Ancient Fossils with Preserved Soft Tissues and DNA
D. Pitman M.D.
Updated March, 2005
One of the earliest published reports concerned DNA extracted from fossil Magnolia leaves (with intact fragments measuring up to 800 base pairs) found in lake bottom sediments of Miocene age, supposedly 17-20 million years old.3 This find was quite interesting because the magnolia leaves were still wet. Of course, DNA disintegrates fairly rapidly when in contact with water. In commenting on the remarkably old DNA in the supposedly 17-million-year-old magnolia leaf, Savante Pääbo exclaimed, "The clay was wet, however, and one wonders how DNA could have survived the damaging influence of water for so long." 24
most of the DNA which has been recovered is from insects and plants preserved in
dry amber, including a termite estimated to be 25-30 million years old,2
a Hymenaea leaf thought to be 25-40 million years old5 and a weevil
estimated to be 120-135 million years old.1 The weevil DNA is
currently claimed to be 80 million years older than any other fossil
DNA ever extracted and sequenced.
Even more amazing than this though are the findings of Dr. Cano, a
microbiologist at California State Polytechnic University. What Dr. Cano
did was dissect a Dominican stingless bee trapped in amber, which was thought to
be 25 to 40 million years old. What he found were very well preserved
bacterial spores inside. In fact they were so well preserved that they
actually grew when placed in the right environment. In other words, they
were still alive! And, interestingly enough, their DNA closely matched the
DNA of modern bacteria that grow inside modern bees. 26 Also, fairly
recently, a viable bacterium was isolated from a primary salt crystal dated at
over 250 million years old. 30
Even more amazing than this though are the findings of Dr. Cano, a microbiologist at California State Polytechnic University. What Dr. Cano did was dissect a Dominican stingless bee trapped in amber, which was thought to be 25 to 40 million years old. What he found were very well preserved bacterial spores inside. In fact they were so well preserved that they actually grew when placed in the right environment. In other words, they were still alive! And, interestingly enough, their DNA closely matched the DNA of modern bacteria that grow inside modern bees. 26 Also, fairly recently, a viable bacterium was isolated from a primary salt crystal dated at over 250 million years old. 30
However, although DNA extracted from amber was maintained in a fairly dry environment, these findings of extremely "ancient" DNA in amber are still problematic. R. John Parkes commented in a fairly recent issue of Nature concerning this and other similar phenomena by noting that, "There is also the question of how bacterial biopolymers can remain intact over millions of years in dormant bacteria; or, conversely, if bacteria are metabolically active enough to repair biopolymers, this raises the question of what energy source could last over such a long period." 29
discoveries have been widely reported by the media. But what has been largely
ignored is the difficulty that these finds present for the standard geological
Lindahl went on to argue for the "contamination" of all such
specimens by modern DNA suggesting that, "The apparent observation that
fully hydrated plant DNA might be retained in high-molecular mass form for 20
million years is incompatible with the known properties of chemical structure of
DNA." 28 In a 1991 issue of Science Jeremy Cherfas
expressed his bewilderment noting, "That DNA could survive for such a
staggering length of time was totally unexpected - almost unbelievable." 25
Lindahl went on to argue for the "contamination" of all such specimens by modern DNA suggesting that, "The apparent observation that fully hydrated plant DNA might be retained in high-molecular mass form for 20 million years is incompatible with the known properties of chemical structure of DNA." 28 In a 1991 issue of Science Jeremy Cherfas expressed his bewilderment noting, "That DNA could survive for such a staggering length of time was totally unexpected - almost unbelievable." 25
a similar vein, Sykes (1991) has commented that in vitro estimates of the rate
of spontaneous hydrolysis imply that no DNA would remain intact much beyond
10,000 years. In his review paper, Lindahl goes on to argue that “it seems
feasible that useful DNA sequences tens of thousands of years old could be
recovered, particularly if the fossil has been retained at low temperature,”
giving as an example DNA from mammoth tissue thought to be 40,000 years old. So,
our knowledge of DNA stability makes it seem highly improbable that this
molecule could be preserved for more than a few tens of thousands of years at
Others have noted that, "Certain physical limits seem inescapable.
