I am forwarding a post from Bryan L. Ford that he sent to Gen-Med today. Anybody want to chip in a get a DNA study of our genes? carole _________________ Bryan L. Ford wrote: >D. Spencer Hines wrote: > > Please do go on into the technical details as they may affect Medieval > and/or Royal Genealogy. > I am certain that the methods as they exist today can be used to answer or at least begin to answer suitably framed genealogical questions. Below I present ideas that D. Spencer Hines may or may not already be familiar with. My intent, with his permission, is to bring the possibility of using molecular genetic technologies in solving certain genealogical questions once again to the attention of other readers of this newsgroup, while at least partially answering the questions D. Spencer Hines presents. I thank him now for this opportunity, and I thank him for the broad questions, and apologize for my lack of time to fully answer them. A bit about one set of tools that are today used commercially, medically and forensically to establish paternity by examining markers on the Y-chromosome (the chromosome whose presence gives a male phenotype). A substantial number of Y-chromosome markers that can be inexpensively and rapidly examined using PCR-based methods are available and in use today. For less than a thousand dollars one can survey an array of markers on the Y-chromosome of any living male, and for perhaps one third to one half that a second and subsequent males can be checked for Y-chromosome identity to that first sample. This method makes use (at last count I have) of some 10 different "short tandem repeats" or STRs that contain between 2 and 13 polymorphisms each. This means that !0 sites within the Y have useful STRs and that these STRs can be readily examined for their length using the Polymerase Chain Reaction or PCR-- the number of length differences seen using PCR on the STRs range from 2 to 13, and total number of possible combinations of over 13 million. Presently the number of STRs characterized and readily PCRed under commercial or forensic lab conditions easily allow one to reject a false claim of paternity at least 99.9% of the time. This can and will be improved in the future. Very high certainty for very important cases (that is were money is not so much the issue) will increasingly be available especially as the Human Genome Diversity Project is completed a few years after the Human Genome Project is completed around 2003. As to applicability to medieval and royal genealogy, let me give outline of one easily perceived general application, for which I will leave it to the readers of this newsgroup to define the specific suitable genealogies. Y-chromosomes follow agnate descents, that is the father passes on to his Y to his sons only, they in turn pass on that same Y genotype to their sons etc. I am sure that there exist well supported claims of agnate descents from medieval times. Of particular interest with respect to the new technologies, even as they exist today, would be any such genealogy (known or suspected) in which the earliest point in such an agnate descent that two brothers give rise to agnate lineages, each with living male representatives. All that is needed are the cheek swabs for DNA from living males in each branch of such a commonly rooted agnate descent. Costs? probably no more than $500 to $1000 to run the tests on one individual and $200 to $400 for any number of willing others suspected of being in the same putative agnate descent. For royal genealogies these methods will be very useful for testing strong genealogical hypotheses that can be framed in terms of agnate lineages... in some cases surely from the early medieval era. All that is needed is for competent genealogists to define one or more solid agnate lineage and then identify hypothetical or suspected parallel agnate descents from the same founder. Similarly, analyses such as these may also be especially productive for genealogy questions involving individuals from lesser social strata for whom records are either less abundant or even nearly non-existent. Numerous genealogical problems seem to crucially involve surname identity, surname similarity or surname transliteration-- with resolution in some cases, for example when surname transliteration accompanies distant migration, being extremely difficult. Questions of ancestry from the era of surnames, which I suppose may be from late medieval for many of the more fortunate families in Europe, and somewhat later for the less fortunate, depending on country and many other factors, can and will be approached using these new methods. But such analysis will not necessarily be confined to sample of DNA from living donors, there is good evidence that the examination of Y-chromosomes from ancient tissues, provided they happen to have been preserved under appropriate conditions (such as in bones under dry conditions) is entirely possible. For ancient royalty and other noteworthy individuals this will no doubt be pursued, since royal corpses are often believed to rest in well marked, and hopefully well-preserved tombs-- and many may actually do so, at least in the absence of mischief or misdeed. I would expect that using such sarcophagi, modern royal agnate Y-genotypes could certainly be compared with their ancestral antecedents. I cite two abstracts here for the curious, I have read the first article, but as yet not the second: Human Genetics 1999 Feb;104(2):164-6 Amplification of Y-chromosomal STRs from ancient skeletal material. Schultes T, Hummel S, Herrmann B Historische Anthropologie und