Forensic Molecular Biology - Term Paper Example

 Forensic Molecular Biology Abstract: Crimes and criminal has become an integrated part of human society and the constant evolution of criminal procedure and sophistication used by criminals makes it huge task for crime investigators to identified criminals. The biggest challenges in front of forensic experts includes very little sample at crime site, mixing up of evidences, and many cases completely burn victims etc DNA based criminal identification and crime detection has revolutionized the forensic investigation due to its huge potential in different kind of crime and highly reliable scientific procedure which can be used directly as evidence or proof in court. Short tandem repeat or STR is polymorphic DNA loci present on chromosome containing repeated nucleotide sequence having two to seven nucleotides repeat. The number of repetition of this nucleotide is unique to particular individual and hence can be use as signature for that particular individual. After realization of STR profiling assay many companies have come out with commercial kit for easy analysis and reproducibility. AmplSTR COfiler PCR kit developed by applied biosystems is one of them. It amplifies 6 somatic STR loci along with one sex determination or amelogenin STR loci. Kit also includes positive and negative standard and amplified STR directly load in to 3100 Avant genetic analyzer and STR profile will be obtained by given software. In case of unknown criminal STR profile is matched with CODIS database to search for probable suspect. Key words: STR, CODIS, AmplSTR Cofiler, Genetic analyzer, Slot blot method etc. 1. Introduction Crimes and criminal has become an integrated part of human society and the constant evolution of criminal procedure and sophistication used by criminals makes it huge task for crime investigators to identified criminals. The science used in crime investigation is known as forensic science which utilizes various scientific methodologies to identify the criminals and establish the links between crime and criminals. The biggest challenges in front of forensic experts includes very little sample at crime site, mixing up of evidences, and many in many cases completely burn victims etc. Development of DNA based identification system has revolutionized the way forensic science was operated. The biggest advantage of DNA based techniques is the requirement of very small amount of biological samples which includes blood, hair, semen or any body parts etc. Similarly, one can obtain DNA sample from highly decomposed or burned victim’s bodies. There are several reports where DNA based techniques are employed to investigate crime which was accord almost 100 years before. Further more invention of polymerase chain reaction (PCR) and thermo stable DNA polymerase has revolutionized these DNA based techniques due to its immense power of DNA multiplication. There are several DNA based techniques that are employed for detection of crime and criminals including Restriction Fragment Length Polymorphism (RFLP), Variable Tandem Repeat (VNTRs) and Short Tandem Repeat (STRs) based Amplified Fragment Length Polymorphism (AFLP) or DNA fingerprinting, Gender ID (Amelogenin), Mitochondrial D-Loop DNA for Matrilineal identification etc. there are many instance where combination of these were used. Short tandem repeat or STR is polymorphic DNA loci present on chromosome containing repeated nucleotide sequence having two to seven nucleotides repeat. Number of repetition varies individual to individual and thus acts as signature for particular individual. For example if one can analyze 13 different loci of STR than possibility of all 13 matching STR will be one in billion or greater than that. Federal Bureau of Investigation of USA has chosen 13 specific STR loci as standard for CODIS. Establishment of CODIS makes uniform procedure of STR analysis and easy sharing of information. The AmplSTR COfiler PCR kit developed by applied biosystems isare widely used for STR typing it amplifies 7 different loci namely D3S1358, TPOX, CSF1PO, and D7S820 , along with that segment of X-Y homologues gene amelogenin is also amplified. Amplification of all these loci in two reactions is done by PCR called multiplex PCR. Detection of amplified product it was carried out by ABI prism instrument. Primers which are used in this process are labeled with three different florescence dye 5-FAM, JOE and NED which gives blue, green and yellow florescence respectively. Table 1 indicates composition of AmpFlSTR Cofiler kit and respective amplification. Table 1 AmpFlSTR Cofiler loci. Locus Designation Chromosome Location Common Sequence Motif Size Range (bp) Dye Label D3S1358 3p TCTA (TCTG)1-3 (TCTA)n 114–142 5-FAM