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October 4 - October 4 2010 Lecture 10 Continued(Molecular...

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October 4, 2010 Lecture 10 Continued (Molecular Genetic Techniques) Dideoxy Chain-Termination Method of DNA Sequencing DNA sequence reaction mix o Reaction One DNA Polymerase Oligonucleotide primer DNA template Usually prepared by DNA polymerase chain reaction dNTPs (100mM) ddATP (1mM) labeled so that it fluoresces at a certain wavelength o in order to extend a DNA chain you need a free 3’ hydroxyl o in dideoxyribonucleoside triphosphate there is a hydrogen rather than a hydroxyl at the 3’ position this makes it a chain terminator o Reactions 2,3,4 are the same except ddATP is replaced by ddGTP, ddTTP and ddCTP, respectively Get a population of labeled molecules of different lengths the length of the molecules depends on the number of nucleotides in the primer plus the distance from the end of the primer to each successive cytosine (or guanine in the newly synthesized strand) o Where theoretically the chain could end Some of the molecules will terminate in each ddNTP Denature the mix Gel electrophoresis o Molecules will migrate through gel of polyacrylamide o Add the reaction mix in a well and subject it to an electric field o The molecules have a negative charge so they will move towards the positive pole o The smaller the molecules are the faster (and further they move)
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o Based on the distance they travel we can build the sequence of the DNA by determining at what base pairs specific ddNTPs end up in the gel Microcapillary tubes separate DNA strands as effectively as gel electrophoresis
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