Humanokologie, Institut fur Zoologie und Anthropologie, Universitat Gottingen, Germany. Abstract: "The adaptation to ancient DNA analysis of a Y-chromosomal STR (short tandem repeat) multiplex comprising the four STR systems DYS19, DYS390, and DYS389I/II shows the suitability of Y-chromosomal STR typing on ancient human remains. A new primer site for the system, DYS389I/II, resulting in products shortened by 94 bp, was chosen to serve the special needs of amplification of ancient DNA. For the first time, it was possible to amplify STR loci of the Y chromosome from historical and prehistorical bones of up to 3000 years old." And the second one I recently discovered but as yet have only ordered through interlibrary loan: Bulletins et Memoires de la Societe D'Anthropologie de Paris 1996 8(3-4):465-479 Sex Determination by polymerase chain reaction using ancient and medieval human bone remains. [in French] Abstract: "The analysis of ancient DNA, amplified by Polymerase Chain Reaction (PCR) offers a molecular method of sex determination which completes morphological procedure. We used this molecular method on 15 Roman and medieval samples. The amplification of a 102 base pair fragment of the Y-chromosome was realised with "Lo" primers. We then examined the sequences of the amelogenin gene which is present on both the X- and Y-chromosomes. We are necessarily prudent; but the concordance of anthropometric and biological data obtained is encouraging." Note: Even though the latter abstract indicates only an attempt to identify the sex of the subjects in question, these researchers are apparently able to amplify a 102 bp fragment successfully. This then implies that they should be able to amplify many, if not all of the STRs now available, and thus we would expect that they could go on to establish a unique Y-genotype for each individual whose tissue is able to be PCRed. Add to this the fact that PCR reagents, equipment and methodologies as well as DNA extraction techniques have improved since this work was done. > Please tell us more about AFLP, in particular. I should not say much about this, since I am not a practitioner of it. Suffice it to say the method supplants RFLPs, it requires nanogram quantities of DNA compared to the micrograms required with RFLP. AFLP, or at least the form I have some familiarity with, does not use radioactive labeling, which is a tremendous advantage from the standpoint of regulatory bureaucracy and technician peace-of-mind. It instead relies on the sensitivity and amplification of PCR technology, and on the use of fluorescent labels. The fluorescent products are typically examined using an $85,000 laser excited scanning fluorescent densitometer. But let me caution that it would not be wise to focus on AFLP, but rather to be aware that tremendously sensitive detection methods are continually being evolved with ever more diverse possible applications. The state-of-the-art in 1988 might have been RFLPs. That state now is definitely not RFLPs but the successor state is difficult to define since now a much larger range of methods is available to identify genotypes. AFLPs, a direct descendant of RFLPs, if you will, work well in certain cases but other techniques, many that do not have familiar acronyms, are available for other specific tasks. The future holds incremental improvements in methods and technology, punctuated by occasional quantum improvements in the technology (much as PCR itself was). AFLP is not revolutionary, but one of many PCR-based incremental advances in methodology and technology. Numerous other more basic methodological advances are more central to the progress being made. Such as the improved isolation of usable DNA from degraded samples, or improvements in detection sensitivity, or the accumulation of genotype databases (e.g. NCBI's Genbank), or improvements in understanding how, and at what rate the polymorphisms (such as the STRs) arise and so on. (Please excuse the lecture--) > Will these techniques allow us to solve some genealogical questions > that may be thought impossible today? Unquestionably. Just imagine a competent genealogist of 50 years ago being told that before the end of the century many highly tenuous genealogical assertions based on surname identity could be conclusively disproved or even substantially supported by using genetics technologies that were not even dreamed of yet. In 1949 only a few lucky individuals knew that DNA was the carrier of genetic information. DNA structure was unknown. PCR was not to be regularly used for another 40 years. The pace of technological innovation has been and will continue increasing at an exponential rate. From my view, it would be hard to imagine that we would not see genealogical analysis revolutionized by the technology available today and even further by the technologies soon to be available. I should add that we may well be amazed, delighted or shocked by the results, not only because many may discover evidence of illegitimacy in the lineages they study, but also surprised in many other ways that are as yet unforeseen. Once these tools are brought to bear on substantial genealogical problems and once they have been shown to give useful results, then genealogists will rightly come to expect much more from such analysis. They will not be disappointed. But, before this can happen, it is very important for at least some competent genealogists to learn what can now be done, and what may soon be done using these technologies. This is the hard part of getting the work underway. Once just a few biotechnology-aware genealogists exist, then