BMB460_Assignment 5 - For the article that I found most...

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Unformatted text preview: For the article that I found most interesting in the field of biochemistry is an in depth article about Rolling-circle amplification. This article I found interesting to me because of the fact that I use Rolling-circle amplification in my research that I am currently conducting in Paul Millards lab. I wish to explore the processes and uses for Rolling- circle amplification in my experiments and why it works in others over PCR. The article consists of information and experiments on the following things. Cloning DNA sequences then inserting them into vectors that can replicate in Escherichia coli is one of the most modern techniques of molecular genetics. This method has limitations due to certain sequences that are difficult to propagate by using available vector host relationships. Some products of these sequences are toxic to the host cell, whereas others are prone to replication errors resulting in a useless sequence. PCR has certain flawed characteristics that make it hard to predict as the basis for a general cell-free cloning of a DNA sequence. The article calls this a mutant jackpots this is the result when mutations arise during the early cycles of a PCR reaction results in all of the copies that are derived form that sequence to have these mutations. The rolling-circle amplification (RCA) reaction carried out by the 29 DNA polymerase at a standard temperature of 30C the ideal length Strands of DNA to be copied are averaging 70 kb in length. Therefore in the experiments outline in this article the products from small circular templates contain many tandem repeats of the starting sequence of DNA molecules. These strands then in turn serve as the templates for new DNA strand synthesis so that the reaction undergoes an exponential phase in which the rate of initiation of new strands is proportional to the quantity of DNA already formed.( Clyde A. Hutchison) Background synthesis that happens is a commonly observed occurrence when 29 polymerase is primed with a random hexamers. This background synthesis, which is believed to be templated by the random hexamer primers has made the amplification of very small amounts of DNA difficult. We reasoned that reducing the volume of a 29 polymerase reaction, while keeping the amount of template constant, would increase the template concentration and suppress background synthesis. This approach led to success in cell- free cloning of individual DNA molecules by using submicroliter reaction volumes.( Clyde A. Hutchison) One application of this method that is described here is the cell-free cloning and sequencing of single synthetic DNA molecules without propagation in bacteria. This method can be used for molecules that are unclonable in E. coli. For example, synthetic X174 genomes containing frame shifts in essential genes can be cloned by using 29 polymerase. These genomes cannot be propagated as phages or, in general, propagated in E. coli by cloning in plasmid vectors either, because several of the X174 gene products E....
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BMB460_Assignment 5 - For the article that I found most...

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