11-16-06 BIMM100 Assignment7

11-16-06 BIMM100 Assignment7 - BIMM 100, FA06 Assignment 7:...

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1. You have just begun a job at Dr. Cerevisiae’s new biotech startup company where your first task is to sequence a mutant gene. You decide to use the Sanger method which makes use of dideoxy chain-terminating nucleotides. a. Draw the chemical structures of deoxyribose and dideoxyribose. Why is a dideoxyribonucleotide called a “chain terminator”? Dideoxyribonucleotide can be incorporated by DNA pol, but since dideoxyribose does not have a 3’OH, DNA polymerase cannot add any more bases onto it. b. You vaguely remember from BIMM100 that the Sanger sequencing reaction somehow utilizes DNA polymerase so you add a DNA template, DNA polymerase, and dNTP’s to a test tube and run the reaction product on a gel. No luck! You don’t see any bands on the gel. What two key components have you forgotten to add to the reaction? 1. Primer 2. ddNTP’s c. Now you are trying to remember how much of each reaction component to add. Your two choices are (A) add 1 μ M of each dNTP (dATP, dCTP, dGTP, and dTTP) and 100 μ M of one ddNTP or (B) add 100 μ M of each dNTP and 1 μ M of one ddNTP. Which choice (A or B) will generate a longer sequence read, and why? B. You want more dNTP’s than ddNTP. Using the amounts in A will probably result in a (very) short read because the ddNTP’s are more likely to be incorporated (therefore terminating the growing DNA chain). d. Finally, you get the sequencing reaction to work and see the following gel. Interpret the gel to determine the sequence of the DNA (hint: you should be able to read 15 bases of sequence). 5’ – acg acg ttc cgc taa – 3’ How many different sequencing reactions (of the type described in parts b and c) were mixed to produce this gel? 4 In a more modern version of this protocol, each ddNTP is labeled with a different fluorescent dye. In this case, how many sequencing reactions would need to be run (resulting in an image like the one shown in lecture 15, 1 of 7
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slide #17)? 1 2. After 2 weeks of hard work, you proudly bring your results to Dr. Cerevisiae, who informs you that PostDoc Bob already sent a sample of the template DNA to a company that sequenced the gene in one day for $5.95. BUT, the good news is that since you now understand sequencing so well you have been promoted to lead a whole genome sequencing effort to sequence the entire genome of a pathogenic species of yeast. You will be sequencing the yeast genome using a technique called Whole Genome Shotgun (WGS) sequencing. WGS was one of the methods used to sequence the human genome, and since then, it has been used to generate the sequences of many other organisms including dog, rice, mouse, poplar tree, and honeybee. The first step in WGS is to create several DNA libraries containing genomic DNA that has been fragmented to different lengths. The clones in these DNA libraries will be used as the template DNA for your sequencing reactions. a. Why would you want to make a genomic DNA library instead of a cDNA library?
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11-16-06 BIMM100 Assignment7 - BIMM 100, FA06 Assignment 7:...

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