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Lecture 22: "DNA Technologies I"

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Biology 1002B
Tom Haffie

Biology Lecture No. 22: DNA Technologies I th Wednesday March 28 , 2012 Unexplained Capabilities Of Elysia: -Most of the 3000 proteins required for energy-transducing organelles to work are found in the nucleus. -What can explain how the chloroplasts in Elysia can remain functional for so long? Could some of the Vaucheria genes have entered the nucleus of Elysia? -Did ancestors of modern-day Elysia pick up some of the genes of Vaucheria in order to sustain chloroplastic function? Horizontal & Vertical Gene Transfer: -Horizontal gene transfer (another term for lateral gene transfer) is the repositioning of genes from on organism to another. -Vertical gene transfer refers to the change in position of genes observed from one generation to the next. -According to an article published in ten National Academy of Science, horizontal gene transfer of a Vaucheria gene in Elysia may be responsible for the maintenance of the sea slug’s chloroplastic function. This is profound as it revolutionizes the understanding of how eukaryotes evolved. Background Information On PsbO: -PsbO is a nuclear gene found in many photosynthetic organisms that codes for a component of the oxygen-evolving complex in Photosystem II. It is a subunit of a protein on the luminal side of the thylakoid membrane. Expression Of PsbO In Elysia: -By using agarose gel electrophoresis, it is possible to amplify (produce enough copies to make visible) the portion (since the sequence includes around 450 base-pairs) of psbO transcript in question. This process can verify the presence of a particular gene in an organism. -What was extraordinary was not only did the transcript of such a photosynthetic gene as psbO become present in Elysia’s genome, but it was discovered 5 months after contact with Vaucheria. -It was then clear that this could not have been just a random RNA molecule (as RNA molecules degrade rather quickly), but it must’ve come from the transcription of a DNA molecule (as DNA is more stable). Polymerase Chain Reaction: -Polymerase Chain Reaction (PCR) is a remarkable major tool used for amplifying preferred DNA sequences. It is used extensively in gene studies, but also for diagnostic studies such as: forensic science, phylogenetic studies and disease testing. The Polymerase Chain Reaction Cycle: -The PCR cycle accomplishes the amplification of DNA in three basic steps: Denaturation, Annealing and Extension respectively. -Denaturation: The DNA strands are heated to extremely high temperatures (94°C) in order to break hydrogen bonds and separates strands. -Annealing: Temperature is lowered (45-65°C) to allow the binding the binding of DNA primers. DNA polymerase needs a 3’ end to catalyze DNA replication. -Extension: Temperature is heated (72°C) for the optimal functioning of taq polymerase, which replicates the specific sequence. -After 30 cycles of PCR, the DNA sequence is amplified a billion-fold. The abundance of a specific sequence is incredibly low under normal conditions in the cell, so it is often necessary to work with the capabilities of PCR. Components For PCR: -Under normal conditions, the preferred DNA template received as a sample is only about 100 – 500 ng, which is not invisible to the naked eye. -In order to amplify the favoured DNA sequence, enough single-stranded DNA primers specific to that desired region/sequence is required for 30 or 40 cycles. - Specified deoxynucleotides are also needed (for synthesis towards the 5’ end) as well as taq polymerase from Thermus aquaticus (which is impressive in its resistance to heat as it takes two hours for half of the proteins to denature at 94°C). -Even though taq polymerase is effective in extension by introduction 1000 nucleotides per minute, 1 about every 9000 bases, it will introduce an error as it doesn’t possess endonuclease proofreading ability – the ability to edit the DNA sequence. PCR & Specificity: -The DNA primers used in PCR are needed to impart extreme specificity in the process as to avoid binding to a sequence that is not preferable for amplification. As there is over one in a bill
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