D16S539 16q24–qter (AGAT)n 234–274 5-FAM Amelogenin X: p22.1–22.3Y: p11.2 –– 107113 JOE TH01 11p15.5 (AATG)n 169–189 JOE TPOX 2p23–2per (AATG)n 218–242 JOE CSF1PO 5q33.3–34 (AGAT)n 281–317 JOE D7S820 7q11.21–22 (GATA)n 258–294 NED 2. Requirements: Equipments: Ice buckets with ice. Large pyrex Dish (2) Sterile 0.2, 0.5,1, and 2 ml tubes. PE 9700 thermo cycler 3100 Avant genetic analyzer ABI capillary array Balanced and Spatula Photographic equipment Razor blade Pipettes (5,10 and 25 ml) Incubator (56 C) Orbital Shaker Rotating water bath (55˚C, 50 rpm) Heat block Mini-centrifuge with adaptors Thermal cycler QIAamp Spin Columns kit ABI CO filer kit Measuring Cylinders Biodyne B membrane (2,12X8cm) Saran Wrap X-ray film X-ray film developer 96 well plates Plate retainer Micropipettes (P10,P100 and P1000) Reagents: Phosphate-buffered Saline (PBS) Protease (kit) AL Buffer (kit) 95% Ethanol AW1 Buffer (kit) AW2 Buffer (kit) AE Buffer (kit) ddH2O K562 DNA (2ng/μl) Mouse DNA (2ng/μl) Pre-wetting solution Spotting solution Wash Solution Citrate Buffer 3% H2O20.1% SDS ECL Detection Reagents 1 & 2 QuantiBlot® Kit: Stnds A – G Calibration Stands 1 & 2 D17Z1 probe Enzyme Conjugate (HPR-SA) TE-4 Buffer (10mM Tris-HCL, 0.1mM EDTA, pH=8.0) ABI AmpFlSTR COfiler Kit: 9947A DNA (+) Primer Mixture PCR Reaction Mixture AmpliTaq Gold Hybe solution. 3. Procedure: DNA purification: Buccal swab was taken and placed into 2 ml labeled sterile tubes and 400μl of sterile Phosphate buffer saline was added to it. After through mixing 20μl of protease was added and incubated for 10 min at 56°C. After incubation sequential buffers were added as per kit instructions and finally loaded on to given QIAamp column. Repeated centrifugation was carried out to perform washing steps as mention in kit instructions and finally DNA was eluted by adding 100 μl of AE buffer in 1.5ml sterile tubes and stored at 4°C till further use. DNA quantification by Slot Blot method: Sterile tubes, 0.5ml were placed and labeled accordingly and 150 μl of spotting regents was added to each empty tubes. The tubes were gently vortexed and were spin down. For each QIAamp sample, that was to be quantified, 1.5 μl of DNA extract was added to each tube. The Biodyne B membrane provided was labeled on the front edge with Slot Blot number. The membrane was placed in the plastic tray with 50ml of Pre-wetting solution for 1-30minutes. The slot blot apparatus was assembled by placing the gasket into the base plate by aligning the gasket tab into the opening in the top left corner of the base plate. The gasket was pushed firmly over the entire surface of the base plate. Using forceps, the wetted membrane was carefully placed on the top of the gasket aligning with the square outline on the gasket. The top plate was placed on the top of the membrane with the letters facing up and aligning with the registration pins on both sided of the base plate. The ‘T’ vacuum adaptor was verified and the tubing was securely attached to the base plate and the vacuum source. A trap was also installed. The unit clamp was turned to ON position and the sample vacuum knob to the OFF position. The vacuum line was turned on and adjusted to 8-10 in. Hg. Each sample was then slowly mixed and pipetted from its spotting tube into the center of the appropriate well. The location of each sample, standard and the control was documented onto the master “QuantiBlot” Worksheet. The sample vacuum knob was switched to the ON position. Samples were drawn through the top plate and thus will adhere to the membrane thus spearing as discrete slot-shaped spots. After all the samples were transferred to the membrane the seal was broken by turning the clamp knob to the RELEASE position, whereas the sample vacuum knob was still ON. The top plate was then removed without allowing it to dry, while processing the membrane, the slot apparatus was soaked on top plate, and gasket and the bottom tub with 0.1% SDS. Hybridization: The membrane was transferred to 100 ml of pre-warmed Hybe solution in small tray with tight-seal lid and 5 ml of 3% hydrogen peroxide was added followed by rotating gently (50 rpm) in a 50°C water bath for 15 minutes. The solution was the poured off. Thirty ml, Hybe solution and 20 μl of QuantiBlot D17Z1 probe were added. Further, was rotated at 50rpm in 50°C water bath for 20 minutes. The solution was then poured off. The membrane was rinsed briefly in approx 100 ml pre-warmed solution by rocking tray for several seconds. The solution was then poured off. Thirty ml of pre-warmed wash solution was added and 90μl Enzyme Conjugate: HRP-SA was added followed by rotating gently (50 rpm) in a 50°C water bath for 105 minutes. The membrane was rinsed for 1 minute in about 100 ml of pre-warmed wash solution