Forensic Molecular Biology Analysis - Lab Report Example

Forensic molecular Biology Lab s) 2008 Forensic investigations play a central role in crime detection and criminal identification. It deals with minute details and clues available from crime site to give leads in investigations. The biggest challenge in crime detection is establishment of criminal and its role in crime. Since 19th century scientists were aware of some unique identity of an individual which distinguish from others, like finger prints. In 1970, DNA was established as key element in human life and which makes individual as unique creature. Variability in DNA sequences among individual to individual have attracted forensic experts to develop technique based on DNA to identify individual or criminal similar to finger prints. Invention of polymerase chain reaction and thermo stable DNA polymerase has revolutionized this concept. Many techniques like 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 Maternal identification etc. have been developed for forensic investigations. PM&DQA1 is the first PCR based typing system, developed by the scientist at Cetus Corporation and commercialized by Applied Biosystems. Inc. PM& DQA1 is simple and rapid technique mainly employed in case of limited DNA sample or in case of highly degraded DNA. The test is performed on 6 different loci to investigate pleomorphism using hybridization based Reverse DOT-BLOT methodology. The major limitation of this technique is its differentiating power due to limited diversity found among 6 loci among different human population. Keywords: VNTRs, PCR, PM&DQA1 loci, Reverse Dot-Blot, Hybridization Introduction Crime investigation obtained new direction after invention of DNA based techniques for identification of criminals. The biggest advantage of DNA based techniques is the requirement of very small amount of biological samples which includes blood, hair, semen etc. 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 many 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. Criminal identification based on human body parts or component has been used since last century as part of forensic investigations. The classical example being Human finger prints which was recognized by British physician Sir Francis Galton (1). In the beginning of 20th century many physiologists and doctors realized that blood cell has distinct antigens and they are different from individual to individuals. Initially blood cell antigens were taken into consideration only in case of blood transfusion and surgery but later on its forensic utilities were realized and it was employed to solve problem of paternity dispute, child mix-up etc. Those days blood samples saliva and semen samples were produced in court as evidence against criminals but it was not taken as conclusive evidence due to its limited distinguished power. In 1970, the things started to change after discovery of DNA structure and the increases in the understanding of its variability between individual to individual. Once DNA was established as target molecule for forensic investigation, many researchers came forward with different methodology and different locus having higher variability like micro and mini satellites. Initially there were many questions that were raised over methodologies and interpretation of DNA based identification systems. But thorough investigation and population genetics based data collection satisfied all those questions. Similarly, there were always comparisons between DNA finger printing and normal finger printings but in many cases a professional criminal have had succeeded to wipe out all fingerprints on crime site but a piece of hair has revealed the identity of that criminal. AmpliType PM&DQA1 is the first PCR based typing system developed by the scientistat Cetus corporation and which was commercialized by Applied Biosystems. Inc (2-3). The system test the pleomorphism in six loci namely (1) Low density Lipoprotein receptor (LDLR), (2) Glycophorin A (GLYPA) (3) Hemoglobin G Gamma Globulin (HBGG) (4) D7S8 (5) Group-specific component (GC) and (6) HLA-DQA1. All the 6 loci are sequence pleomorphic and are detected by hybridization with allele specific oligonuclotied (ASO) probes. The hybridization procedure follows in this procedure is known as reverse dot-blot method where probes were impregnated on membrane strips at specific place. In commercial kit two strips are given one for DQA1 and second for remaining 5 loci. All the probes are tagged with Horseradish peroxidase along with streptavidin while PCR amplicon is generally biotinylated. After hybridization color development is observed using chromogenic substrate for Horseradish peroxidase. The major disadvantage of this technique is its discriminatory power due to the limited number of alleles that exist in population. But it is the primary tool to start investigation where DNA sample is too less (up to 0.5ng of DNA) or it is highly degraded. Similarly, extreme care has to be taken against contamination of external DNA which can leads to false results (4) Requirements: Equipment Ice buckets with ice. PE 9700 Thermal cycler. Sterile 0.5 and 0.2 ml tubes. Mini centrifuge with adaptors. Gel Electrophoresis rigs Measuring Cylinders Bottles,250 ml Balanced and Spatula Orbital Shaker Thermal cycler 250 ml water weights (1/Tray) Minicentrifuge with adaptors Automatic pipettor Photographic equipment Rotating water bath (55C, 50 rpm) Pipettes (5,10 and 25 ml) Incubator 55C Typing Trays and Aluminum foil Polaroid Camera Polaroid Type 665 film Chemicals Double distilled water. Bovine serum albumin (BSA), 8 mg/ml. AmpliType PM and DQA1 kit: Positive control DNA, Primer Mixture, PCR Reaction Mixture. 