DNA REPLICATION (BASIC)
=The Nature of DNA Replication= Experimentation in 1958 that provided evidence for the semi-conservative nature of DNA replication. It was already suspected that DNA replication was semi conservative, but alternatives had been suggested. Some of these are outlined below: [image:http://i.imgur.com/VYUCtMs.png] =Meselson-Stahl Experiement= The Meselson-Stahl experiment was relatively straight forward and has even been called "the most beautiful experiment in Biology". It highlights how a series of relatively simple procedures can provide insights into the unseen world of molecular biology. The experiment can essentially be broken down into the following [image:http://i.imgur.com/Mfa4L8s.png?1] 0) Bacteria ('E.coli') were grown on a substrate (food) that contained an isotope of nitrogen (N15) that is heavier than than most nitrogen atoms normally found in the environment. The base pairs in DNA contain nitrogen (that is why they are referred to as nitrogenous bases). After several generation the bacteria had incorporated the isotope into their DNA making it slightly more dense (heavier) than normal. The DNA was extracted and centrifuged in a salt density gradient, where the DNA separates out at the point at which its density equals that of the salt solution. As expected, the DNA formed a band that was significantly lower than normal, indicting the bacterial DNA was in fact more dense (heavier). 1) The bacteria were then returned to substrate (food) containing the most common form of nitrogen (N14). The bacteria grew and divided. DNA was extracted from this first generation and centrifuged through the salt gradient. The DNA formed a single band that was slightly higher when compared with the parental generation (0). This essentially ruled our the 'conservative' model of DNA replication which would have produced two bands one corresponding to heavier DNA containing only N15 and one corresponding to lighter DNA containing only N14. These results showed that all the DNA molecules now contained a combination of both 'heavy' DNA (N15) and 'light' DNA (N14). The question that now remained was whether or not the original (heavier) DNA that contained the N15 had been conserved as one continuous strand or was it now dispersed in sections across both strands? 2) The bacteria continued to grow and the second generation had their DNA extracted and centrifuged. Two distinct bands were now visible. This is consistent with the semi conservative model of DNA replication. The original bacteria had 'heavy' DNA that contained only N15. Once these bacteria had divided the first generation all had DNA that consisted of one 'heavy' strand and one 'light' strand. Once these bacteria divided their of offspring would either inherit a 'light' strand (and therefore have two 'light' strands) or a 'heavy' strand (and therefore have one 'heavy' and one 'light' strand just like their parents). The higher band contained DNA from those bacteria that had inherited a 'light' strand and the lower band contained DNA from those bacteria that had inherited a 'heavy' strand. 3) As the bacteria continued to grow and divide, DNA was extracted and centrifuged. Each time two distinct bands were seen corresponding to those bacteria that either had / had not inherited a 'heavy strand'. However, because the newly synthesised strands were always made from 'light' DNA the proportion of bacteria that would inherit a 'heavy' strand decreased with each generation. This is why the higher band became more pronounced with each subsequent generation and the lower band became less pronounced. This was strong evidence for the fact that each DNA strand is conserved in it's entirety. If the 'heavy' DNA had been conserved in sections across both stands then all offspring would inherit some 'heavy' DNA. In this case there would have been a single band that became less dense (lighter) with each generation as the 'heavy' DNA continued to be divided up between offspring. The appearance of two distinct bands in the same position after each successive generation proved this wasn't the case.
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