Lec13_W11

Lec13_W11 - 2. DNA Sequencing Most sequencing methods are...

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Unformatted text preview: 2. DNA Sequencing Most sequencing methods are based on DNA replication so you need a primer and a DNA polymerase But If you dont know the sequence, how do you design a primer? That is why we have to clone pieces of DNA of unknown sequence into a vector you can then design the primer to a sequence in the vector to start replication pGEM Unknown insert Primer Sanger or chain termination DNA sequencing The type of sequencing that we are doing is known as the Sanger, or chain termination method. It uses a mixture of normal deoxynucleotides (dNTPS) and dideoxynucleotides (ddNTPs) that are missing the 3OH on the sugar. There are many other new methods of DNA sequencing on the market, but most of these are used for sequencing whole genomes Sanger sequencing is still the most cost effective for a smaller sequencing jobs What you need: Template DNA (the Vibrio DNA insert) Primer that hybridizes to a sequence in the plasmid upstream or downstream of the insert sequence T6 promoter sequence - see pGEM map DNA polymerase Unlabeled deoxy nucleotides (dNTPs) Fluorescently labeled dideoxy nucleotides (ddNTPs) Each ddNTP is labeled with a different color dye http://www.wiley.com/college/pratt/0471393878/stu Sequencing via Sanger chain termination method 1. Denature the double stranded plasmid containing the insert DNA 1. Lower temperature so that the primer anneals to known sequence just upstream or downstream of insert sequence 1. Carry out extension with DNA polymerase and the mixture of ddNTPs and dNTPS....
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Lec13_W11 - 2. DNA Sequencing Most sequencing methods are...

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