ࡱ> NPM7 bjbjUU "7|7|l         .000000$e  T     T  i   . .P..  v..0.  .Dr. Tonkyn The Real CSI 04/02/2007 What is a Forensic Scientist? -Holds a BA of Science usually in Chemistry, Biology, or Forensic Science -There are city, county, state, federal and private labs where they can work at. -Is a criminalist who answers the who, what, where and when. -They identify and collect evidence at crime scenes. -Perform scientific analysis- anything that can be measured and compared to a standard. -Considered expert witnesses in court and are allowed to provide opinion and hearsay evidence. Forensic vs. Clinical Analysis -Forensics deals a lot with the unknown. Samples can include anything, are usually from an unknown history, uncontrollable environmental effects, can be from multiple individuals, usually sample size is very limited and results can and most likely will be present in court. -Clinical analysis, unlike forensics, deals with known samples, known history, usually not degraded sample due to a controlled environment, from a single individual, enough of sample for multiple analysis. -The similarities was that they can both get speciems from other species, can be contaminated and can result in life or death situation. DNA Basics- Deoxyribonucleic Acid -No two people have the same DNA, exceptions are Identical twins. It is the 0.1% that is variable between individuals. -What distinguishes DNA from one person to another is the order of the bases -Nuclear DNA is unique, has 2 copies per cell, inherited from both parents, non coding region is of interest, found in nucleus of a cell. -Mitochondrial DNA is not unique, has more than 1000 copies per cell and is maternally inherited. HV1 and HV2 are the regions of interest when testing. Found outside of cell in the mitochondrion. Advantages- high amount available, less prone to degradation, highly variable. mtDNA analysis is similar to nucluear DNA analysis with PCR Reaction. -Compimentarity makes bases pair in specific way- At with T and G with C. It is the basis of replication, hybridization and PCR Reaction. -Limits to DNA testing-can identify donor, but it doesnt mean that its the perpetrator. DNA does not say when the sample was deposited, only that it is at the scene. It doesnt say how it got there or who else was present, for example, in a rape case it could have been a consentual partner that deposited the sample, but not the killer. PCR Reaction -Done within 32 cycles, in a Thermal Cycler. First cycle denature the original DNA strand at 95 degrees, Taq is also activated, the primers attach at 59 degrees during the annealing process. The temperature is then raised to 72 degrees where extension then takes place. This happens about another 30 times. Then the final extension at 60 degrees takes place, this is for the attaching of the extra base pair. -Multiplex PCR-Copies multiple markers in one PCR Reaction. Uses different fluorescent dyes, with distinguish the different STR alleles with overlapping sizes. After thermal cycles are finished, the extracted DNA samples and controls are put there Capillary Electrophoresis and computer software then reads the profile. DOJ DNA Lab Programs Programs include: Criminal Casework, Method Development and training, post conviction testing, missing persons, mass disasters, human rights violations, convicted felon DNA databank, and cold hit program. Cold Hit cases -Deals with cases that go unsolved. DNA from crime scenes are submitted to the databank and sometimes there might be a hit to a suspect, then they are compared and can possible lead to a match, that then leads to an investigation and charges are usually filed. Missing Persons Cases -Legally accepted methods of Identification- Visual, Dental, DNA, Medical/Anthropological, and fingerprints. -When bones are found, they are pulverized with liqued nitrogen and then put through quantification, quantitation, put through DNA Extraction, amplification and capillary electrophoresis and the DNA profile is created. Profile Plus or COfiler Amplification Set-Up For this lab all teams set up the PCR reaction. We set up the work areas. We took the substrate control, K562, and negative control from the last DNA extraction lab. We also hand thawed all materials for the master mix. We labeled 6 eppendorf tubes, 1 was the negative control from last lab, 2 was the substrate control from last lab, 3 was K562 DNA that was extracted in the last lab, 4 new K562 sample given by Dr. Lee, 5 was the 99479 and 6 was the negative control dH20. We then proceeded to make a master mix enough for 7 sample tubes. This mix included 7ml of AmpliTAQ gold DNA polymerase, 77ml of Profiler Plus or COFiler Primer Set, 147ml of Reaction Mix, mix was then put vortexed and centrifuged. Then 30ml of the master mix was placed into the 6 ependorf tubes labeled in the beginning. Then 50 ml of mineral oil was added to the tubes ( 2 drops). We then added the proper amounts of the samples from the previous lab and those supplemented by Dr. Lee to the appropriated tubes, deposited below the oil. Then all sample were centrifuged. After we gave Dr. Lee our sample tubes and then Dr. Lee placed them in the Thermal Cycler. It was set to the appropriate cycle temperatures and times. We will later run the Capillary Electrophoresis on the sample.  #A%? M ,A#9CJ 56CJ5CJ#AN%8* w  ^ ? L +,A#9/ =!*"v#$% i4@4 NormalCJaJmH sH tH <A@< Default Paragraph Font" z#AN%8*w^? 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