1X TBE Buffer 6X Blue/Orange loading Dye 100 bp DNA ladder Agarose(PCR Grade) Hybe solution (55C) Wash Solution (55C) 30% H2O2 dH20 AmpliType PM+DQA1 Kit: DQA1 strips, Enzyme conjugate, PM strips, TMB chromagen. Citrate buffer and color development solution H2O2, 30%. Chromagen solution Deionized water. Methodology: PCR analysis The program for PCR in the thermal cycler (PE 9700) was saved as "FRSC-PMDQA". The steps were as follows: i. 95C for 5 minutes ii. 32 cycles: 95C for 30 seconds 63C for 30 seconds 72C for 30 seconds iii. 72C fro 10 minutes iv. 4C hold. The samples were vortexed and then were spin down using a microcentrifuge for 10 sec. The PM+DQA1 Master Mix worksheet was used to set up a master Mix in a labeled 0.5 ml tube. Master Mix each of 82 l was added in the PCR tubes (0.2ml). Chelex DNA (Concentration was determined according to QuantiBlot) and ddH20 was added to the corresponding PCR tube (used in step 3). For Positive control 20 l of DNA was added whereas in the negative control 20l of ddH20 was added to the corresponding PCR reaction tube. The PCR was run according the programme (mentioned in step 1). The products were stored at 4C for further analysis by gel electrophoresis and Dot blotting. Gel Electrophoresis Agarose gel solution (2%), 75 ml was prepared in 1X TBE using PCR grade agarose. There 0.5ml tubes were labeled for each amplified sample, control and as well as for the 100 bp DNA ladder. Three l of 6X blue/orange dye was added to each sample, control and the ladder tube. The entire volume of the ladder, sample and the control was loaded on the gel and was run at 75 volts for approx. 45 minutes. Care was taken that the yellow dye front does not run off. The gels were stain for approx. 20 min in ethidium bromide. The gel image was taken on the UV box. Dot Blotting, Color Development and Photography of the strips. The rotating water bath was set to 55C and the temperature was maintained continuously. The hybridization solution and the wash solution were warmed to 55C until all the solids were completely dissolved. Using filter forceps, the pole strips were removed from each tube and were labeled carefully. Each strip was placed in each clean well of the typing tray, in the same orientation. The thermal cycler temperature was set to 95C to denature the amplified DNA. The tubes were incubated at this temperature for 3-10 minutes. Pre-warmed hybridization solution, 3 ml was added to each well at the labeled end of the strips. Each tube was then removed from the thermal cycler and was spin down for complete solubilization. Twenty micro liter of the amplified DNA was withdrawn and was immediately added below the surface of the hybridization solution into the well of the corresponding strip. Only hybridized CHELEX DNA sample were kept for hybridization. Care was taken that each strip was completely wet throughout the color development and the photography step. The amplified samples were hybridized to the probe strips at 55C for 15 min at 50 rpm. Five minutes before the above incubation, conjugate solution was prepared using prewarmed hybridization solution and enzyme conjugate according the requirements. The solution was mixed thoroughly and was kept at room temperature. The contents of each tube were aspirated out followed by rinsing each well thoroughly with 5 ml of the prewarmed solution. Enzyme conjugate solution, 3 ml was added to each well and was covered with clear plastic lid. The tray was transferred to rotating water bath at 55C, 50 rpm. The enzyme conjugate solution was incubated with DNA probe strips at 55C for 5 min. After the completion of the incubation the contents of each well was aspirated and the wells were again was rinsed with prewarmed solution. The DNA probe strips were incubated at 55C for 12 mins. The contents were again aspirated and 5 ml of the wash solution was added into the well. The tray was rocked from several seconds and slowly the contents were pour off. Color Development Citrate buffer, 5ml was dispensed in each well and the tray was kept in an orbital shaker at 50 rpm at room temperature for 5 mins. In the meantime, color development solution was prepared taking acre that it is protected from light. After incubation with citrate buffer, the contents were poured off and 5 ml of freshly prepared color development solution was added to each well. The strips were allowed to develop at room temperature by rotating on orbital shaker at 50 rpm for 30 mins. After incubation, the contents were poured off and the color development process was stopped by washing the strips with deionized water. The washing step was performed twice with 3 deionized water washes. Photography The wet strips were wrapped in plastic wrap to minimize the exposure of the strong light. The strip was then photographed