Mitochondrial DNA Sequencing and its Application in Forensic Sciences

DNA (deoxyribonucleic acid) has significantly impacted the scientific world since it was fully decoded by the Human Genome Project in 2003. The Human Genome Project was a 13 year project to fully sequence the human genome, and find the similarities and differences throughout the human race. The value of the project [ ] appears beyond doubt. Genome research is revolutionizing biology and biotechnology, and providing a vital thrust to the increasingly broad scope of the biological sciences (US Department of Energy, 1996). Through full decoding of the human genome, scientists were able to discover that less than 1 percent of human DNA is unique to the individual (Pollard, 2009). Despite the microscopic margin, this difference creates millions of variations specific to an individual. The ability to determine, or disregard a suspect due to a slight difference in DNA has become a significant help to science, but especially in forensic applications. Originally, sequencing DNA was extremely difficult.

First generation sequencing was mostly done using the Chemical Cleavage Sequencing method, introduced by Maxim and Gilbert or the Chain Terminating or Sanger Sequencing method introduced by Sanger and Associates. Both approaches rely on the separation of the mixture of DNA fragments of various sizes on polyacrylamide (PAA) slab gels (Guzvic, 2013). This would separate the DNA into a single strand to make multiplication of the bases easier to complete, before introducing an alkali treatment that breaks the strand. The breaking of the strand was recorded on the gels by how light or dark the slab became. Unfortunately these two methods created excessive redundant information that could easily cause confusion on which strands had broken, and both needed copious amounts of DNA to begin synthesizing. Towards the end of first generation sequencing, Polymerase I was used to complete the human genome. The easier to synthesize Polymerase I reaction was more effective, yet it had shortcomings. Short mono - nucleotide repeats were hard to interpret, as the bands were often not separated and correct reading of the sequence mostly depended on the experience of the operator (Guzvic, 2013). In 2005, 454 sequencing ushered in the second generation of DNA sequencing.

Developed by 454 Life Sciences, 454 sequencing relies on pyrosequencing. Pyrosequencing [ ] is based on the detection of light signal upon incorporation of nucleotide by polymerase. [the] amount of light is proportional to the number of ATP molecules, which is proportional to the number of released pyrophosphates, or, in other words, incorporated nucleotides (Guzvic, 2013). First, the DNA is nebulized and double strand adjusters are added to the ends of the DNA sequence. The adjusters bind any fragments before the mixture is added to a Polymerase Chain Reaction (PCR) solution. This mixture is then put into a Thermal Cycler which clones the DNA sequence. These sequence fragments are then put onto a PicoTiter Plate, which then run through an instrument's chamber to produce specific light patterns that show the DNA sequence. Unfortunately, this method is costly and has a high margin of error in repeat sets. Though it is a more costly method, 454 sequencing can do the work of first generation synthesis in a fraction of the time. Third generation sequencing is just beginning to come forward. It's main purpose is to reduce the time and cost of DNA sequencing. Scientists believe that if the amplification and preparation of DNA could be cut from the process, the subsequent sequencing would be faster, more effective, and less costly.

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