It is this fossil that will be the main focus of DNA extraction in this paper.
These are the main culprits in the sudden race among geneticists to be the one to extract and process the oldest DNA. To date, the oldest piece of isolated DNA came from a 125 million year old insect trapped within a bit of Lebanese amber by California Polytechnic Institute at San Luis Obispo researcher Raul Cano (C.F., 1993). Analyzed, the now extinct insect was found to resemble closest the modern day pine cone weevil. However, research is underway to extract protozoa from a 225 million year old piece of amber obtained by Robert Poinar at
University of California at Berkely (Richardson, 1994). Extraction The extraction of DNA from a fossilized organism or piece of an organism must be a completely sterile procedure. The contamination of any other type of organism, including bacteria, could result in a faulty sample. The popular way of eliminating such potential contaminants is using ultraviolet (UV) light. The UV rays mess up some of the chemical components of DNA, effectively eliminating potential contaminating DNA. The sample is shielded from such rays(DeSalle and
Lindley, 1997). The ideal specimen would be a piece of an animal, insect, or other organism preserved by its natural surroundings. Examples of this would be the Mastodon dung discovered in Florida in 1993 that was effectively preserved in sedimentary layers beneath a river bed (AP, 1993), or the preserved remains of a saber-toothed cat that was recovered from the La Brea tar pits in Los
Angeles (Grimaldi, 1993). Both of these animals went extinct somewhere between ten and fifteen thousand years ago. Unfortunately, in both cases, no adequate
DNA samples were recovered. Finding a fossilized specimen in these states with intact DNA, as stated before, due to the natural degradation processes of organic material is slim (Lewin, 1997). The main focus of DNA isolation is on the various organisms found preserved within amber. In the Crichton book, the suggested way of extracting DNA from an organism is to drill a hole to the organism, and insert a needle (1991). However, this process in reality would be very inefficient (Desalle & Lindley 1997). By doing this, the needle could inadvertently pick up DNA from something else contained within the amber, or something on the surface of the organism itself. A much more efficient way would be to crack the amber in half at the site if the specimen. One would then proceed to remove pieces of the organism (Cano 1996). Upon dividing the specimen into individual cells, the cell and nuclear membranes must be broken to get to the DNA contained within the nucleus. To accomplish this, the cells are added to a solution with a soapy like detergent substance to dissolve the lipids in the membranes and the enzyme proteinase to break down the proteins allowing access to the DNA itself. The genetic material is then isolated using an ultracentrifuge. With this done, the isolated DNA is entered into a thermocycle, fluctuating first hot then cold, along with certain polymerase buffers and individual nucleotides. By fluctuating the heat, the DNA breaks apart, then reforms. Through a process known as the polymerase chain reaction which strings together the nucleotides creating a mirror image of the original DNA, the DNA is multiplied exponentially until it reaches a desired amount (Gibbons,1994). The multiple strands of DNA can be used to study evolutionary trends by comparing them to the DNA of related modern organisms, or even attempting to clone a once extinct species. Research Bacteria Bacteria are simple, unicellular organisms and are often used in genetic research because of their haploid strand of DNA, and method of binary fission reproduction (Cano, 1996). In binary fission, bacteria reproduce by exactly replicating their DNA and then splitting in half.
So in essence, bacteria clone themselves to reproduce. George and Roberta Poinar discovered bacteria cells in the remains of the alimentary canal of nematodes preserved in Mexican amber (Poinar, 1994). Bacteria would be a simple starting step for determining a process for, isolating, testing, and replicating DNA of higher organisms in order to eventually clone or study them. Unfortunately attempts to isolate ancient bacteria have been inconclusive. The chief concern in isolating ancient bacteria is the contamination of the sample by modern bacteria through fractures in the amber. Despite the extensive sterilization techniques, scientists cannot be sure whether the bacteria isolated are truly ancient bacteria (Poinar, 1994). For instance, Bacillus subtilis bacteria were cultured from an amber from an amber specimen of a stingless bee from the
Dominican Republic, but these bacteria are commonly found in both the alimentary canal of the modern-day stingless bee and in the soil. Also problems arise in extracting the DNA from the single-celled organisms without accidentally destroying the small amount of genetic material present (Richardson, 1994). Raul
Cano continues studies of ancient bacteria at California Polytechnic State
University in San Luis Obispo (Poinar 1994). Cano became famous recently for his reviving of a 600 thousand year old bacteria that was in an endosporic state which kept it alive(Cano, 1996). Before this, Cano was brought into the spotlight for extracting bacterial cells off an extinct bee which is estimated to be 40 million years old (Lewin,1995). Insects Insects are commonly preserved in amber after being caught in the sticky resin (sap) emitted by some trees as a defense mechanism (Morell, 1993). In 1982, George and Roberta Poinar identified intact cellular components such as nuclei, ribosomes, and chromosomes in insects embedded in amber, but were unable to isolate DNA at that time (McAuliffe,
1993). The first successful DNA extraction was from an extinct termite,
Mastotermes electrodominicus, by a team at the American History Museum headed by
David Grimaldi (Grimaldi, 1993). These termites were found in amber from the
Dominican Republic. This species is defined by the large, fan-like lobe at the base of its hind wings and by its many wing veins. The perplexity is that these characteristics are also given to cockroaches, which evolved before Mastotermes electrodominicus; thus evolutionary lines cannot be defined on such simple attributes and need to have more exclusive traits to the species in order to establish the evolutionary unit. Another puzzle was the "missing link" between termites and cockroaches: Is the Mastotermes closer related to termites or cockroaches (Grimaldi, 1993)? Scientists are able to establish such links by doing evolutionary comparison between ancient and modern DNA (Morell, 1993).
