Mitochondrial DNA Analysis

by Alyssa Braver

Introduction:
Mitochondrial DNA (mtDNA) analysis is essential to forensic laboratories. This technique allows scientists to use evidence to create DNA profiles in cases where other types of analysis such as RFLP or STR are unsuccessful. Rather than extracting nuclear DNA, mtDNA analysis uses the DNA taken from the mitochondria. All maternal relatives have the same mtDNA. This is because when a sperm fertilizes an egg, the midsection and tail remain outside while the DNA-containing head fuses with the egg. The mitochondria of the sperm are found in the tail and midsection, so these mitochondria fail to reach the egg. The egg destroys the part that gets through after fertilization, and because of this degradation of sperm mtDNA, all the mitochondria in an embryo is of maternal origin.
Diagram depicts the mitochondrion and mtDNA. Diagram taken from the National Institute of Justice.
Diagram depicts the mitochondrion and mtDNA. Diagram taken from the National Institute of Justice.


History:
In the 1960s using electron microscopy, Margit M. K. Nass and Sylvan Nass discovered a DNA sensitive thread inside the mitochondria, mtDNA. Ellen Haslbrunner, Hans Tuppy and Gottfried Schatz developed mtDNA analysis by performing biochemical assays on fractions of highly purified mitochondria. In the late 1980s, the FBI Laboratory initiated feasibility studies on mtDNA analysis. In 1992, a protocol was begun at the lab in forensic casework using mtDNA sequencing. After the sequencing technique was certified, analysis on samples was started in June of 1996.

By 2002, the FBI established the National Missing Persons DNA Database and had utilized mtDNA analysis in over 500 case. 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 technique proved so successful, that the FBI decided to to open four new laboratories solely for the purpose of mitochondrial DNA analysis.
timeline.jpg
Timeline showing the history of mtDNA. Created by Alyssa Braver.


Background:
DNA is a double stranded molecule in which each nucleotide always associate 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, its complementary nucleotide, C ,is across from it on the other strand. Similarly, if A is on one strand, T will be across from it on the opposite strand. Base pairs (bp) are the pair of nucleotides on the two strands, and there sequence forms the nucleotide sequence.

The human mtDNA genome is approx. 16,569 base pairs long, and the genome is usually found in a circular formation. 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 controlling the number and types of products produced in the coding region. Within the control region, there are two highly variable regions in the human population. These two regions are termed Hypervariable Region I (HV1), which is approx. 342 base pairs long, and Hypervariable Region II (HV2), which is approx. 268 base pairs long. Since these areas contain a lot of variability from one individual to the next, Forensic mtDNA examinations are performed using these two regions.
Diagram depicts the two regions of the control region. Diagram taken from the Federal Bureau of Investigation.
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 confusing and difficult if all of the sequences were listed. Therefore, mtDNA sequence information is compared to a reference DNA sequence and only the differences are listed. The Anderson sequence (1981)was the first sequence to be completed and published, and it serves as this reference sequence. Each base pair in the sequence is given a number. Variations 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 is 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.
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.
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
Prior to the mtDNA sequencing process the specimen is cleaned of any adjoining or adhering material that might contaminate the sample. Cleaning is particularly significant because handling might introduce extraneous cells that could easily taint a sample. To become clean, the sample is usually immersed in detergent and given an ultrasonic bath.
Step 3: DNA Extraction
To release cellular material, including the mtDNA, the specimen is first ground to a powder and then it is placed in an extraction solution. This solution is a mixture of organic chemicals that separates the DNA from the cells other biological molecules. After a spun in a centrifuge,the DNA remains soluble in the top water-based layer. This layer is then filtered.
Step 4: Amplification by the Polymerase Chain Reaction
PCR is a procedure that allows the lab to make a lot copies of DNA. In the first step of the process, heat is used 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, excess reagents are removed, purifying the DNA. It is the quantified by using a technique called capillary electrophoresis. This compares the DNA in the PCR product to a known standard.
The PCR is used to make loads of copies of specific regions of  DNA. With each cycle, the amount of DNA theoretically doubles. Diagram taken from the Federal Bureau of Investigation.
The PCR is used to make loads of copies of specific regions of DNA. With each cycle, the amount of DNA theoretically doubles. Diagram taken from the Federal Bureau of Investigation.

Step 6: Sequencing
Sanger’s Method (1977) is used for mtDNA sequencing. It is similar to the PCR, but with different chemical reagents. In addition to normal bases, specialized terminator bases enable the newly created strand of DNA to elongate. These terminator bases lack a chemical group which prevents the enzyme from placing another base after them. They also contain a fluorescent dye that is easily detected. The terminator bases compete with the standard bases for incorporation into the DNA strand. This results in 2 different DNA products that differ in size by a single base. Those that have incorporated the terminator base are also fluorescently labeled at the end. The different DNAs are then separated on the basis of length by gel electrophoresis. Finally, computer software reconstructs the mtDNA sequence while a fluorescence detector reads the labels at the end of each strand of DNA.
The diagram depicts the cycle sequencing process. Diagram taken from the Federal Bureau of Investigation.
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
  • Cases where STR analysis will not work such as samples of hair without a root.
  • Samples that have been exposed to environmental extremes like high humidity, heat, 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. 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 were not of Panula. 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.”
  • An older case solved by mtDNA analysis was the murder of a 4-year-old little girl. A mother left her daughter at a friend’s 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 adhering to her body. There was also proof of sexual abuse. The FBI Laboratory performed mtDNA testing on the hairs, both those found on the girl's body and those at the crime scene. The results were then 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:
The number of individuals performing mtDNA analysis at the FBI Laboratory has tripled since 1996. 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 an important technique for identifying remains. In the years ahead, mtDNA analysis will continue to be an essentiall tool for law enforcement officials.

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-unknown-child-identified-110426.html>.