Purpose
There have been sighted European freshwater turtles (Emys orbicularis) in Denmark, modalities by Salten Å (we look at two here) and Igelsø. The question which arises is, whence comes this? We were presented with two theories, but a third exists:
1st A natural teacher 's belief that swamp turtle has survived in central Jutland, and is not extinct 2000 years ago, as we thought it would. They were after his testimony to a small population of the original Danish (North European) subspecies. This survival was achieved without genuine described observations.
2nd That it is purchased pet is either put out a few copies, or that they have been systematically deployed from the genus would then be of those from the Balkans, namely Serbia and Ukraine, as it is these you can buy in pet shops.
3rd A third theory, which we do not get directly out, it is that it is a new subspecies or race of the vulnerable in the years which should have put animals out (which can not be more than 100 years, if at all) should have evolution to a sub-race. This would occur only if the individual mutated, this is likely, but the probability is said to be so small that this theory can be readily dismissed.
You could say much about the first theory, if I should come with a comment, I would immediately call it pop-biology, as this theory attract much media interest but not directly by the large in itself. Since that Nature which we are presented with does not come with any concrete evidence but just a lot of circumstantial evidence and postulates. However, one can say that it has been widely accepted, for example may be mentioned that in a otherwise very informative online dictionary as wikipedia, you can read, "European Pond Turtle (Emys orbicularis) was rediscovered in Denmark, Jutland in 1997." Here we must say that they indicate that it is not just a few exposed specimens, but an actual population.
Our aim with this exercise is to determine where these turtles observed really from. We have DNA from Salten Å, Poland, Ukraine, Greece and Serbia. If their DNA matches with those from the Balkan regions, we must conclude that they are exposed, whereas, they are different, one can say that they are surviving copies of the 2000 years old Midtjyske subspecies.
Theory
There is some theoretical substance which must be here, so I chose to break it down into these four sub-themes:
· European pond turtle:
The European pond turtle, Emys orbicularis is a turtle found in South and Central Europe, West Asia and North Africa, but since this has such widespread areas describing often Europe as a undeart named Emys orbicularis orbicularis to differentiate between it and another also widespread subspecies Emys orbicularis orientalis.
It lives in slow running water, and hibernation for up to seven months. It is eggs, as many other reptiles and for them to be able to hatch, the warmest summer month's average temperature exceed 18 ° C.
· Electrophoresis and restriction enzymes:
An electrophoresis is a kind of purification of DNA pieces. But before we can proceed with electrophoresis, you should have cut its DNA into pieces. To this user Mon restriction enzymes that are specifically designed to cut after a special code. The enzymes we used is called ECO R1 and R1 PST, and these rocks, respectively, by G Eftf. of AATTC and CTGCA Eftf. of G, this looks like this:
When you have cut the DNA into smaller pieces add, a heavy marker buffer so that later in the experiment can see how much DNA piece is horizontal.
The gel, called the sieve is made of agarose, deionized water and gel buffer. Agarose is as the name suggests a sugar (carbohydrate), extracted from Japanese red algae. The volume of agarose determines how close the filter is, you have small sequences of DNA wants a dense filter, and you add therefore very agarose. When we were producing the dissolved 0.5 grams we agarose in 50 mL of TAE buffer (see below) and heat it in microwave oven so that the two substances melted together. Then pour the still liquid gel into the bathtub where it solidifies.
When one has devoted his DNA material in the gel sets Mon flow to each end of the bath. At one end is the anode (positive electrode) and the other is the cathode (negative electrode). It has reversed the gel such that the end was allocated mix of facing away from the anode. This is because DNA molecules are negatively charged, so they will migrate towards the anode. Travel through the gel takes place at different speeds depending on molecule size. The larger molecules are the slower they move through the gel. The reason we have added markørbufferen is that it will always move faster than some of the DNA molecules, so when the cursor has come down to the other end off the Mon stream.
· PCR, color marker and TAE buffer:
One critical problem in this study is the amount of DNA material to do a proper electrophoresis, which will give a realistic picture, you need very DNA material. If you do not, you can use a molecular biological reproduction method called PCR, Polymerase Chain Reaction (polymerase chain reaction) which were invented as early as 1983 by Kary Mullis, who 10 years later received the Nobel Chemistry Prize for precisely this discovery. As the name suggests, it is an artificially produced replikationsmetode. It is relatively simple, I have tried to illustrate in the figure below:
· Step 1: DNA is poured into a container, together with polymerase and free nucleotides.
· Step 2: The entire substance is heated to slightly above 65 ° C (at BIO RAD machine is more than 90 ° C) at this temperature break hydrogen bonds between nucleotides in the DNA for. And there is no risk of damage to nucleotides since they first melts at +300 ° C
· Step 3: The substance cooled again to below 65 ° C, after which free nucleotides are committed to the free seats in the DNA, and in this way has now produced two identical DNA strands, this sequence can be done again, and each time doubled the number of DNA pieces.
The color marker I mentioned in the theory section on electrophoresis, which must say indicate how far the DNA pieces is reached in the gel, they would not want to walk completely through it. This implies that the pieces of the cursor is less than the smallest pieces of DNA. But it is also made such in our experiments, so the cursor has a speed of 500bp corresponding to a sequence of 500 base pairs. The highlights of this marker are doing will be visible to the naked eye, so that as said on when to turn off.
