BCHM 4116 1st Edition Lecture 12Outline of Last Lecture I. Optimizing PCR conditions II. Other PCRIII. LimitationsIV. RT-PCR Outline of Current Lecture I. Digital droplet PCRII. Applications III. DNA sequencing Current LectureI. Digital droplet PCRII. Applicationsa. Diagnostics b. Quantificationc. DNA fingerprintingd. Molecular evolutionIII. DNA sequencinga. Sanger sequenchingi. 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 sizediv. Regular/ contains 3’ OH so will continue1. dATP, dGTP, dCTP, dTTP v. irregular / does not contain 3’ OH so will not continue1. ddATP, ddGTP, ddCTP, ddTTP IV. 2nd Sequence a genomea. No need for gels i. Size differentiation b. 1 dNTP at a time to see which will match and provide florescentV. 3rd sequencing techniquea. 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 dNTPa. so only 3’ on original primer, this ensures that only the correct dNTP will be attached2. wash away all non specific dNTPs3. replace 3’ OH onto that 1st correct dNTP4. then submerge with all dNTPsa. so the correct dNTP will be attached5. wash away all non specific dNTPs6. replace 3’ OH7. repeatVI. othersa. ion
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