Fragments of mitochondrial DNA of Mastotermes were amplified using the polymerase chain reaction and then linked to the modern-day termite, Mastotermes darweinis (McAuliffe, 1993). Ancient DNA has also been extracted from stinglees bees being studied by Raul Cano and a 123 million year old extinct insect examined by George Poinar (Morell, 1993). The fossilized insect that inspired the book and movie Jurassic Park has yet to be thoroughly examined. This insect being a 125 year old biting midge found in a piece of Lebanese amber. This insect could potentially have intact dinosaur DNA preserved within it ("Jurassic Bug", 1993). Dinosaurs Michael Crichton’s book Jurassic Park introduced the idea of making dinosaurs from ancient DNA preserved in amber to the public. In the words of Washington biotechnology correspondent Jeremy Rifkin,
"Jurassic park is the most massive exposure of biotechnological research ever!" (Hamilton, 1993). Many scientists have done research into the possibility of accomplishing this. Some say that it is impossible to recreate dinosaur DNA because of the many gaps in the strands. Furthermore, any DNA recovered would have to be from the gut of a blood-sucking insect that happened to perish in a pool of sap almost immediately after feeding off of a dinosaur, (for it is very unlikely that a dinosaur would be preserved in amber itself).
Plus, the amount of DNA extracted would be quite minuscule compared to what it takes to make a complete organism (DeSalle & Lindley, 1997). In the book and movie, the holes in the DNA sequence are filled using frog DNA, yet like critics say "too much frog DNA and your T-Rex Croaks" ("Are Movies Science",
1996). In addition, the much more realistic gene donor would be the closer related bird (Monastesky, 1994). The easier way of extracting intact DNA would be to find preserved fossilized remains with reliable DNA (Svitil, 1995).
However, efforts to isolate DNA from fossilized bones have been unsuccessful because most organic material is converted to inorganic compounds in the fossilization process, and because of the exposure to air and water. Scott
Woodward of Brigham Young University in Provo, Utah, claimed to have extracted
DNA from a bone of a dinosaur from the cretaceous period 134 base pairs long of cytochrome b, but controversy remains as to if the DNA belongs to a contaminant or to the actual dinosaur (Gorman 1994). Using the amino acid racemization test, scientists found that the percentage of D-enantiomers had reached 21 percent, which cast further doubt on the authenticity of the DNA found (Monastersly
1996). Other claims, such as those made by scientist Jack Horner, of the Montana
State University Museum, who oversaw the extraction of red blood cells from the fossilized leg of a Tyrannosaurus Rex which could contain viable DNA by graduate student Mary Schweitzer have been widely disputed (Breo, 1993). No claims have been greeted with a "warm welcome". This is mainly due to the fact that since dinosaur DNA is unknown to science, being absolutely sure of what is being extracted is almost impossible (Kiernan, 1993). Assuming that scientists could actually obtain and isolate actual dinosaur DNA, and even fill in the gaps in the DNA with that of another organism, with the intention of creating an actual dinosaur, the problem that remains is how? In the cloning of "Dolly", scientists, after inserting the genetic material into a fertilized egg, could input the genetically altered egg into the womb of a surrogate sheep ("Are
Movies Science", 1996). To create a dinosaur, one would need to implant the material into an egg, derived of the same species or at least similar. Since nothing is known about dinosaur DNA, it would be impossible to distinguish a species. Also the lack of information would make it hard to choose a host egg that provided the proper environment for the dinosaur embryo (DeSalle &
Lindley 1997). Even if one were able to get past this somehow, and a dinosaur were hatched, how would we take care of it? So little is known about dinosaur diets and behavior, that it would be very hard to accommodate the creature (Waters, 1995). In addition, there are so many new and altered diseases in the atmosphere at present time than there were in the times of the dinosaurs, that it would be next to impossible to keep a free-roaming dinosaur healthy. The most probable place a dinosaur would be kept would be in a sterile lab facility, much unlike the park Michael Crichton created in his book (Lessum, 1993). Yet however improbable, scientists continue on in their quest to create a dinosaur. "The risk is well worth the end result," states George Poinar (Poinar, 1994).
Indeed, the recreation of a dinosaur would lead to remarkable new discoveries about their behavior, eating habits, disease resistance, and quite possibly determine the reason for their extinction, not to mention, amazing millions of little kids around the world. Conclusion Cloning ancient life forms like in the movie and book, Jurassic Park is a sequence of "long shot" chances. The path from finding and sequencing suitable DNA, as well as providing a host for growth and a suitable environment for it to function is beset with many obstacles.
Maybe after decades of extensive research in each of these areas, such a project as recreating a dinosaur may be attempted, but most scientists agree that their"extinction is permanent" (Paabo, 1993). Thus, cloning dinosaurs or any ancient organism, remains a frontier of the future. However, as David Grimaldi writes, "While it is a long way from amplifying a bit of DNA to reconstruct a whole dinosaur - or even a termite - these new developments open up many exciting scientific possibilities" (1993).