Bi1X_Assignment3_PCR_Digestion_solution_2011

Bi1X_Assignment3_PCR_Digestion_solution_2011 - Bi 1X,...

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Page 1 of 9 Bi 1X, Spring 2011 Assignment 3: PCR and Restriction Enzymes Polymerase Chain Reaction (PCR) One of the key steps in our experiment is amplifying the environmental genomic DNA that we have extracted from the Caltech ponds. We will achieve this amplification using the ubiquitous process of Polymerase Chain Reaction (PCR). PCR is a powerful technique that, along with restriction enzymes and a couple of other "tools," has transformed modern molecular biology through its ability to amplify specific pieces of DNA, even if there is only a small amount of "template" DNA to start with. As shown in Fig. 1 below, through a repetitive series of temperature cycles, DNA polymerase acts as a molecular Xerox machine copying molecules again and again. The questions of this problem set will familiarize you with the basic breakthrough concept of PCR as well as the restriction enzymes. References on PCR: http://www.idtdna.com/support/technical/TechnicalBulletinPDF/Polymerase_Chain_Reaction.pdf http://www.idtdna.com/support/technical/TechnicalBulletinPDF/A_Basic_PCR_Protocol.pdf Problem 1: Understanding PCR A. Templates, amplicons and anchored products in a PCR While it is true that the total number of molecules in a PCR reaction increases exponentially as a function of the cycle number, closer inspection reveals that this population is actually the sum of three distinct populations at any given moment in the PCR tube: there is the "original template", the "amplicons" and the "anchored products", all of which are targets for further amplification. The original template is simply the original molecule(s) from which we are trying to amplify a certain sequence. The amplicon is the double stranded DNA that stretches from the sequence of the forward primer to the sequence of the reverse primer; this is the desired end product. The anchored product is different from the amplicon in that only one side is bounded by the forward or reverse primers—there is no cue to tell the DNA polymerase when to stop replicating on the other side. Therefore, anchored products have an undetermined length that will be longer than the amplicon length. Note that anchored products are amplified off of the original template, whereas amplicons are actually formed from replication of either anchored products or other amplicons and not of the original template. Figure 1 below depicts the PCR amplification for the first three cycles and shows the original template, the anchored products and the amplicons. i. How many original templates, anchored products, amplicons and total molecules exist in the PCR tube at the start of the reaction and at the end of the first, second and third cycles (i.e. n=0,1,2 and 3) in Figure 1? For example, for n=1 (at the end of the first
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Page 2 of 9 cycle) there is one original (double stranded) template molecule, one anchored product (comprised of two single stranded DNAs) and zero amplicons, for a total of two molecules. You may assume that the amplification efficiency is 100%, i.e., at the end of
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Bi1X_Assignment3_PCR_Digestion_solution_2011 - Bi 1X,...

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