Innovative advances in deoxyribonucleic acid (DNA) technologies over the past few decades have brought about remarkable changes in the ancient DNA research which is the game changer of molecular phylogenetic analysis. The field of ancient DNA research which has a history of 28 years, involves the retrieval, amplification, sequencing and analysis of DNA sequences from archaeological and paleontological remains to unravel the evolutionary relationship between extant and extinct species (Millar, Huynen, Subramanian, Mohandesan & Lambert, 2008). Ancient DNA research has undergone major phases of development through improvements in DNA sequencing techniques, provided new insights and holds a ...view middle of the document...
PCR enables routine amplification of extremely low concentration of short or single-copy of mtDNA sequences (Pääbo, Higuchi, & Wilson, 1989). In addition, PCR avoids cloning artifacts caused by misrepair of damaged DNA during molecular cloning. This was evident from the sequencing results of PCR products of quagga that showed two nucleotide positions of the sequence to be different from that of a molecularly cloned quagga, but similar to the orthologous zebra sequence (Pääbo & Wilson, 1988). Furthermore, the sequence of mammoth bone that was the oldest dated vertebrate remains could be produced from the PCR amplified DNA extract (Hagelberg et al., 1994). This shows great potential of ancient DNA sequencing in phylogenetic studies due to the presence of PCR.
Unfortunately, the high sensitivity of PCR in detecting minute amounts of DNA not only allows efficient DNA amplification, it also increases the risk of DNA contamination. Contaminant DNA can be easily amplified which results in false positive results if the template ancient DNA amount is insufficient to allow the target sequence to compete with the contaminant DNA for amplification (Yang & Watt, 2005). Contamination is a concern especially when dealing with ancient DNA because ancient DNA is highly susceptible to degradation; and thus, resulting in damage-free modern DNA to easily outcompete the authentic ancient DNA. The drawbacks of PCR cause the ancient DNA sequencing results to be less accurate and convincing.
With the advent of first generation sequencing that uses automated Sanger sequencing strategy, computer algorithms can be incorporated to allow accuracy in determining DNA sequences (Slatko, 2011). An example of the Sanger sequencing system is Applied Biosystems 3730XL genome analyzer (Kircher & Kelso, 2010). It comprises of capillary electrophoresis which allows more accurate sequencing results at a faster speed. However, Sanger sequencing faces the issue of low throughput and extremely high cost of performing large-scale sequencing. Therefore, Sanger sequencing is not well-suited for whole genome sequencing in ancient DNA studies. This can be observed from the Human Genome Project in which the first human genome that was sequenced via the Sanger method, took a long period of 13 years and incurred a high cost of $2.7 billions (Voelkerding, Dames & Durtschi, 2009). Hence, Sanger sequencing is eventually superseded by the evolution of next generation sequencing (NGS) that overcomes the limitations associated with Sanger sequencing.
The emergence of NGS transformed and launched a new era in the field of ancient DNA studies. It opens up new possibilities of employing ancient DNA to produce a more reliable phylogenetic analysis. NGS has the capability of performing simultaneous sequencing reactions known as massive parallel sequencing. It generates high throughput of DNA sequencing read from 100 megabases to gigabases in a single instrument run (Voelkerding et al., 2009). Two...