TAE Buffer (Tri-Acetate-x) key role is to keep DNA stable at pH 7.6. This must be done because a pH value of 7.6 ensures that the DNA remains negatively charged. The image on the right shows AMP Adenosine monophosphate, a nucleotide, and one can see the negative end with O-.
· Size marker:
In this study, it is important to compare the size of the cut DNA pieces, so to have a reference using a so-called size marker, which in this case is a DNA size marker. The DNA we used came from a virus and we must assume that it is a virus that scientists have determined its entire genome, and therefore all base sequences. So when we know the size of all fragments, measuring at how far they have walked in the gel, we can make a graph in a Semi-ordinates. This shows the number of base pairs up the second axis and number wandered mm. in the gel out of the first axis. The results seen in the section entitled "Results".
Method
see Exercise Guide.
Error Sources
During the experiment we came to pour TAE buffer into the tube at the end. For this reason, our DNA samples slightly smudged. But this did not alter the outcome, which ultimately was the best, compared to the other groups. A fault is it, but maybe an ameliorative one of its kind.
Another possible source of error, although not documented, it could be that gel is not completely homogeneous but the concentration of sucrose could be just a fraction higher in some areas of the gel, this would then lead to variations in travel.
Results
I have chosen to use the manufacturer's (BIO-RAD) image of the gel, as this will give the most accurate results.
1: Size marker
2: Salten 1
3: Salten 2
4: Poland
5: Ukraine
6: Greece
7: Serbia
Here are the results from the regression of our viral DNA:
The blue line as seen, exponential regression, and the function you get out of it is drawn with thin dotted line.
I then made a rule so you can calculate the number of base pairs from the number of migrated mm. However, I have chosen to exclude the first point with 23,130 base pairs, since it differs from the otherwise straight lines in the Semi-coordinates, but with the other results are the rule to look like this: where f (x) is the number of base pairs, and x is the number of migrated mm. From this model and the measured distances from the gel, you can run a table showing the different band base pairs for the various turtles DNA:
Salten 1
Salten 2
Poland
Ukraine
Greece
Serbia
3648
2820
2970
3821
3189
2969
2969
1112
1,773
3078
2620
2070
740
862
1117
740
1061
1116
Discussion
Looking at the table of turtles and their base pair sizes, you can relatively easily determine that the turtle "Salten a" face, and that it originated from the Ukrainian subspecies. The biggest difference in base pairs between the two, is at 173 base pairs. One must say that it is relatively little when 173 base pairs almost nothing is, and we found that there was an uncertainty marker around. 100-200 base pairs. When we look at Salten 2 see it immediately worse. It has no immediate similarities with any of the others. This is a strong indication that the strains from the original northern European subspecies, although they were counted as extinct 2000 years ago. But you must tell the result's defense that if only the population is small enough, it will have been very difficult to spot in the wild. Turtle overwinters as I said in seven months, and there are otherwise here vegetation around lakes and rivers are partially gone, so that one could easily spot it. It should also be said that it is a nocturnal animal, and she will feel even remotely threatened, it glides quietly and silently into the water.
Another fact which you can see from the gel, is that they are all very closely related, however, we knew this beforehand, but it is clearly illustrated in the table in it that they all have a DNA sequence approximately 3000 base pairs. More specifically, the average of the band of 3016, and the largest deviation from this is Greece with 173 base pairs, and again this is within the uncertainty of the cursor.
We were initially told of that, "... if this summer's hottest month average temperature is below 18 °, their eggs do not hatch" This fact, I examined and considered from DMI's website a depiction of Midtjyllands climate from 1961 to 1990:
And, as evidenced by the dark line indicating the mean temperature has gennemsnitstemperatu-clean in the hottest months (July to an average of 15.4 ° C) was not near the required 18 ° C, so it provides a dilemma. Either we must wholly reject our results, which otherwise strongly suggesting that they originated from the original Danish subspecies, or may choose to ignore the fact that in a number of years at 29 years, there has been a temperature of 18 ° C.
If our restriction enzymes had not worked our gel would have given us a very bad image to work with, for in that there would not be cut anywhere in the DNA, it would become one very long base sequence that would move very slightly in the gel because agarosemængden adapted very small pieces. Therefore we would only see one big line straight out of the well, and we would not be able to use this result to something.
Conclusion
Now that I have come to the conclusion that I should have one definitive answer. This is just not true. I have two contradictory arguments: If you choose only to look at the gel, I find that swamp turtle Salten 1 is an exposed "poor" because the composition of DNA sequences recalls hitting a lot about it from Ukraine, which also is available for purchase in Danish animal dealers. And Salten 2 is a surviving subspecies, who has lived in semi-hiding in 2000 years.
If you choose the other hand also to look at the graph from DMI, we see here the contradictory argument. There have in the period 1961-1990 was no one in July with an average temperature of the required 18 °. This indicates that it is not a survivor and childbearing population, as it simply would not have a month which is warm enough to hatch its eggs.
However, I would say that should this trial be successful, we must throw away the argument with the necessary 18 °, as this completely spoils the otherwise fine performance; that there is a surviving wild swamp tortoise in Denmark. Or should we turn it on and find a solution to this problem. And a possible theoretical solution to the problem could be that the turtle has been exposed to a selective pressure that would penalize the ability to lay eggs which can hatch at lower temperatures, so that there simply has been an evolution in relation to the freshwater turtles in southern Europe. This would give very good sense.
