Here is the full citation for the first paper I’m going to review.
Campana, Michael G., Bower, Mim A., Bailey, Melanie J., Stock, Frauke, O'Connell, Tamsin C., Edwards, Ceiridwen J., Checkley-Scott, Caroline, Knight, Barry, Spencer, Matthew, and Howe, Christopher J. 2010. "A flock of sheep, goats and cattle: ancient DNA analysis reveals complexities of historical parchment manufacture." Journal of Archaeological Science 37 (6) 1317-1325.
You can read and download the entire paper at ResearchGate You are not allowed to view links.
Register or
Login to view. although I don’t recall if I had to give them my e-mail in order to download.
1. What research question was Campana et al. asking?
Can standard DNA sequencing, with reliance on Polymerase Chain Reaction (PCR) amplification, then cloning, followed by sequencing of the results, provide identification of what species was used to make the parchment used for group of documents created in the 1700-1800s?
Some important points to note:
(i) What is being tested --
relatively new parchment documents, compared to the Voynich, which were considered
not valuable;
(ii) How the samples were prepared – “1-200 mm2” square pieces were removed using a scalpel or scissors” (e.g.
destructive sampling).
(iii)
The use of PCR (repetitive copying of preselected target DNA sequences from what DNA was isolated from the sample and cloning and sequencing of those results) You are not allowed to view links.
Register or
Login to view. is more information about PCR.
(iv) What genetic sequences were the targets of the PCR?
(A)
Cytochrome B coding sequence – cytochrome b is an enzyme encoded on mitochondrial DNA. You are not allowed to view links.
Register or
Login to view. is a link to the first group to propose using this gene
to distinguish one species from another. You are not allowed to view links.
Register or
Login to view. is a link to a full paper that is free that discusses this.
A few things to note about mitochondrial DNA – inherited only from female (from mitochondria in the egg) so therefore is haploid (only one copy) and found in a circular double helix form. There has been and continues to be controversy about the use of mtDNA in identification work.
If you’re interested in learning more about the pros and cons of mtDNA You are not allowed to view links.
Register or
Login to view. is an article. Please note that citations of this article indicate
a continued uncertainty even to 2021 about the appropriate use of mtDNA particularly for taxonomy relationships – which could complicate how the data can be used to move backwards and forwards in time with data within a single species.
(B)
D-loop – this is another area of the mtDNA that has been
commonly used to figure out the specific populations within one species from another.
( C)
Short Tandem Repeats (STR) – these are autosomal (standard, nuclear, chromosomal) sequences. There will be two copies of this genetic location (called an “allele”) in each cell – one inherited from male parent and one inherited from the female parent.
Because of the presence of many repeats these gene locations are highly polymorphic (lots and lots and lots of variation) so they are used
to identify one individual’s DNA from another. Thus, the point of this analysis was to see if any of the parchment sheets were from the exact same animal but would not (at this time) be used to identify the species.
You are not allowed to view links.
Register or
Login to view. is a paper which discusses the issues with using STR analysis for animal species identification, in this case, badgers. Note that the test described in You are not allowed to view links.
Register or
Login to view. could maybe work for something like the Voynich with expansion to include goat – so there has been significant work on this since 2010.
2. What were Campana et al.'s results?
a. The species for
only three of the parchments were able to be identified using either the cytochrome b or D-loop results (see Table 1). Campana et al. said these three were definitely bovine and likely the species Bos taurus. One of the parchments only had sequences identified as goat – but four different goat sequences were isolated from that single specimen – calling this result into question.
b. The remaining eleven parchments contained sequences identified to be from
multiple species (including humans)
and a large amount of what Campana et al. termed PCR “artifacts.” No conclusions as to even which species could be reliably drawn for these parchments.
c.
The cytochrome b and D-loop results did not confirm each other as they had hoped. All identifications were based on consistent results for EITHER cytochrome b OR D-loop.
d.
The STR results were all inconclusive – i.e., they could not draw any conclusions about the relationships of any of the individual animals used for the parchments to each other – there was simply too many “peaks” (results) to be able to see the relationships. In other words, all the parchments in this test looked like they came from
multiple animals of different species.
Although the reasons for these results could be multiple laboratory errors or contamination (e.g. it is well known that modern bovine sequences are common contaminants because of the widespread use of fetal bovine serum for common lab practices), BUT Campana et al. did many control experiments to rule the great majority of these issues out –
including sending all new samples to a second genetic lab that confirmed their results.
****Note I am greatly simplifying the mess of these results and if there is any part of it you want further details about, just ask!****
What was left was the likely conclusion that the method of making parchment resulted in so much cross-contamination of the resulting sheets with so many species’ DNA that it is impossible to focus on only the DNA that actually came from the cells present in the parchment.
TLDR –
The Campana et al. results reflect an unhelpful mixture of bovine, ovine, caprine, and human DNA sequences which appear to be cross-contamination from the parchment production process.
Although this could be a result of using later more “industrially” produced parchment this isn’t entirely true because at least one other group testing leather had a similar result to theirs. At this point in the development of the testing process, it looks like significant modifications of the testing method will have to be made for parchment to get useful results.
As you can imagine, this was not a particularly propitious start for the use of genomic analysis for parchment.
I’ll talk about whether this conclusion from 2010 has held up over time as I go through what has been done since. Spoiler alert -- things are a little better -- but there are still many, many questions.