In approximately 50,000 years, water alone strips bases from the DNA and leads
to the breakage of strands into pieces so small that no information can be
retrieved from them. Oxygen also contributes to the destruction of DNA. Even in
ideal conditions–in the absence of water and oxygen and at low
temperature–background radiation must finally erase all genetic
Others have noted that, "Certain physical limits seem inescapable. In approximately 50,000 years, water alone strips bases from the DNA and leads to the breakage of strands into pieces so small that no information can be retrieved from them. Oxygen also contributes to the destruction of DNA. Even in ideal conditions–in the absence of water and oxygen and at low temperature–background radiation must finally erase all genetic information," 27
the fossils from which DNA has been recovered are thought to be, in some cases,
tens of millions of years old. There
is obviously a problem here. It is
for this reason that some scientists are now viewing some of the reported finds
(especially those that do not involve preservation in amber) skeptically.
It has been argued that some of the detected residues were the result of
contamination by modern DNA, so more recent workers have been conscientious in
arguing that they have eliminated this possibility. In any case, the data, as
presently known, does not sit comfortably with the accepted millions of years
Very Old Protein
Despite the reproducible evidence that DNA as well as many proteins are rather unstable and decay relatively rapidly, the positive reported findings of such existent material in fossils supposedly millions of years old, seems rather intriguing. For example: In 1992, Dr. Muyzer, et al., used polymerase chain reaction (PCR) to amplify a protein that they suspected to be osteocalcin from two Cretaceous dinosaurs identified as “Lambeosaurus F38” (which they believe to be 75.5 million years old), “Pachyrhinosaurus F39” (supposedly 73.25 millions years old), and a third dinosaur sample identified only as “F33”. They used two different methods to determine if this protein really was osteocalcin or not.
The first method used an immunological reaction. Here is how it works: When a few molecules of a foreign substance are injected into an animal, that animal’s immune system will naturally produce antibodies to fight it. The kind of antibodies it produces depends on the kind of foreign material introduced. Furthermore, the animal’s immune system will produce lots of antibody cells in response to just a few foreign molecules, so the antibodies are much easier to detect than the foreign material itself.
The researchers took some osteocalcin from alligator bones and injected it into a rabbit to see what kind of antibodies the rabbit produced to fight off the osteocalcin. Then they took some powdered dinosaur bones and injected it into the rabbit, and it produced the same kind of antibodies, indirectly indicating that there was osteocalcin in the powdered dinosaur bones. The second method used a direct measurement of Gla/Glu ratios “detected by high-performance chromatography.” 7
Their conclusion was that both methods showed osteocalcin was still present in the three different dinosaur bones they analyzed. This was published in October, 1992. The literature search found that all the articles on organic material still present in dinosaur bones were published from April 1990 until November 1994 (As far as I can tell, there has been nothing published in Nature or Science on the subject since then). In the same publication as Dr. Muyzer 7, Dr. Matthew Collins made the following statement:
"Dinosaurs hold an enduring fascination. We reported the detection of a protein in a dinosaur bone, published at around the same time as the release of Steven Spielberg's blockbuster, Jurassic Park, [so it] was bound to receive the full media treatment. Our report claimed to have detected osteocalcin immunologically and also to have found an unusual amino acid g-carboxyglutamic acid (Gla) in a dinosaur bone from immature (unheated) sediments. Osteocalcin is peculiarly suited to such spectacular survival, it is very abundant in bone, binds strongly to it and has the distinction of being the only ancient protein ever to have been sequenced."
Some articles suggested that the finding had brought forward the chances of successfully turning the science fiction of Jurassic Park into scientific fact. Elsevier magazine (2/10/93) stated "[The detection of osteocalcin] has set other scientists thinking, if it is possible for a protein, perhaps it is also possible for DNA". The Daily Telegraph even suggested that trend-setting restaurateurs may start serving dinosaur soup! The scientific community was more skeptical. Jeff Bada (an experienced protein geochemist) warned in a 1992 interview in Science News "I worry greatly about the stability of Gla. Why would it remain unaltered for tens of millions of years?".
Scientists are asking, "How can this protein be so fresh when it is contained in such old bones?" We should consider the possibility that they will never find the answer because they might be asking the wrong question. Maybe they should ask, "How can these bones be so old when they contain such fresh protein?" That throws a whole new light on the subject. They will not ever figure out how protein and DNA can last for tens of millions of years without breaking down if protein and DNA cannot really last for tens of millions of years. They might just as well be wasting their time.
