molgenchp4 - Chapter 4: Sequencing Genomes Molecular...

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Sequencing Genomes Molecular Genetics Lecture Outlines We will be covering only the chain termination (Sanger) method, and not the chemical degradation (Maxam & Gilbert) method of sequencing. Fig. 4.1: What is the resolution of a polyacrylamide sequencing gel? 10 to 1500 nucleotides resolve SS DNA that differ by 1 nucleotide Tech Note 4.1: Why not use agarose for sequencing? Because its resolving power is not good for SS DNA that differ by 1 nucleotide What is the range of best resolution for acrylamide? 10-1500bp For agarose? 200-20kb What is bis? methylenebisacrylamide What is its purpose? Ratio of this to acrylamide is what determines pore size in the gel bis-acrylamide is the linker in the gel What are ammonium persulfate and TEMED used for? Polymerization of acrylamide:bis solution What is urea used for? A denaturant that prevents intrastrand base pairs from forming in the chain- terminated molecules (straightens out DNA) Why use glass plates to form the gel; why not just pour it like an agarose gel? O2 ruins the gel Why do you have to use radioactivity with nonautomated sequencing? need the radioactivity to see the small bands especially since there probably is a low concentration What’s a capillary gel? Same PAGE except in a capillary (1 well) Fig. 4.2: Name all the components in a Sanger sequencing reaction. Primer, template, dNTPs, polymerase, fluorescence labeled ddNTPs Why do you need a primer? Start synthesis on the template DNA What is the structure of ddNTP? No –OH on 3 rd C, means no more bases can be added What is the function of ddNTPs in the sequencing reactions? Terminates the adding of bases to a sequence What is common to all the members of the "A family"? All end in ddATP What does the length of each A family member represent, in terms of the template? There are Ts at each of these lengths How many reactions per DNA sample sequenced? 4 How do they differ in recipe? Different ddNTP in each reaction How do you read a sequencing gel? Bottom to top Where in the gel, top or bottom, do you find sequences closest to the primer? bottom What would happen if you added too much ddNTP? you would get only short products Fig. 4.3: How are the bases called if all four are in one lane? How are each of the 4 families of products (G,A,T,C) labeled a different color? Different color fluorophore What does the "peaks" readout mean? That ddN is present at that length What you want in a DNA polymerase for sequencing. What is high processivity and why is it desirable? Able to produce long polynucleotides before terminating through natural causes; want it to proceed long enough to incorporate ddNTP to determine sequence Why must 5' --> 3' exonuclease activity be eliminated? Removal of nucleotides from 5’ end of new strands would alter length making sequence determination impossible What is it's function in nature? Replacing RNA primers and connecting lagging strand in replication
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molgenchp4 - Chapter 4: Sequencing Genomes Molecular...

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