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Biochem Brandl 10

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Biochemistry 2280A
Eric Ball

Biochem - Lecture 10 - Nov. 29th Case Study (con’t) Chain Termination Sequencing: • two methods were developed in the early 1970’w to sequence DNA • chemical sequencing (Maxam and Gilbert) • dideoxy or chain termination sequencing (Sanger) • involves the enzymatic synthesis of a DNA strand in the presence of base specif- ic chain terminations 2’, 3’ Dideoxynucleotides: • Figure 10.20 • dideoxy NTPs act as chain termination • dideoxy lacks 3’ OH so it can’t add another nucleotide • stops the chain - chain terminators • key to chain termination sequencing Materials Required to Sequence a DNA: • DNA to be sequences (template) • oligonucleotide primer that anneals to the template • DNA polymerase • dNTPs (G, A, T, C) • ddNTPs (small amount - 0.2% of dNTP) Extension Reaction in the Presence of the Dideoxy: • Figure 10.20 • 1 in 500 chance dideoxy will be incorporated at that spot, and chain will be termi- nated • ie - at each base where a dA should be inserted into the growing chain, there is a 1/500 chance that a ddA will be inserted and the chain termi- nated • a collection of DNA molecules is made which all ahve the same 5’ end, but they differ at the A at which they stop • denature DNA molecules, analyze the products by gel electrophoresis and read the DNA sequence as a ladder from 5’-3’ • autoradiography of a sequencing experiment; typically about 300 bases can be read on sequencing gel • automated DNA sequencing: each ddNTP is labeled with a different dye that fluo- resces at a different wavelength • Question: If you added too little of the ddNTPs to a sequencing reaction what would happen? • Answer: the chains would “never” stop and no sequence could be read • Question: To sequence YFG in the plasmid the primer(s) can have a sequence that anneals to the plasmid? • Answer: true • Question: Which of the following need to be considered when expressing your fa- vorite human protein in E. coli? • Answer: cDNA does not have a promoter, different mechanisms of trans- lational initiation, and different protein modification occurs in prokaryotic and eukaryotic cells • cDNA not having a promotor and the different mechanisms are handled through the design of the plasmid used for cloning Plasmid for Expressing YFP in E. coli: • Figure 10.24 • promotor driving your gene; bacterial promotor that you insert before inserting YFG • contains strong promotor • ribosome binding site and ATG downstream from promotor • right after ATG, there is a useful cloning site into which you can insert YFG Production YFP: • purify protein from E. coli • check protein for purity, activity and toxicity • YFP may be subject to proteolysis in E. coli
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