Really "Old" Dinosaur Soft Tissues, Blood Cells, and Protein
Many different kinds of intact proteins are being found in "ancient" fossils that are not completely fossilized. Some scientists seem to have found intact hemoglobin molecules in the bones of 65 million-year-old T. rex fossils! How fairly large portions of such a seemingly delicate molecule could survive intact over many millions of years is quite a mystery.
"The lab filled with murmurs of amazement, for I had focused on something inside the vessels that none of us had ever noticed before: tiny round objects, translucent red with a dark center. Then a colleague took one look at them and shouted, 'You've got red blood cells. You've got red blood cells!'. It was exactly like looking at a slice of modern bone. But, of course, I couldn’t believe it. I said to the lab technician: 'The bones, after all, are 65 million years old. How could blood cells survive that long?'" 13,14
This account was given by Mary Schweitzer, a PhD student at the time, from Montana State University. A well preserved Tyrannosaurus rex skeleton had been found in 1990 and brought for analysis too Montana State University. During microscopic examination of the fossilized remains, it was noted that some portions of the long bones had not mineralized, but were in fact original bone. Upon closer examination it was noted that within the vascular system of this bone were what appeared to be red blood cells (note retained nucleus in the center of the apparent RBCs and the fact that reptiles and bird generally retain the RBC nucleus while mammals, like humans, do not). Of course, this did not seem possible since the survival of intact red blood cells for some 65-million years seems very unlikely if not downright impossible.
Further testing of these cells was done to attempt to disprove the notion that they could possibly be red blood cells. Several analytical techniques were used to characterize the material to include nuclear magnetic resonance (NMR), Raman resonance and Raman spectroscopy (RR) and electron spin resonance (ESR). These techniques did identify the presence of the heme group molecule, but the detection limits of these methods were not able to rule-out or rule-in the presence of hemoglobin or myoglobin proteins due to the small amount of specimen available. So, Schweitzer and her team decided to use a more sensitive detection method, the immune system. They injected some of the T. rex extract into laboratory rats to see if these rats would mount an immune response to the foreign T. rex material. And, the rats did mount a very specific immune response against hemoglobin. This immune response was not only against heme, but hemoglobin, and not just hemoglobin in general, but against a certain type of hemoglobin.42 The reaction was strongest against pigeon and rabbit hemoglobin. There was also a weak reaction against turkey hemoglobin, but there was no reaction against snake hemoglobin. The specificity of these reactions were further confirmed by the lack of reactivity with plant and sandstone extracts.
Consider the conclusions that Schweitzer and her team made concerning these findings:
"The production of antibodies specific for hemoglobin in two rats injected with the trabecular extract is striking evidence for the presence of hemoglobin-derived peptides in the bone extract. . . That the antisera did not react with snake hemoglobin shows that the reactivity is specific and not artifact. . . When considered as a whole, the results support the hypothesis that heme prosthetic groups and hemoglobin fragments were preserved in the tissues of the Late Cretaceous dinosaur skeleton." 16
These results are quite interesting since they indicate a very specific immune response, not just against hemoglobin, but certain types of hemoglobin molecules. Note again that the antibodies formed did not react against snake hemoglobin indicating that the antibody reactivity was "specific and not artifact." The question is, how much of the original T. rex hemoglobin molecule would need to be intact to elicit such a specific immune response in the laboratory rats?
Schweitzer goes on to suggest that "Immunogenicity is not dependent on fully intact protein, and even very small peptides are immunogenic when complexed with larger organic molecules . . . even after extensive degradation has occurred."42 But how extensively, roughly, could the hemoglobin molecules have degraded and yet retain their ability to elicit a fairly strong and quite specific immune reaction in laboratory rats? In order to obtain such strongly specific immunogenicity it would seem that a significant percentage of the globin portion of the hemoglobin molecule would need to be intact. But, how could a protein of any significant size large enough to elicit such a specific immune response be maintained over the course of 65 million years? One might very reasonably conclude that natural decay, over this amount of time, would completely destroy the ability of hemoglobin or the required larger fragments of degraded hemoglobin from being antigenic in such a specific way.
