Mitochondrial DNA Analysis By Alyssa Introduction: Mitochondrial DNA analysis is essential to forensic laboratories as it allows scientists to create DNA

profiles from evidence not suitable for RFLP or STR analysis. Rather than extracting nuclear DNA, mtDNA analysis uses the DNA extracted from the mitochondrion, another cellular organelle. It is important to note that all maternal relatives have the same mtDNA. This is because when a sperm fertilizes an egg, the DNA-containing head of the sperm fuses with the egg. However, the tail and midsection are left outside. The mitochondria of the sperm are found in the tail and midsection, so it never reaches the egg. The egg destroys the part that does get through after fertilization, and because of this degradation of sperm mtDNA all the mitochondria in an embryo come from the egg. http://www.dna.gov/rawmedia_repository/c0d646c8_a499_415a_be58_3c61ad8c47cd Diagram depicts the mitochondrion and mtDNA. Diagram taken from the National Institute of Justice. History: Mitochondrial DNA was discovered in the 1960s by Margit M. K. Nass and Sylvan Nass by electron microscopy as DNase-sensitive thread inside mitochondria. Ellen Haslbrunner, Hans Tuppy and Gottfried Schatz did biochemical assays on highly purified mitochondrial fractions and are also credited with developing mtDNA analysis. The FBI Laboratory began conducting studies on the feasibility of mitochondrial DNA (mtDNA) analysis in the late 1980s. In 1992, laboratory research began on a protocol for using mtDNA sequencing in forensic casework. Examination on samples began in June 1996, after the sequencing technique was certified. By 2002, the FBI had processed more than 500 cases using mtDNA analysis and had established the National Missing Persons DNA Database. A second database of mitochondrial DNA was also created and can be accessed through the FBI's CODIS (Combined DNA Index System) software. In 2005, the FBI decided to expand its mitochondrial DNA work and planned to open four new facilities focusing directly on mitochondrial DNA analysis. TIMELINE Created by Alyssa Braver. Background: DNA is a double stranded molecule and its nucleotides always associate themselves with a complementary nucleotide. The four nucleotides are adenine (A), guanine (G), cytosine (C) and thymine (T). For example, if G is on one of the strands, G is across from it on the other strand. Similarly, if A is on one strand, T will be across from it on the other strand. Base pairs (bp) are the pair on the two strands, and the nucleotide sequence is the sequence of base pairs. The human mtDNA genome is approx. 16,569 base pairs long, and the genome is usually found in a ring-like conformation. It consists of two major parts: a coding region that accounts for the majority of the molecule, and the control region, which is responsible for regulating the production of products from the coding region. Within the control region, there are two regions that are highly variable within the human population. These two regions are termed Hypervariable Region I (HV1), approx. length of 342 base pairs, and Hypervariable Region II (HV2), approx. length of 268 base pairs. Forensic mtDNA examinations are performed using these two regions because of large amount of variability found among individuals. http://www.fbi.gov/about-us/lab/forensic-sciencecommunications/fsc/july1999/images/dnaf3.gif Diagram depicts the two regions of the control region. Diagram taken from the Federal Bureau of Investigation.

Approximately 610 base pairs of mtDNA are currently sequenced in forensic mtDNA analysis. Comparing sequences would be difficult and confusing if all sequences are listed. Thus, mtDNA sequence information is recorded by listing only the differences with respect to a reference DNA sequence. The Anderson sequence (1981) serves as this reference as it was the first complete sequence to be published. Each base pair in the sequence is given a number. Deviations are recorded by a number and a letter. The number demonstrates the position and the letter designates the different base. For example, a transition from T to C at Position 222 would be written as 222 C. Deletions and insertions are also denoted. Analysis Procedure: Step 1: Primary Visual Analysis The sample is examined under a microscope and compared to reference samples. If it does not share the same microscopic characteristics as the known sample, then mtDNA analysis is not performed. http://www.fbi.gov/about-us/lab/forensic-science-communications/fsc/july1999/images/dnaf4.gif Microscopic characteristics of hairs from known sources are compared to hairs from questioned sources. This is to determine if they can be associated with each other. Diagram courtesy of the Trace Evidence Unit, FBI Laboratory. Step 2: Sample Preparation Samples are cleaned prior to the mtDNA sequencing process. This is to remove contaminating materials adjoining or adhering to the sample. Cleaning is particularly significant because extraneous cells from handling can easily taint a sample. Usually the sample is immersed in detergent and an ultrasonic bath. Step 3: DNA Extraction The sample is ground to a powder and placed in an extraction solution to release the cellular material, including the mtDNA, from the cells. The extraction solution is a mixture of organic chemicals that separate DNA from other biological molecules. It is spun in a centrifuge, and the DNA remains soluble in the top water-based layer. The top layer is filtered and concentrated. Step 4: Amplification by the Polymerase Chain Reaction PCR is a procedure that makes many copies of DNA. In the first step of the process, the sample is heated to separate the two strands of DNA. A new strand is then made from each template with help from a special enzyme. The enzyme copies the existing DNA molecules, and the process is repeated multiple times until there are millions of copies. Step 5: Postamplification Purification and Quantification The DNA created by the PCR is purified and quantified. Thanks to filtration devices that remove the excess reagents, the DNA is purified. It is quantified by using capillary electrophoresis. This technique compares the DNA in the PCR product to a known standard. http://www.fbi.gov/about-us/lab/forensic-science-communications/fsc/july1999/images/dnaf5.gif The PCR is used to make many copies of specific regions of interest in DNA. Only the hypervariable regions are amplified from the extracted material, and with each cycle of the reaction, the amount of DNA present theoretically doubles. Diagram taken from the Federal Bureau of Investigation. Step 6: Sequencing