they should be in good position to guide the technologists to the most important genealogical cunundrums likely to be illuminated by applying the new molecular genetics tools. > > It's refreshing to hear from someone who appears actually to know what > he's talking about --- a quondam rarity here. I hope my comments above do not dissuade you from this perhaps undeserved judgement. Cheers, Bryan L. Ford > D. Spencer Hines --- "Black care rarely sits behind a rider whose pace > is fast enough." --- Theodore Roosevelt (1888) > > Bryan L. Ford <bryan.ford@orst.edu> wrote in message > news:37675C05.21A6@orst.edu... > > Todd A. Farmerie wrote: > > > > > > William Addams Reitwiesner wrote: > > > > > > > > "Todd A. Farmerie" <taf2@po.cwru.edu> wrote: > > > > > > > > >Yes, they have, but the approach used for those studies > (PCR/sequencing > > > > >of mtDNA) is more tollerant of degeneration of the source DNA, > by a > > > > >factor greater than 10,000, than the RFLP analysis perfomed on > > > > >Y-chromosomes. Except in extremely rare circumstances, the > latter is > > > > >not practical for dead dudes. > > > > > > > > Y-chromosome analysis is what was used to > [prove/suggest/confirm/pick one] > > > > the Thomas Jefferson/Sally Hemmings connection. > > > > > > Yes, but using DNA from living individuals (I was refering to the > source > > > of the DNA, not the individual the study is addressing). It could > not > > > have been done using DNA from Jefferson himself (if it could, they > would > > > not have done Y-chromosome analysis - they would have been able to > test > > > TJ and Eston Hemmings for autosomal markers and determine > unambiguously > > > whether TJ was the father or just a male relative). All of the > other > > > prominant studies which have been discussed in this thread, and > several > > > that have not, have been mitochondrial studies using material > extracted > > > from the remains of deceased individuals. > > > > Nuclear DNA from ancient sources has been examined using PCR, and > > specifically Y-chromosomes have been examined. The results have been > > published. To give one example: > > > > Hummel S, Herrmann B (1991) "Y-chromosome-specific DNA amplified in > > ancient human bone." Naturwissenschaften 78:266-267. > > > > The major present weakness of Y-chromosome analysis may be the > > relatively poor degree of gene mapping and sequence information > > available, especially for the Y-chromosome, long thought to be > largely > > functionless other than in sex determination. This absence of > detailed > > sequence information for the Y will soon change as the human genome > > project proceeds. The mitochondrial genome on the other hand has > long > > been completely sequenced and numerous variant markers are > characterized > > within it. > > > > An expert in this field (one of the researchers who did the > > Neandertal/modern human mitochondrial marker analysis) has told me > that > > he suspects that ancient nuclear DNA may actually be more accessible > to > > recovery and analysis than mitochondrial DNA. There are good reasons > for > > this which I can go into in a bit of detail if anyone here is > > interested. > > > > > I have heard of a few instances where RFLP analysis (like that > done on > > > the Y-chromosome) has been used on the very-recently (and > unembalmed) > > > > Formaldehyde (as formalin solution) amongst available histological > > fixatives, is often preferred when a histological sample may be > > eventually be used as a source of DNA template for PCR. AFLPs, the > > current technology largely supplanting RFLPs, are readily done on > > embalmed tissue samples. > > > > > dead (to identify victims), but not on anyone dead more than 30 > years or > > > so (Vietnam MIAs). > > > > It may not yet have been tried yet. There is no fundamental reason > why > > such samples cannot be successfully analyzed both at the > mitochondrial > > and nuclear genome level. Thirty years of storage or burial is not > > necessarily a challenge provided that the right tissue sample is > used > > (for example bone) and that the storage conditions were not horrific > > (such as incineration). Again, if anyone here cares I would be happy > to > > discuss the technical details, or at least those I'm familiar with. > > > > > By comparison, there is at least one report of > > > mitochondrial DNA analysis (using PCR and sequencing) on a plant > 10 > > > million years old, > > > > This result has been reviewed very carefully and is now considered > to be > > an artifact of PCR contamination. Under certain conditions DNA is > > surprisingly rugged but apparently not that rugged. More likely to > > contain really ancient DNA are from insects etc. entombed in samples > of > > certain types of amber. > > > > > and it has been used on the type specimen of > > > Neandertal Man, which is several hundred thousand years old. > > > > Actually this sample was around 50,000 years old. The 450,000 years > > represents the inferred divergence time between this Neandertal and > > modern humans, that is the approximate date for the last common > female > > ancestor of these two species of human. > > > > The conditions of storage of tissues is the most important factor > > determining whether DNA markers can be read. The samples taken from > a > > 50,000 year old neandertal femur show substantial degradation. The > > sequences has to be pieced together from a series of short, > overlapping > > PCRs. Such difficulties do suggest limits are present, at least for > a > > given sample from a given provenance. > > > > Cheers, > > Bryan L. Ford