using the Polaroid camera with type 665 film and an orange filter with settings at f8 for 1/8 secs. Results and Discussion: PM-DAQ1 analysis of given sample were carried out as per the manufacturers protocoll with slight modifications mentioned in the method. Thereafter, the PCR samples were loaded on to the 2% agarose gel for determination of PCR efficiency. Gel analysis clearly indicates successful PCR methodology (Figure:1). Positive control gives prominent band while in case of negative control there was no band observed. Experimental sample showed exactly identical band of around 200 bp to positive control indicating proper amplification of DQA1 while in case of other five PM markers amplification was not effective. Figure: 1 Gel Image displaying PCR amplified products. In the final stage of analysis reverse Dot-blot analysis of PCR amplified product was performed. Based on the results obtained for negative and positive controls it was concluded that the assay was successful in terms of all parameters. The nail scrap DNA was found to be belonging to 1.2, 4.1 DQA1 type while other allelic types were LDLR- BB type, GYPA-AB type HBGG-AB type, D7S8-AB type and GC-AC types. The Analysis of suspect revealed that Janel Conti and Justin Liverman have similar type of DQA1 loci while in case of Jenel Conti GYPA loci was not amplified and rest 4 loci was found to be have similar type of fingernail scraping. In case of Justin Liverman except GC loci rest 4 were exactly matching the evidence. Based on these results it is clearly established that these two suspects have some role to play and further investigation should be focused on these two suspects. But the obtained results only indicate their role, which needs to be investigated in depth using other techniques to conclude the investigation. The given experiments have demonstrated use of DQA1-PM based technique for crime detection. It was also established that this method gives indicative results and has to be confirmed by other methodology techniques like AFLP, RFLP or minisatellite bases fingerprinting to conclude the investigation. Reference: 1) Cole S. Suspect Identities: A History of Fingerprinting and Criminal Identification. Cambridge, MA, Harvard University Press, 2001. 2) Saiki, R.K., Walsh, P.S., Levenson, C.H., and Erlich, H.A.. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc. Natl. Acad. Sci. U.S.A. 1989 86;6230-6234. 3) Reynolds, R., Sensabaugh, G., and Blake, E. Analysis of genetic markers in forensic DNA samples using the polymerase chain reaction. Anal. Chem 1991; 63:2-15. 4) Bieber FR, Miles HL (eds). Forensic DNA Evidence in the Courtroom. A Comprehensive Review of the Science and Practice. Boston, MCLE, 1999 Abstracts of referred papers. 2) Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes R K Saiki, P S Walsh, C H Levenson, and H A Erlich Abstract: The analysis of DNA for the presence of particular mutations or polymorphisms can be readily accomplished by differential hybridization with sequence-specific oligonucleotide probes. The in vitro DNA amplification technique, the polymerase chain reaction (PCR), has facilitated the use of these probes by greatly increasing the number of copies of target DNA in the sample prior to hybridization. In a conventional assay with immobilized PCR product and labeled oligonucleotide probes, each probe requires a separate hybridization. Here we describe a method by which one can simultaneously screen a sample for all known allelic variants at an amplified locus. In this format, the oligonucleotides are given homopolymer tails with terminal deoxyribonucleotidyltransferase, spotted onto a nylon membrane, and covalently bound by UV irradiation. Due to their long length, the tails are preferentially bound to the nylon, leaving the oligonucleotide probe free to hybridize. The target segment of the DNA sample to be tested is PCR-amplified with biotinylated primers and then hybridized to the membrane containing the immobilized oligonucleotides under stringent conditions. Hybridization is detected nonradioactively by binding of streptavidin-horseradish peroxidase to the biotinylated DNA, followed by a simple colorimetric reaction. This technique has been applied to HLA-DQA genotyping (six types) and to the detection of Mediterranean beta-thalassemia mutations (nine alleles). 3) Analysis of genetic markers in forensic DNA samples using the polymerase chain reaction. Reynolds R, Sensabaugh G, Blake E. Abstract; The ability to extract and type DNA from forensic evidentiary samples has revolutionized the field of forensic serology. Previously, genetic marker typing was limited to the analysis of blood group markers and soluble polymorphic protein markers. Because the number of suitable markers expressed in particular fluids and tissues is relatively small, and because mixtures of fluids cannot be separated for conventional genetic marker typing, a suspect frequently cannot be included or excluded as a fluid donor in a case. However, the development of methods to extract DNA from virtually all biological specimens has greatly expanded the potential for individual identification. Of particular importance was the ability to extract mixtures of sperm cells and epithelial cells found in. Read More