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Elias Saber-Khiabani / Instructor M. Villani / Forensic Science / PD. 2
all organisms are either multi-cellular or unicellular a single eukaryotic (protist, fungus, plant, animal) cell is comprised of multiple membrane-bound organelles the nucleus is an organelle in eukaryotes which houses the primary genetic material (DNA) mitochondria are organelles which are responsible for cellular respiration (ATP production) mitochondria have a double membrane, cristae (folds), a matrix, and their own DNA mitochondrial DNA (mtDNA) codes for proteins and enzymes used by the mitochondria nuclear DNA also codes for enzymes used in the mitochondria
Nuclear DNA vs. Mitochondrial DNA
found in nucleus of the cell 2 sets of 23 chromosomes used with evidence such as saliva, semen, blood maternal and paternal can "discriminate between individuals of the same maternal lineage“ double helix bounded by a nuclear envelope DNA packed into
found in mitochondria of the cell each mitochondria may have several copies of the single mtDNA molecule used with evidence such as hair, bones, and teeth maternal only cannot "discriminate between individuals of the same maternal lineage“ circular free of a nuclear envelope DNA is not packed into chromatin
Nuclear DNA vs. Mitochondrial DNA
the sperm only contributes its nucleus (23 chromosomes) mitochondria of the sperm cell are located at the mitochondrial sheath which is destroyed upon fertilization the only available mitochondria (mtDNA) is that of the mother's. this is why mtDNA is of maternal origin .Maternal Inheritance of mtDNA during fertilization.
Maternal Inheritance of mtDNA .
Key Facts mtDNA of siblings will match each others and that of their mother mtDNA is found as a single. circular chromosome in the cell mitochondrion may contain multiple copies of mtDNA a human cell may contain hundreds or thousands of mitochondria mtDNA may be useful when nuclear DNA is limited because of its abundance .
Rickettsiales" this has mostly likely occurred as a result of phagocytosis.The Endosymbiotic Theory mitochondria "originated as separate prokaryotic organisms which were taken inside the cell as endosymbionts (any organism that lives within the body or cells of another organism)" it is believed that "mitochondria developed from the order of proteobacteria. the process of a cell engulfing it's food and creating a .
3. Evidence for Endosymbiosis mtDNA is circular. it resembles a nucleoid 2. it resembles a nucleoid (bacterial DNA in the form of a single circular chromosome) mitochondria and bacteria both reproduce via binary fission mitochondria are semi-autonomous (the nucleus contains genes which originated from mtDNA) mitochondrial ribosomes resemble bacterial ribosomes mitochondria have a double membrane. 5. mtDNA is circular. possibly created through phagocytosis . 4.1.
13 proteins. 22 tRNAs.569 base pairs (bp) in length encodes 37 genes. 16. and 2 rRNAs two general regions: The Mitochondrial Genome coding region: "responsible for the production of various biological molecules involved in" cellular respiration control region: "responsible for the regulation of the mtDNA molecule" two regions that are highly polymorphic (variable) in the human population: Hypervariable Region I (HV1): 342 bp Hypervariable Region II (HV2): 268 bp HV1 and HV2 are used in mtDNA examinations .
Lab research began for using mtDNA in criminalistics June 1996 . Paul Ware .FBI Lab conducted studies to test the usefulness of mtDNA analysis for human identity testing 1992 .Forensics and mtDNA History: Late 1980s .Examinations on mtDNA evidentiary samples began in the case of State of Tennessee v.
Uses for mtDNA in Forensics mtDNA will be used when "biological evidence may be degraded [i. charred remains] or in small quantity" Cases in which evidence consists only of: hairs bones teeth Missing Persons Cases (use of skeletal remains) Establishing Individuals as suspects .e.
T. or the Oxford sequence to denote deviations from this sequence. “a transition from A to G at Position 263 would be recorded as 263 G” mtDNA sequencing is more rigorous. i. G. the Cambridge reference sequence.e.More on Forensics and mtDNA 610 bp of mtDNA are sequenced for forensic analysis (from the control region) this 610 bp sequence can be referred to as the Anderson Sequence. time consuming and costly in comparison to nuclear DNA profiling . or C) designating the different base pair. the assigned number of the different bp is recorded with a letter (A.
4. 2. Primary Visual Analysis Sample Preparation DNA extraction Polymerase Chain Reaction (PCR) Amplification Postamplification Quantification of the DNA . 5. 3.Forensic mtDNA Analysis Steps of mtDNA Analysis Process to obtain a mtDNA Sequence from a Sample: 1.
Primary Visual Analysis Hair: the hair sample is microscopically compared with a sample “population of reference hairs” with a comparison microscope if the questionable hair “exhibits similar microscopic characteristics as hair from a known source” then mtDNA analysis will be performed to see if the hairs are .
then mtDNA analysis “can be used in conjunction with medical. odontological examinations to assist in the identification process” .Primary Visual Analysis Bones and Teeth: forensic anthropologists or odontologists inspect the tissue to determine if it’s of human origin if it is of human origin. anthropological.
this ensures that the mtDNA obtained is from the sample itself and not from “exogenous human DNA” Hair Samples: undergo “detergent treatment in an ultrasonic water bath” are placed in an “extraction solution” are ground “using a small mortar and pestle” this results in a homogenate of “cellular material and the released DNA .Sample Preparation evidentiary samples are cleansed to remove any contaminants.
