Reading Ancient DNA
Reading ancient DNA is very hard and expensive to read. Here's why:
Chromosomal shredding
Every cell contains enzymes that shred DNA molecules to reuse its components somewhere else in the cell as a part of a recycling process. However, these enzymes continue to work after a cell dies, breaking down a molecule several million base pairs long into scraps just a few dozen letters long in a time period of a few thousand years.
Water and oxygen can also react with DNA's backbone and degrade it further. That's why the best samples of ancient DNA ever found were taken from cold and dry environments like tundras or caves. These kinds of conditions also slow down the activity of the DNA-shredding enzymes.
Contamination
In ancient DNA samples, 99% of DNA comes from invading fungi or bacteria. Scientists remove these using "RNA bait". They develop strips of human RNA with a chemical Velcro on it. These are then released to attach to only the pieces of human DNA. Then beads of a metal are poured in, locking the DNA and RNA pieces together. They are then held down by a magnet and everything that isn't human DNA gets washed away.
This technique gives very good results, but is also very expensive, particularly the RNA developing part, which can cost $300 000.
Corruption
also called deamination. This process happens when a water or oxygen molecule snips off a NH2 (amine) group from a cytosine base. This base then chemically changes into a uracil base, which, however, doesn't normally occur in DNA. The enzymes that read DNA so it can be copied read this modified base as thymine, which results in a substantial mutation the final DNA molecule. It contains too few Cs and too many Ts, and the newly synthesised complementary DNA strand differs greatly from the unmutated mother DNA molecule.
Scientists haven't solved this problem yet. They know how to remove DNA with uracil in it, but often they can't afford to do so, because many samples contain very little usable DNA.
To demonstrate one of the newer methods used now, here is one of them. It was used to sequence a major milestone in paleogenetics, Neanderthal DNA. This code was then compared to codes of other ancient hominins to outline the human evolution family tree.
1. Extracting
DNA is extracted from the sample of tissue (often bones) using the silica extraction method. Fine silica powder is used to bind the DNA molecule so it can be separated from the unwanted substances in the tissue sample. What is left is a mixture of human DNA contaminated by microbial or modern human DNA. The scientists wanted to remove the contamination by increasing the so-called signal to noise ratio, or, sequencing a lot of ancient human DNA versus little other DNA. This is achieved by releasing restriction enzymes which cut up bacterial DNA which is then more easily degraded. Human contamination is prevented by strict sterile conditions in the lab, including bleach, ultraviolet light, hair nets, and special lab coats.
2. Sequencing
DNA sequencing means taking the DNA molecules from the sample and running chemical reactions that identify the types of nucleotide and their order. Originally, this was done by pyrosequencing, which read a few long strands of DNA at the same time. Scientists started used a different method instead, because Neanderthal DNA is usually found in short strands anyway. The new method allows sequencing millions of shorter strands at the same time in a sequencing machine much faster.
3. Aligning
Alignment is essentially joining the short pieces of DNA strands together. The fact that there is an enormous number of tiny pieces that need to be joined into one million-nucleotide-long strand in the right order makes this a very challenging task. That's where bioinformatics step in. Bioinformaticians developed clever algorhithms from human and chimp genomes that serve as scaffolds when aligning ancient DNA. Bioinformatics has also found the answer to solving the above described corruption of ancient DNA.
Discoveries
Many discoveries and a lot of research in this field were made by a Swedish scientist, Dr Svante Pääbo, who is considered to be the founder of paleogenetics. It took him and his team decades to perfect these methods. The oldest samples human DNA are a 430 000-year-old fragment of Neanderthal genetic code and the oldest sample of DNA ever found is from a horse unearthed from Yukon, Canada, between 560 000 and 780 000 years old. In 2006, Pääbo decided to sequence the whole Neanderthal genome from from a toe bone found in Denisova Cave in the Altai Mountains in 2010. In 2014, a complete, high quality, 52x coverage Neanderthal genome was published.
But even this field of research has its limits. Irrespectively of how much scientists perfect the methods of extracting, sequencing and aligning ancient DNA, they can't use them if there is none left. Calculations have shown that DNA kept in optimal conditions could survive around 1 million years. Beyond that boundary, it is far too decomposed and contaminated.
For a brief atlas of ancient DNA samples collected up to this point, click here.
References:
1. Kean, S 2014//2015, 'Ancient DNA', Distillations, Fall 2014/Winter 2015, accessed 2 January 2019, available at <https://www.sciencehistory.org/distillations/magazine/ancient-dna>
2. Wolford, B 2016, 'Interpreting ancient DNA: Not so easy a caveman could do it', Misciwriters, 30 August, accessed 2 January 2019, available at <https://misciwriters.com/2016/08/30/interpreting-ancient-dna-not-so-easy-a-caveman-could-do-it/>
3. Alex, B 2017, 'Everything Worth Knowing About ... Ancient DNA', Discover., 19 June, accessed 2 January 2019, available at <http://discovermagazine.com/2017/jul-aug/ancient-dna>
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