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In October of last year, an interesting reanalysis of the current progress in Neandertal genome sequencing project appeared in the journal PLoS Genetics. And with a title like this, “Inconsistencies in Neanderthal Genomic DNA Sequences,” it is no secret what the authors found in their reanalysis.

The authors of this reanalysis, Wall and Kim, scrutinized both the Noonan et al. and Green et al. approaches to sequence the Neandertal genome. The Noonan team use the tried and true method of cloning fragments into vectors and batch culturing to begin the sequencing whereas the Green team deployed the hot new technology, large scale parallel sequencing approach provided by 454 Life Sciences.

In a nutshell, Wall and Kim found big discrepancies in the the Neandertal contribution to modern European ancestry. The biggest discrepancy was when they attempted to estimate the population split times between modern humans European and Neandertals. The Green data revealed population split time of 35,000 years ago while the Noonan data was almost 10 times as old, a calculation of a 325,000 year old population split. The authors write,

“Given the large discrepancies in the parameter estimates from the two studies, it is clear that the conclusions reached by at least one of the studies are incorrect.”

A new paper from today’s issue of the American Journal of Physical Anthropology suggests a way to overcome sequencing error rates using ancient DNA, such as Neanderthal DNA, use ion-exchange columns. The paper is titled, “Technical Note: Improved Ancient DNA Purification for PCR Using Ion-Exchange Columns.”I’m currently using ion-exchange columns to purify proteins, and they basically work by passing a solution down the column where ions and small particles are trapped within the beads. To elute the trapped particles, solutions of salts and buffers are passed over the column and then trapped particles wash out in fractions, which end up purifying the sample.

In this current application proposed, ion exchange columns are used to remove PCR inhibitors such as humic acid, fulvic acid, biologically degraded products, and collagen that are all often found in soil and sediment… which have housed ancient DNA samples for thousands of years. When one usually begins to isolate DNA from ancient bones, “a brownish discoloration in many of the DNA extracts, which is a marker of insufficient PCR inhibitor removal,” is noticed. These inhibitors prevent the amplification of ancient DNA by competing or distracting the polymerase enzyme from doing its thing.

To validate the efficiency, or success, of ion exchange in purifying ancient DNA, the authors took

“Thirteen PCR-resistant ancient bone samples aged 500–3,300 years were tested to extract aDNA using a recently reported, silica-based aDNA extraction method and an ion-exchange column method for the further purification. The PCR success rates of the aDNA extracts were evaluated for the amplification ability of the fragments of mitochondrial DNA, a high-copy DNA, and amelogenin, a low-copy DNA. The results demonstrate that the further purification of silica-based aDNA extracts using ion-exchange columns considerably improved PCR amplification.”

When compared to just passing the ancient DNA down a silica based column to passing it down a silica based column and then an ion-exchange column the PCR results were like night and day. The authors provide photos of gels loaded with different amounts of template and the 6ul reaction was the most outstanding. It clearly documents how well ion exchange was at removing PCR inhibitors.

Agarose gel electrophoresis analysis of PCR products

What you wanna see in any PCR reaction is a single bright band. That indicates you’ve most likely amplified one product. Seeing a smear, as shown in the silica based method, indicates either polymerase wasn’t targeting the correct area because the primers were annealing at random targets, or you have some sort of inhibitor that’s altering your polymerase enzyme’s fidelity and accuracy. As you can see, the ion exchange method, shows bright sing bands, for most of the wells loaded, and the difference between the silica based method for purifying ancient DNA is like night and day.

The authors did not get into any crazy discussion on how the ion-exchange matrix is purifying away the inhibitors. But it works, and it is a major milestone for ancient DNA as well as many other applications of extracting and amplifying DNA when there’s a lot of inhibitors around. I imagine people working with DNA samples from fecal material, such as the guys behind the blog, DNApes, or people working with forensic material would also benefit from this. In my personal experience, this method seems much better than the expensive PCR inhibitor removers, a.k.a. Perfect Match that have never worked for me.

    Kim, K., Kim, K., Jeon, E., Togloom, A., Cho, Y., Lee, M., Lkhagvasuren, G., Choi, J., Tumen, D., Ja Park, A., Kim, K., Park, K., Kim, J., Noh, M., Yoo, K., Lee, K. (2008). Technical note: Improved ancient DNA purification for PCR using ion-exchange columns. American Journal of Physical Anthropology DOI: 10.1002/ajpa.20782

    Wall, J.D., Kim, S.K. (2007). Inconsistencies in Neanderthal Genomic DNA Sequences. PLoS Genetics, 3(10), e175. DOI: 10.1371/journal.pgen.0030175