by rocking at room temperature. The process was repeated once again. The membrane was then washed with 100 ml pf pre-warmed solution and rotated at 100 rpm at room temperature for 15 minutes and the solution was pour off. The membrane was rinsed with 100 ml of citrate buffer by rocking the tray. Detection: The film developer was turned on to allow at least for 15 minutes for warm –up time prior to film development. The citrate buffer was poured off the membrane. Five ml of ECL reagent was mixed with 5 ml ECL reagent 2 in small 15 ml tube. The solution was poured in the membrane and was shaken briskly for exactly 1 minute. The membrane was wrapped in a piece of Saran wrap such that there are no wrinkles or bubbles on the top of the membrane. The membrane was placed into a film cassette. The cassette was closed such that the film was allowed to expose for 15 minutes at RT. The film was developed and was labeled with the Quantiblot run name and date. Multiplex PCR Amplification of STR Loci: In a 0.5ml tube, each DNA sample was added to a concentration of 0.15 ng/μl with TE-4. A set of clean 0.2ml tubes were labeled for both sample and control. An additional tube was also labeled for master mix. The master mix was prepared according to the literature provided. Ten μl of MM was added to each PCR tube ensuring that all the components are in the bottom of the tube and the tubes were then placed on ice. Five μl of sample, positive control DNA were added to the appropriate 0.2ml tube. Five μl TE-4 was also added to the negative control tube and kept on ice. Thermal cycling: The samples were placed in a Thermalcycler and were run on “FRSZ-COflier”program as follows: 95°C for 11 minutes. 28 cycles: 94°C for 1 minute 59°C for 1 minute 72°C for 1 minute. 60°C hold for 90 minutes 4°C Hold. CE analysis of STR loci: For CE analysis of amplified STR, the amplified product was taken into a new 0.2ml PCR tubes including ladder standard. Similarly, fFormamide: size standard were prepared for calibration. After mixing and gentle centrifugation 12.5 μl formamide: size mixture was added to each of the 0.2ml tubes. Sample, 1.2 μl of sample and 1.0 μl of ladder were added to respective PCR tubes. All the samples were then transferred to a labeled 96 well plates and covered with plate septum. Plates were briefly centrifuged and incubated for 5min at 95°C for denaturation. After incubation immediately plates were kept on to ice for 5-10 min and than placed in ABI 3100Avant system for analysis. The spectrum was analyzed by given software. 4. Results and discussion; STR loci analysis of given sample were carried out using AmpFlSTR Cofiler loci profiling systems. Rape kit containing vaginal swabs clearly indicates there was no rape as all 6 loci was fund to be exactly matching with victim’s reference while, Amelogenin analysis of bed sheet indicates presence of male during the crime. Amplification from red satin 1 resultant in to failure as there was no amplification. Red stain 2 which was obtained from bed indicates that blood is belong to victims only as all the 6 loci have exact match with victim’s reference. Amplification obtained from hair sample 1 indicates presence of another female having different STR index than the victims. Based on the above results one can strongly established the fact that along with victim there was one male and another female was present at time of crime. Unfortunately STR analysis of boyfriend and male companion was failed and thus role of these two individual can not be established. Secondly STR analysis of alleged girlfriend was not matching with Female sample obtained from crime site, thus ruling out involvement of alleged girlfriend in crime. When sample STR index was matched against student database lead to an emergence of new suspect. The male sample obtained from site was not matching with any of the male student while female sample STR loci was exactly matching with one of the student namely Hayley Dean and made her one of the primary suspect. To establish the role of male, criminal investigator can start investigation by taking charge of Hayley Dean and her interrogation will lead to further revelation. There were many samples where STR profiling was failed and thus it is necessary to re-run the whole process with extreme care. 5. References 1) Bieber FR, Miles HL (eds). Forensic DNA Evidence in the Courtroom. A Comprehensive Review of the Science and Practice. Boston, MCLE, 1999 2) https://products.appliedbiosystems.com/ 3) Peter Gill, Colin P. Kimpton, Andrew Urquhart, Nicola Oldroyd, Emma S. Millican, Stephanie K. Watson, Terry J. Downes Automated short tandem repeat (STR) analysis in forensic casework - a strategy for the future, Electrophoresis. Vol. 16, Page: 1543-1552. Read More