Sangers Method (1977) is used for mtDNA cycle sequencing. It is similar to the PCR, but with different chemicals. In addition to normal bases, a set of terminator bases is used to elongate the growing strand of DNA. (They lack a chemical group that allows the enzyme to place another base after them, and they carry a fluorescent dye that can be easily detected.) The normal bases compete with the terminator bases for incorporation into the DNA strand. This results in a collection of DNA products that differ in size by one base and have a fluorescently labeled base at the end. The products are then separated on the basis of length by gel electrophoresis. Finally, computer software reconstructs the mtDNA sequence as a fluorescence detector reads the labels at the end of each strand of DNA.

http://www.fbi.gov/about-us/lab/forensic-science-communications/fsc/july1999/images/dnaf7.gif The diagram depicts the cycle sequencing process. Diagram taken from the Federal Bureau of Investigation. Usefulness: MDNA analysis is beneficial to use on: Older remains and biological samples such as hair, bones, and teeth that lack nucleated cells Samples like shed hair which do not contain a root and will therefore not work using STR analysis Samples that have been exposed to environmental extremes like heat, humidity, etc Improperly stored samples MDNA analysis is helpful when it comes to: Identifying missing people When there are unidentified remains Determining lineage Notable Cases Solved: In Us vs. MacDonald, Mr. MacDonald was charged with murdering his family. In 1979, he was found guilty and sentenced to consecutive life terms, despite the fact that he never wavered from his claim of innocence. In 1997, the courts finally acquiesced to his requests to test crime-scene evidence, which had now been buried away for some three decades. The results of the mitochondrial DNA testing broke open the case. Human hairs found under his wife's body, under the fingernails of one of his daughters, and in her bedding where she was killed did not match that of Mr. MacDonald. MtDNA proved his innocence. Another case that has been solved thanks to mtDNA is that of the unknown child. A little boy who died on the Titanic has finally been identified. According to Ryan Parr, Vice-President of Research and Development of Genesis Genomics in Ontario, the remains of the young boy are most likely those of an English child, Sidney Leslie Goodwin. The child was previously believed to be Eino Viljami Panula, a 13-month-old Finnish infant who drowned with his parents in the disaster. However, after researchers carried out more extensive mtDNA analysis, sequencing both the HVS1 and the HVS2 region, it was positively confirmed that the unknown remains are Goodwin's. An older case solved by mtDNA analysis was the murder of a 4-year-old little girl. A mother left her daughter at a friends house, and returned to find her lying on the floor dead and naked. The girl was taken to the hospital where a medical examiner found several hairs clinging to her body and evidence of sexual abuse. The FBI Laboratory performed mtDNA testing on the hairs found at the crime scene and on the victim's body. The results were compared to the mtDNA profile of a family friend, and all sequences were the same. The friend was sentenced to life without parole for felony murder and two terms of 25 years for rape.

Conclusion: Since 1996, the number of individuals performing mitochondrial DNA analysis at the FBI Laboratory has tripled. Hundreds of mtDNA cases have been completed, and many cases have been solved. More people are learning of the value of mtDNA sequencing for obtaining information from small or degraded samples and mtDNA sequencing has also become a complementary technique for use with other human identification procedures. It will continue to be a powerful tool for law enforcement officials in the years to come. References: Alice, Isenberg R. "Forensic Mitochondrial DNA Analysis: A Different Crime-solving Tool." The FBI Law Enforcement Bulletin, July-Aug. 2002. Web. Cormier, Phillip G., Andrew Good, Barry C. Scheck, and Harvery A. Silverglate. "Federal Habeas Corpus & Actual Innocence." The National Law Journal (2011). Print. "DNA Forensics." Oak Ridge National Laboratory. Web. 15 May 2011. <http://www.ornl.gov/sci/techresources/Human_Genome/elsi/forensics.shtml>. "DNA.gov: Mitochondrial Analysis." The DNA Initiative. Web. 15 May 2011. <http://www.dna.gov/basics/analysis/mitochondrial>. Isenberg, Alice R., and Jodi M. Moore. "Mitochondrial DNA Analysis at the FBI Laboratory." Forensic Science Communications 1.2 (1999): 1-10. Print. Lorenzi, Rossella. "Titanic's 'Unknown Child' Identified." Discovery News: Earth, Space, Tech, Animals, Dinosaurs, History. Mar.-Apr. 2011. Web. 15 May 2011. <http://news.discovery.com/history/titanic-unknownchild-identified-110426.html>. \