The explanation for this phenomenon, given later by Dr. Horner (Schweitzer's boss) and even Schweitzer herself, was that the tougher heme molecule survived the 65 million years with maybe three or four amino acids of the original globin molecules attached to it. Consider the following statement Schweitzer made in a response to an inquiry by Jack Debaun:
"But the heme itself is too small to be immunogenic [only about 652 daltons]. We believe that there were possibly 3-4 amino acids from the original protein attached to the heme, and that was what may have spiked the immune response." 17
Now, it just seems quite unlikely that just 3 or 4 amino acids stuck onto a heme group is going to give rise to an immune response as specific for a certain type of hemoglobin as was found in this case (Note that a fully formed globin molecule ranges from 141 to 146 amino acids in length with specific folding characteristics that antibodies detect). As far as I have been able to tell, the degree of immune response specificity noted by Schweitzer et al. has never been realized in any confirming experiment with so few hemoglobin amino acids stuck to a heme group and I doubt that such an attempted experiment will ever be successful.
There are several reasons why I feel this way. For one thing, a certain minimum antigen size is required before it can elicit an immune response. The most potent immunogens are macromolecular proteins with molecular weights greater than 100,000da (~740aa - Note: the average amino acid weighs ~135da). Substances weighing less than 10,000da (~75aa) are only weakly immunogenic, and those foreign proteins/antigens weighing less than 1,000da (~7aa) are usually completely non-immunogenic. Homopolymers (repeats of the same amino acid) are pretty much non-immunogenic regardless of size. Co-polymers of glutamic acid and lysine must be ~35,000da (~250aa) to be immunogenic. It seems then that, in general, immunogenicity increases with structural complexity. Also, aromatic amino acids, such as tyrosine or phenylalanine, contribute much more to immunogenicity than do non-aromatic amino acids. For example, the addition of tyrosine to a co-polymer made up of glutamate and lysine reduces the size limitation to ~15,000da (~100aa) and adding tyrosine and phenylalanine together reduces the minimum to 4,000da (~30aa). Also, it is all four levels of protein structure (1o, 2o, 3o, & 4o) that influence immunogenicity - not just a short linear sequence of amino acids.18-21
Of course, a rather specific immune response can be elicited by relatively few amino acids as part of an epitope on a larger protein molecule, but they usually are not immunogenic without first being part of a larger molecule. Also, epitopes are not usually sequential in nature but are assembled by protein folding. This means that a rather large portion of the original molecule usually needs to be intact in order for most epitopes to remain intact. Epitopes with definite three-dimensional shapes and charged amino acids are particularly well recognized by antibodies. The average epitope probably involves about 7 to 15 contact amino acid residues and a few of these may be critical to the epitope's specificity and the avidity of the antibody-antigen reaction.18-21 But, in order to make an epitope antigenic, it must be processed first.
Antigen presenting cells (APCs) like macrophages, dendritic cells, and even B-cells are responsible for antigen processing and the presentation of epitopes/antigens to the T-cells. T-cells do not recognize the initial foreign antigen directly. They only recognize processed parts of antigens, consisting of no more than 15 or so amino acids, presented to them by APCs in association with MHC (major histocompatibility) molecules. So, in order to activate T-cells (required for cellular immunity and very helpful in humoral immunity), the foreign antigen must first be recognized as "foreign" by the APC cells. This initial APC recognition requires more than just a handful of amino acids floating around or else there would be complete meltdown of the immune system. In fact, generally speaking, molecules with a molecular weight less than 10,000da (~75aa) are only weakly immunogenic when picked up by APC cells. Significant potency usually requires antigens to be rather large at over 100,000da (~750aa).22,23
Given all this, it seems quite difficult for me to imagine how "3 or 4" amino acids stuck to a heme group could elicit an immune response that was so specific for a certain type of hemoglobin. Recall that the heme molecule, by itself, only has a molecular weight of around 652da. To make a strong as well as specific immunogen (such as the strongly specific hemoglobin immunity developed in rats exposed to T. rex extract in this case) one might expect the immunogenic hemoglobin molecule to be at least 10,000da (~75aa or so) in size.18-21 Certainly then, a heme group with 3 or 4 amino acids attached to it (just over 1,000da) would not seem to give rise to the relatively strong and specific immune response (specific to a certain type of hemoglobin) observed by Schweitzer et al. in rats exposed to T. rex bony extract.
