BCHM 4116 1st Edition Lecture 12 Outline of Last Lecture I Optimizing PCR conditions II Other PCR III Limitations IV RT PCR Outline of Current Lecture I Digital droplet PCR II Applications III DNA sequencing Current Lecture I Digital droplet PCR II Applications a Diagnostics b Quantification c DNA fingerprinting d Molecular evolution III DNA sequencing a Sanger sequenching i Chain termination or dideoxy method ii Involves synthesis of DNA strands by DNA polymerase from a template in the presence of dideoxy nucleotide triphosphate tha tcause termination of synthesis when incorporated iii Terminated products are run on gel and sized iv Regular contains 3 OH so will continue 1 dATP dGTP dCTP dTTP v irregular does not contain 3 OH so will not continue 1 ddATP ddGTP ddCTP ddTTP IV 2nd Sequence a genome a No need for gels i Size differentiation b 1 dNTP at a time to see which will match and provide florescent V 3rd sequencing technique a illumina sequencing i reversible 3 OH These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute ii steps 1 take off 3 on dNTP a so only 3 on original primer this ensures that only the correct dNTP will be attached 2 wash away all non specific dNTPs 3 replace 3 OH onto that 1st correct dNTP 4 then submerge with all dNTPs a so the correct dNTP will be attached 5 wash away all non specific dNTPs 6 replace 3 OH 7 repeat VI others a ion torrent
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