Sample Preparation Bone and Tooth Samples: are sanded to remove material adhering to the surface have a tissue sample removed and ground into a fine powder (in teeth samples. the tissue may be obtained from the dentin and the pulp) are placed in an .
DNA Extraction cellular homogenate is “exposed to a mixture of organic chemicals that separate the DNA from other biological molecules. such as proteins” DNA settles in the top water-based layer spun in mixture is top layer is filtered and concentrated a centrifuge DNA sample is now purified .
” DNA is heated at 94° C to separate the two strands of the DNA double helix in the sample new DNA strands are then made from the template (initially separated strands) of DNA by using DNA polymerase.Polymerase Chain Reaction (PCR) Amplification PCR is a “procedure that makes many copies of a small amount of DNA. primers. and free nucleotides the process is repeated multiple times. doubling .
Polymerase Chain Reaction (PCR) Amplification .
” which compares the amount of DNA in the PCR product to “a known DNA standard to determine the concentration of the DNA in the PCR-amplified sample.” Postamplification Purification and Quantification Quantification is performed “using capillary electrophoresis (CE).” . Purification is performed “using filtration devices that remove the excess reagents used in the PCR from the sample.
resulting in a .Automated DNA Sequencing Dideoxy Terminator (Sanger’s) Method: similar to the PCR amplification process terminator bases are added in addition to free nucleotides terminator bases lack a chemical group which allows DNA polymerase to place another base after it terminator bases are tagged with a fluorescent dye “normal bases compete with the [terminator bases] for incorporation into the growing DNA strand.
Gel Electrophoresis: Automated DNA Sequencing DNA products are separated by length of bp pore size of the gel influence how far the DNA fragments will travel when placed in an electric field smaller fragments will travel faster and appear further from the wells in the gel larger fragments will travel slower and appear closer to the wells in the gel fluorescent detector “records the emitted wavelength of the fluorescent dyes on each base as the fragments travel past the detection area of the instrument” a chromatogram is generated. showing the colors of the labeled fragments .
Data Analysis mtDNA sequences are generated by a computer and edited by a DNA examiner to obtain the final sequence difference(s) is/are recorded by comparing the finalized sequence to the Anderson reference sequence if sequence concordance (“the presence of the same base or a common base at every position analyzed”) is observed. then both mtDNA samples could be considered as originating from the same source .
Data Analysis Because the human body has trillions of cells each with thousands of copies of mtDNA. it is impossible for there to exist complete homoplasmy (the same mtDNA sequence) in all these cells Heteroplasmy “(the occurrence of more than one mtDNA type at a particular position or region in a DNA sequence) is expected to be present at some level” .
the Armed Forces DNA Identification Laboratory. and other laboratories collaborated to compile a mtDNA population database (called SWGDAM (Scientific Working Group on DNA Analysis Methods) .mtDNA Population Database The FBI Laboratory.
and Hispanics contains 4.142 mtDNA sequences from unrelated individuals in a forensic index contains 6.mtDNA Population Database SWGDAM Database (as of August 2002): includes HV1 and HV2 sequences from Caucasians. Asians. Native Americans. etc… “too small to provide estimates of the frequency of occurrence of most mtDNA . Africans.686 mtDNA sequences from paternity-testing labs. blood banks.
[and] UV irradiation” instruments are routinely .Quality Assurance Guidelines of the American Society of Crime Laboratory Directors-Laboratory Accreditation Board and the FBI Standards for Forensic DNA Testing Laboratories: precautions to minimize contamination (use of gloves. laminar flow. and aerosol-resistant pipette tips) only one item of evidence from a case is opened at a time preamplification areas are separate from postamplification areas workspaces and instruments are “subjected to isopropanol washings. lab coats. 10% bleach washings.
FBI Laboratory mtDNA Case Acceptance Policy Hairs: analysis is done on human head hairs and pubic hairs these hairs must be “associated through comparison microscopy and confirmed according to FBI Laboratory Hairs and Fibers Unit (HFU) protocols other human hairs will be “subjected to mtDNA analysis as deemed appropriate by the FBI Laboratory examiner” .
FBI Laboratory mtDNA Case Acceptance Policy Bones and Teeth: individuals providing bones or teeth for mtDNA analysis must “telephone the FBI Laboratory prior” to their submitting a letter must be sent along with the bone or teeth evidence referencing to the telephone conversation in which “approval was granted by FBI personnel for the acceptance of a case consisting of bone or teeth evidence for requested mtDNA analysis” .
FBI Laboratory mtDNA Case Acceptance Policy Bodily Fluids: bodily fluids “will be subjected to mtDNA analysis at the discretion of a qualified FBI Laboratory examiner” any “questions regarding mtDNA analysis should be directed at the FBI Laboratory Hairs and Fibers Unit (telephone: 202-324-4344)” .
Pennsylvania Bode Technology Group in Springfield. 5. Virginia Armed Forces DNA Identification Laboratory BioSynthesis. 6. FBI Laboratory Laboratory Corporation of America in Research Triangle Park. 4. 7. Louisiana . 3. Inc. there are 7 American laboratories conducting mtDNA examinations: 1. 2.mtDNA Examination Labs Currently. Texas Reliagene in New Orleans. North Carolina Mitotyping Technologies in State College. in Lewisville.
sampling. and quality of Forensic mtDNA databases of a “National Missing Enhanced Development .mtDNA and the Future Future Developments for mtDNA analysis: Improved detection methods for Heteroplasmy size.
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