However, the argument is sometimes used that Schweitzer failed to identify any specific size of hemoglobin fragment by gel electrophoresis. What happened is that the electrophoretic pattern observed by Schweitzer when she ran the T. rex proteins through the gel was a diffuse or smeared pattern. This means that there were no discrete clusters of proteins that were the same size. But this is only to be expected since a wide range of protein sizes would only be expected after an extended period of degradation. The fact of the matter is though that hemoglobin fragments ranging between 30 and 200 amino acids in size where definitely present in the T. rex extract (per NMR analysis filtering).16
Another argument often used is that the molecules that elicited the immune response were indeed quite large, but they were made up of fragments of smaller hemoglobin molecules and other organic and inorganic molecules to form a new collective molecule. The problem here is that there is that this hypothesis has not been tested or demonstrated. Beyond this it doesn't seem very likely. For one thing heme is non-covalently bonded to the globin portion of the hemoglobin molecule. If the much stronger covalent bonds were broken and rearranged so much between the remaining amino acids, how is it reasonable that the relatively weak non-covalent bond between the amino acids and the heme group would be maintained? Also, such rearrangement of covalent bonds would have distorted the covalent bonds within the amino acids themselves as well as between the covalent bonds they share with other amino acids. This level of decay would alter the type of amino acids or destroy them as amino acids completely. The specificity of the immune response against hemoglobin in particular speaks strongly against this degree of change having taken place.
If this is not already enough, Schweitzer recently made an even more startling discovery. About three years ago (2002) she and her team had to divide a very large T. rex thigh bone in order to transport it on a helicopter. When the bone was opened flexible, even elastic, soft tissue "meat" was found inside. This is incredible because this bone was supposed to be some 68 million years old. Microscopic examination revealed fine delicate blood vessels with what appear to be intact red blood cells and other type of cells like osteocytes - which are bone forming cells. These vessels were still soft, translucent, and flexible. Subsequent examination of other previously excavated T. rex bones from this and other areas have also shown non-fossilized soft tissue preservation in most instances.31
This find calls into question not only the nature of the fossilization process, but also the age of these fossils. How such soft tissue preservation and detail could be realized after 68 million years is more than miraculous - - It is unbelievable! Schweitzer herself comments that, "We may not really know as much about how fossils are preserved as we think . . .” 31 Now, if that is not an understatement I'm not sure what is.
So, it seems rather clear, despite the objections of many evolutionists, to include Schweitzer herself, that a 1,000da molecule would elicit an extremely weak response at best and would not necessarily elicit a specific response to a certain type of hemoglobin molecule since surface epitopes are generally more specific in their antigenic nature than are buried epitopes (i.e., heme is somewhat hidden within a cleft of the hemoglobin molecule so 3 or 4 amino acids attached to it would also be somewhat hidden). How then is it remotely logical to suggest that a molecule weighing just over 1,000da (a heme group plus 3 or 4 amino acids) could elicit such a strong as well as specific immune response as Schweitzer et al. observed? In light of the additional recent finds of even more striking soft tissue and blood cell preservation, it seems much more likely that such an immune response so specific for certain types of hemoglobin could only be elicited by a larger portion of intact hemoglobin than many scientists seem to even consider. Of course, one can't really blame them because explaining how delicate soft tissue vessels (with obvious red blood cells inside containing relatively large portions of hemoglobin molecules) could remain intact for over 65 million years seems just a little bit difficult.
Such finds are much more consistent with a fairly recent catastrophic burial within just a few thousand years of time. Non-catastrophic burial would allow for rapid biodegradation of such delicate soft tissues. Time itself destroys soft tissues as well as DNA and proteins in short order. Current real-time observations suggest that bio-proteins could not remain intact more than a few tens of thousands of years - 100,000 years at the very outside limit of protein decay. The fact that such proteins are found, intact, in bones supposedly older than 65 million years is simply inconsistent with such an assumed age - by many orders of magnitude.
I think that one further study should be done. This study should be a Carbon 14 dating of this organic material as well as other “fossilized” organic material. If any convincingly non-contaminant carbon 14 remains in any detectable amount in organic specimens supposedly millions of years old, then a real problem arises that is equivalent to finding a hominid in the Cambrian. This, combined with the fresh DNA and protein problem seems to me to be quite a quandary for the theory of evolution. At least I have not found a good solution advocated in the scientific literature to explain these problems as of late.
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