are separated by size based on their total charge)

DNA fragments travel through capillary according to their size & reach the window which coincides with the Laser device in the instrument. (Shortly before reaching the positive electrode, the fluorescently labeled DNA fragments, separated by size, move across the path of a laser beam. The laser beam causes the dyes attached to the fragments to fluoresce.)

Laser excites the fluorescently labelled DNA fragments. (The laser beam causes the dyes attached to the fragments to fluoresce.)

CCD Camera behind the window records the excitation peaks. (The dye signals are separated by a diffraction system, and a CCD camera detects the fluorescence.)

Excitation peaks for 16 different loci are obtained.(Because each dye emits light at a different wavelength when excited by the laser, all colors, and therefore loci, can be detected and distinguished in one capillary injection.)

For each set of sample standard allelic ladder is run.

DATA COLLECTION software collects the data of these excitation peaks. (The fluorescence signal is converted into digital data, and then the data is stored in a file format compatible with an analysis software application.)

The DNA extracted from saliva detected on cigarette butt found at scene of crime and blood sample of suspect was typed at 15 STR LOCI and gender specific Amelogenin locus using PCR Amplification technique.

Table No.5 shows that the DNA profile obtained from saliva detected on cigarette butt found at scene of crime and DNA profile obtained from blood sample of suspect is identical and from one and same source of male origin. The DNA profile of saliva detected on cigarette butt found at scene of crime and DNA profile of blood sample of suspect are matched with the maternal and paternal alleles in the source.

In this case a complainant, who worked as senior clerk in election commission office, filed a complaint. According to this complaint, material records and memory chips of voting machines of 2012 election were sealed in small boxes and all these boxes were kept in locked room. It was very well confirmed that all this election related material was kept in completely locked room, but it was found that this locked room was break open by pushing the door very strongly by unknown person. This unknown person broke the sealed boxes and succeeds in taking very important election material. He also removed paper seal of main hall door where election boxes were kept. By doing all this, the unknown person pretended it is a scenario of robbery to fool the people and police. During investigation, police found only cigarette butts at scene of crime. So police send these cigarette butts along with the blood sample of suspect for DNA fingerprinting.

DNA present in body fluids such as blood, semen, saliva, sweat, nasal secretion etc. get preserved after drying on any object at 37 0C forever. Due to stability and sensitivity of DNA it is possible to extract DNA from various materials which are found at scene of crime like cigarette butts, chewing gum , glass bottles and any unclaimed articles used by the used by accused . This is a DNA analysis from only cigarette butts as evidence collected from scene of crime. DNA profile of salivary epithelial cells adheres to the cigarette butt matched with the DNA profile of suspect control blood sample. So we can say that, small evidence collected from scene of crime is also very much useful to trap the criminal, only because of the sensitivity and accuracy of DNA fingerprinting technique.

Author thanks to Director General (Legal & Technical) Home Dept.Govt.of Maharashtra and Forensic Science Laboratory, Mumbai, for the facilities to do this analysis.